EINFÜHRUNG Das CCR Blickt Nun Auf Sieben Jahre Seines Bestehens

Einführung

EINFÜHRUNG

as CCR blickt nun auf sieben Jahre seines rité auf durchaus hohem Bestehens zurück. Gegründet wurde es im Niveau betrieben, aller- D Jahre 2003 als ein interdisziplinäres kardio- dings nicht in wünschens- vaskuläres Forschungszentrum an der Charité, einer wertem Maße koordiniert. renommierten, traditionsreichen Stätte medizinischer Verschiedene Hintergründe und Traditionen der ein- Forschung, die dieses Jahr auf ihr 300-jähriges Beste- zelnen Fakultäten und Klinika sowie die mangelnde hen zurückblickt. inhaltliche und strukturelle Vernetzung innerhalb der Zum Gründungszeitpunkt des CCR hatte die „alte“ Berliner kardiovaskulären Forschungsinstitutionen, Charité, also das Klinikum in Berlin-Mitte, das als zwischen Kardiologen, Nephrologen sowie kardio- Medizinische Fakultät der Berliner Humboldt Universi- vaskulär orientierten Physiologen und Pharmakolo- tät bis zur deutschen Wiedervereinigung im Jahr 1990 gen, zwischen Grundlagenforschern und Klinikern der medizinische Leuchtturm der Deutschen Demo an verschiedenen Standorten der Charité kann als kratischen Republik (DDR) eine Ursache dafür angesehen werden, dass es in war, bereits eine Fusion mit Berlin - mit Ausnahme eines „Transregio“-Verbundes dem Westberliner Virchow- der Deutschen Forschungsgemeinschaft (DFG) zum Klinikum hinter sich; eine Thema Myokarditis - bisher nicht zu grösseren insti- weitere Fusion wurde in der tutionellen Forschungsverbünden kam, wie etwa zu folgenden Dekade mit dem einem kardiovaskulären Sonderforschungsbereich Universitätsklinikum „Ben- der DFG. jamin Franklin“ der Freien Universität (West-) Berlins Mit der Gründung des CCR war die Hoffnung ver- vollzogen. Die Charité wurde bunden, diese Lücke zu schliessen, die teilweise damit als eigenständige Körperschaft mit beinahe divergierenden kardiovaskulären Forschungsaktivi- 15.000 Mitarbeitern zum grössten Universitätsklinikum täten einzufangen und zu gemeinsamen Projektan- Europas. Ähnliche Entwicklungen hat es in anderen strengungen zu bündeln. Dies ist bisher zum Teil europäischen Hauptstädten gegeben, z.B. in London gelungen. und Paris. Die Hintergründe dieser Fusionen waren im Wesentlichen ökonomischer, nicht unbedingt wissenschaftlicher oder Patienten-ori- entierter Natur.

Kardiovaskuläre Forschung im Grundlagen- und klinischen Bereich wurde an allen Standorten der Cha-

5 Einführung

Eine Initiative für einen Sonderforschungsbereich zum Eine weitere Bereicherung des CCR stellt die Auf- Thema Vaskulärer Stress ist auf den Weg gebracht. Sie nahme von Dr. Michael Schupp im August 2010 dar, wird wesentlich von Mitgliedern des CCR bestritten. eines ehemaligen Doktoranden aus dem Institut für Vielfache räumliche Umstrukturierungen sowie zeitrau- Pharmakologie im CCR, der nach einem fünfjährigen bende Vorbereitungen für einen neuen medizinischen Forschungsaufenthalt in den USA als Stipendiat der Studiengang an der Charité, beide insbesondere den Emmy-Noether-Stiftung der DFG ans CCR zurückge- Bereich der Vorklinik betreffend, haben dieses Projekt kehrt ist (siehe spezifischer Text). verzögert. Parallel dazu beteiligt sich eine Reihe von Diesen erfreulichen Entwicklungen steht ein grosser Wissenschaftlern des CCR an einem Projektantrag Verlust entgegen: die Arbeitsgruppe von Prof. Patricia der Charité für ein vom Bundesministerium für Bildung Ruiz Noppinger hat Ende 2009 das CCR verlassen, und Forschung (BMBF) in Zusammenarbeit mit der da sich Frau Prof. Ruiz anderen Aufgaben im wissen- Helmholtz-Gesellschaft initiiertes deutschlandweites schaftlich-administrativen Bereich zugewandt hat. Vernetzungsprojekt namens „Deutsches Zentrum für Herz-Kreislauf-Forschung (DZHK)“. Die Initiativanträge Die organisatorische Struktur des CCR hat sich im zu diesem Projekt sind eingereicht; eine erste Aus- Berichtszeitraum nicht wesentlich verändert; sie wahl unter den Antragstellern wird Ende des Jahres ist durch die im Jahre 2008 vom Fakultätsrat der stattfinden. Charité verabschiedete neue Satzung des CCR vorgegeben. Der CCR-Vorstand setzt sich wie folgt Im Mai 2010 hat das CCR eine wichtige Berei- zusammen: cherung erfahren durch Auf- nahme des von Prof. Pontus CCR-ORGANISATION: Persson geleiteten Institutes für Vorstand

Vegetative Physiologie an der Cha- • Prof. Thomas Unger (Direktor) rité. Die Umsiedlung dieses Institutes • Prof. Axel R. Pries (stellvertretender Direktor) • Prof. Ulrich Kintscher war erforderlich geworden, da die • Prof. Harm Peters (kooptiert im Januar 2010 nach Charité sich von einer Vielzahl von Ausscheiden von Prof. Ruiz Noppinger) • Dekan/Dekanin der Charité Immobilien ausserhalb ihrer eigent- lichen Standorte getrennt hat. Die Satzungsgemäß gibt es weiterhin ein wichtiges Gremium, den Nutzerrat, der sich aus Projektleitern und technischen Mitarbeitern zusammensetzt. Einsicht in den gemeinsamen wis- In jüngster Zeit hat sich ein weiteres Gremium etabliert, die Arbeitsgruppen- und senschaftlichen Vorteil der Vereini- Projektleiterkonferenz, die zwar in der CCR-Satzung nicht vorgesehen ist, sich gung sowie die Bereitschaft beider aber zu einer wichtigen Informations- und Austauschbörse entwickelt hat, die einmal im Monat tagt. Seiten zu räumlichen Einschränkun- gen haben die Eingliederung der Seit 2010 hat das CCR eine wissenschaftliche Koordinatorin in der Person von Frau Dr. rer. nat. Karin Effertz. Sie kümmert sich um die Belange der Öffentlichkeits Physiologie in ein bereits voll beleg- arbeit und unterstützt Verbundanträge und Verbundprojekte am CCR. tes Forschungszentrum erleichtert.

6 Einführung

Die einzelnen wissen- Frank Zollmann entsteht die Firma CCR-Pharma schaftlichen Schwer- GmbH (siehe spezifischer Text zu diesem Projekt). punkte des CCR haben sich in den vergangenen Grosse Fortschritte hat ein weiteres Projekt aus diesem Jahren unterschiedlich Bereich gemacht: die Entwicklung eines Agonisten: entwickelt: des Angiotensin AT2-Rezeptors zum Medikament. Die von Dr. Ulrike Steckelings geführte Arbeitsgruppe hat Die kardiovaskuläre Geschlechterforschung ist den ersten Vertreter dieser neuen Substanzgruppe, ihren Weg konsequent weiter gegangen. Die von Compound 21, erfolgreich in einer Vielzahl von expe- Prof. Regitz-Zagrosek geleitete DFG-Forschergruppe rimentellen Ansätzen in vitro und in vivo getestet. zum Thema geschlechtsspezifischer Mechanismen Zahlreiche Kooperationen im In- und Ausland sind im der Myokardhypertrophie, die wesentlich von CCR- Rahmen dieses Projektes entstanden. Compound 21 Wissenschaftlern getragen wird, sieht einer zweiten wird nach Abschluss gegenwärtiger toxikologischer Förderperiode entgegen (siehe gesonderter Textbei- Untersuchungen voraussichtlich Anfang 2011 in die trag); das DFG-Graduiertenkolleg 754-Myokardiale Phase I der klinischen Erprobung gehen. Genexpression- und Funktion Das in Zusammenarbeit mit der Firma Vicore in befindet sich in der Auslaufphase. Göteborg/Schweden sowie der Universität Uppsala/ Dieses Kolleg hat mit seinen zahl- Schweden durchgeführte Projekt wird seit neuestem reichen Stipendiaten/innen die aus Europäischen wissenschaftliche Arbeit am CCR Fördermitteln im seit seiner Gründung bereichert. Rahmen des Euro stars-Programms Der relativ neue Forschungsschwerpunkt Drug Deve gefördert (siehe spe- lopment hat sich besonders rapide entwickelt: Die zifischer Text zu die- der Arbeitsgruppe von PD Heiko Funke-Kaiser vom sem Projekt). BMBF im Rahmen des Go Bio-Programms sowie von der Investitionsbank Berlin (IBB) im Rahmen des Pro Die Arbeitsgruppe um Prof. Ulrich Kintscher beschäf- fit-Programms bereitgestellten Mittel in Millionenhöhe tigt sich im Rahmen ihrer Untersuchungen zellulärer für das Projekt zur Entwicklung eines Hemmstoffs des Transkriptionsfaktoren weiterhin mit der Entwicklung vor einigen Jahren entdeckten (Pro)-Renin-Rezeptors von Modulatoren des Transkriptionsfaktors PPARγ zu wurden erfolgreich zur Findung von Molekülen mit den Medikamenten mit metabolischer Indikation auf der gewünschten Eigenschaften eingesetzt. Die Forde- Basis bereits bestehender Strukturen von Arzneimit- rung des BMBF, mit Hilfe des Go Bio-Projektes eine teln. Start-Up Firma aus der Charité heraus zu gründen, wurde erfüllt: Mit den Gründern PD Heiko Funke- Die Entwicklung von Arzneimitteln, in der Vergangen- Kaiser, Dr. Jan Schefe, Prof. Thomas Unger und Dr. heit fast ausschliesslich eine Domaine der Pharma- zeutischen Industrie, hat sich, wie am Beispiel CCR

7 Einführung zu sehen, zum Teil in den akademischen Bereich ver- den verschiedenen Charité-Standorten, als auch aus lagert, insbesondere, was die frühen Phasen der Ent- dem Max-Delbrück Centrum für Molekulare Medizin wicklung angeht. Schlagworte in dieser Hinsicht sind (MDC) in Berlin-Buch. die sog. Innovationslücke bei der Industrie, aber auch Im Bereich der vaskulären Forschung am CCR hat die sog. Translationslücke bei der Umsetzung von sich weiterhin das von PD Ivo Buschmann initiierte und neuen, der akademischen Forschung entstammen- geleitete internationale Arteriogenese Netzwerk (Art. den, Substanzen in die industrielle Weiterentwicklung Net) mittlerweile fest etabliert. Diese Arbeitsgruppe, zu Arzneimitteln. Angesichts der grossen Bedeutung die ein gemeinsames Labor mit derjenigen von Fer- dieses aktuellen Umbruchs für die Weiterentwicklung dinand LeNoble am MDC betreibt, hat erfolgreich des CCR findet sich in diesem Report ein Artikel von Fördermittel von Seiten öffentlicher und industrieller Prof. Garret A. FitzGerald, Direktor des Institute for Drittmittelgeber eingeworben und bietet ein gutes Bei- Translational Medicine and Therapeutics an der Uni- spiel translationeller Forschung von den theoretischen versity of Pennsylvania, zum Thema „Drug, Industry Grundlagen bis zur erfolgreichen klinischen Anwen- and Academia “. dung (siehe spezifischer Text).

Der dritte Forschungsschwerpunkt Vascular Plasti Der Forschungsschwerpunkt Metabolism ist weiter city hat in den vergangenen Jahren eine Modifikation verstärkt worden. Mit der Einbindung der Abteilung erfahren, im Wesentlichen bedingt durch die schon für Endokrinologie, Diabetes und Ernährungsmedizin erwähnte SFB-Initiative Vascular Stress. In dieser Ini- (Prof. J. Spranger) ist es gelungen, eine DFG-Nach- tiative, die zu mehr als einem Drittel von Mitgliedern wuchsgruppe im Emmy-Noether Programm an das des CCR getragen und von Prof. Axel Pries und Prof. CCR zu rekrutieren. Dr. Michael Schupp hat nach sei- Thomas Unger geleitet wird, bündeln sich jetzt die nem Postdoc-Aufenthalt an der University of Penn- ursprünglichen vaskulären Projekte des CCR zum sylvania in Philadelphia, USA, im August 2010 seine Thema Angiogenese, Arteriogenese, Aneurysma und Arbeit am CCR aufgenommen. Transplantationsvaskulopathie, bereichert durch neu dazu gekommene Forschungsprojekte etwa aus dem Der Bereich der angewandten Kachexieforschung um Bereich Genetik und genetisch modifizierter Tiermo- Prof. Stefan Anker konnte mit der SICA-HF Studie eine delle sowie der mikro- von der EU unterstützte Multizenterstudie zur Präva- vaskulären Funktion lenz, Persistenz, und Phänotypisierung von Adiposi- bis zu neuen system- tas, Kachexie und Typ 2 Diabetes Mellitus bei Patien- biologischen Ansät- ten mit chronischer Herzinsuffizienz einwerben und zen. Hervorzuheben übernimmt die Studienleitung. Hierdurch konnte ein ist, dass in dieser Ini- wesentlicher Baustein der metabolischen Forschung tiative viele kardiovas- des CCRs gestärkt werden. kulär tätige Arbeits- Die Integration des Forschungsscherpunktes Meta- gruppen in Berlin ver- bolism in thematisch verwandte Forschungsverbünde eint sind, sowohl aus der Charité ist ebenfalls erfolgreich fortgeführt worden.

8 Einführung

So bestehen nun enge aktive Kooperationen mit den ser), das Berliner Excellenzcluster Neurocure (Ivo DFG-geförderten klinischen Forschergruppen 218/1 Buschmann) oder die wissenschaftliche Koordination Hormonal Regulation of Body Weight Maintenance, der NIH-WARCEF-Studie und der oben erwähnten und 192/2 Skeletal Muscle Growth and Regulation, SICA-HF-Studie durch Stefan Anker. und ermöglichen eine transdisziplinäre angewandte Weitere Einzelheiten sind der Zusammenstellung wei- Metabolismusforschung am CCR. ter unten zu entnehmen und in den Berichten der einzelnen Arbeitsgruppen angeführt. Forscher aus dem CCR, insbeson- Geplant ist, unter der Leitung von Ulrich Kintscher und dere der jüngst PD Kai Kappert, wieder ein EU-finanziertes Graduier dazu gestosse- tenprogramm ans CCR zu holen als Fortsetzung der nen Vegetativen bisher so erfolgreichen Graduiertenprogramme der Physiologie unter Prof. Pontus Persson sind weiter- DFG und der Europäischen Union (EU) (Graduierten- hin massgeblich an einer neuen nierenphysiologisch/ kolleg 754; EU Graduiertenprogramm Cardiovasc / klinisch-nephrologischen Forschergruppe der DFG Marie Curie Early Stage Training Program) sowie das beteiligt (FG 1368, Mechanisms of acute kidney injury), gemeinsame Graduierten- und Trainingsprogramm in die vor kurzem im CCR begutachtet wurde mit gros- Vascular Medicine der Universitäten Padua, Gdansk sen Chancen auf Förderung. Ob sich daraus ein CCR- und Charité/CCR weiter zu führen. Die oben erwähnte spezifischer Forschungsschwerpunkt entwickeln SFB Initiative Vascular Stress beinhaltet ebenfalls ein wird, bleibt abzuwarten; der Vorstand des CCR und Graduiertenprogramm. die an der FG 1368 Beteiligten würden eine solche Entwicklung begrüssen. Am 15./16. Februar dieses Jahres fand die zweite wissenschaftliche Vor-Ort Begutachtung des CCR Netzwerke und Forschungsverbünde innerhalb durch den Wissenschaftlichen Beirat des CCR statt, des CCR und mit weiteren Berliner Institutionen ein Gremium international renommierter deutscher und innerhalb und ausserhalb der Charité sind mittlerweile ausländischer Wissenschaftler (siehe Kasten). selbstverständlich geworden. Darüber hinaus sind die am CCR tätigen Wissenschaftler natürlich auch CCR – EXTERNE GUTACHTER 2010 bemüht, sich mit Forschern anderer Einrichtungen national und international zusammen zu schliessen. • Prof. Matthias Blüher, Leipzig, Germany • Prof. Thomas Eschenhagen, Hamburg, Germany Beispiele sind u.a. Anträge bei der transatlanti- • Prof. Jürgen Floege, Aachen, Germany schen Fondation Leducq (Thomas Unger, Ulrike • Prof. Xavier Jeunemaitre, Paris, France • Prof. Michael Mulvany, Aarhus, Denmark Steckelings) zusammen mit der US-amerikanischen • Prof. Jeremy Pearson, London, UK Arbeitsgruppe um Irvin Zucker (University of Neb- • Prof. Thomas Philipp, Essen, Germany raska) und einer Reihe weiterer Wissenschaftler aus • Prof. Eberhard Ritz, Heidelberg, Germany • Prof. Heikki Ruskoaho, Oulu, Finland dem In- und Ausland; das Europäische Forschungs- • Prof. Bernward Schölkens, Frankfurt, Germany netzwerk Ingenious HyperCare (Heiko Funke-Kai-

9 Einführung

An dieser Begutachtung nahm auch der Prodekan in Bad Saarow / Brandenburg. Ein Auszug aus dem für Forschung der Charité, Prof. Rudolf Tauber, teil. Bericht der Gutachter an die Medizinische Fakultät der Vorbereitet wurde die Begutachtung seitens des CCR Charité ist im Folgenden abgedruckt: durch eine Klausurtagung aller Projektleiter im Januar

"General Founded in 2003, the Center for Cardiovascular Research (CCR) is an interdisciplinary center with 12 research groups and about 130 projects. These impressive numbers are echoed by the scientific output, multiple graduate and training programs, participation in comprehensive and collaborative scientific projects, the external funding record, the structural organization representing all research groups and the rich stream of research proposals. Overall, there was a unanimous feeling of an excellent centre of cardiovascular research that should be encouraged to continue based on its remarkable performances and possibly reinforce its activities.

Strengths The committee was favourably impressed by the high quality and the diversity of research performed at the CCR, from basic science and experiments to clinical trials and drug development. There was a consensus that this centre conducts research which is really original, innovative and productive.….The activities related to education and training of young scientists and students are also one of the strengths of the centre. Nine students got their Ph.D. through the Marie Curie program in the last 4 years. This program as well as the Graduate course coordinated by Prof Vera Regitz-Zagrosek are important instruments not only for the students, but also to stimulate discussions and collaborations between the groups of the CCR.

Limitations Cardiovascular research in Berlin probably suffers from not being sufficiently visible and structured. As a first step, Prof Unger informed us that he would submit an application to the SFB programme, joining 16 teams of cardiovascular research, many of them being in the CCR, others being outside, especially at the MDC (Prof Bader, Prof Hübner). The committee unanimously supported this initiative, hoping that it would get funded – the project being already well ranked in 2009. Success would provide a platform for a complementary application in 2011 for a Graduate School in Cardiovascular Medicine. Another limitation is the very limited amount of financial and structural support given by the Faculty to the CCR…. The committee believes that it is essential for the viability of the CCR that it has at least some central funds to allow coordination. Even though the committee understands that the Charité has to face difficult times with a 25% reduction in its overall budget, it was highlighted that the CCR on its own provided approximately 8% of the external funding of the Faculty. This should be taken into account, and closer support and connections are strongly requested…

10 Einführung

The third limitation that appeared…was the extent of new joint research projects between groups of the CCR. Links and participation to common projects exist, but there is still room for more formal established and previously discussed common projects. In that regard, the number of scientific publications involving several groups at the CCR appeared to be still too limited.

Finally, the advisors also agreed that some very few projects did not really fit into the overall concept. It would be an option for the CCR to further focus the activities and to redistribute resources and space to support the more active and collaborating groups. In that regard, the expectation that a young researcher, Dr Michael Schupp at the moment in the USA, who has obtained funds for the DFG on a project on metabolism, will return and join the CCR, perfectly fits with the renewal of the structure and research foci. Clear procedures for reallocating space in the light of scientific development should however be developed…

Concluding remarks During the past three years, the CCR has successfully continued to be a unique model of an interdisciplinary centre for cardiovascular research with very good productivity and performance. The output of the last 3 years is the most appropriate justification for continued support, development and growth. The scientific direction towards 4 research foci and a greater impact on drug development has been favourably appreciated by the committee. Since its start in 2003, the CCR has developed into an innovative and productive entity at the university. The advisors recommend that the university Charité takes actions to further expand the CCR to strengthen interdisciplinary and translational approaches in cardiovascular research in Berlin. Core functions and infra- structures, in particular, deserve active support from the university. In addition, some improvements in the CCR governance (especially increased networking between groups, increased common budget, better possibilities for space reallocation with the Faculty) should improve the visibility and the performance of this excellent research centre. CCR should under all circumstances to be continued based on the excellent track record, the past performance and future plans”.

Soweit zum Bericht des externen Wissenschaftlichen dieser Stelle nochmals für ihre Arbeit und die Zeit, die Gutachtergremiums über das CCR im Februar 2010. sie für unser Zentrum aufgebracht haben, ganz herz- Der Vorstand des CCR möchte den Gutachtern an lich danken.

11 Einführung

Um dem Gutachterurteil noch Substanz hinzuzufü- Schliesslich gilt unser gen, seien im Folgenden einige Zahlen und Fakten Dank wiederum auch angeführt: der Firma Boehringer Ingelheim, die nun schon im siebten Jahr kontinuierlich die CCR Seminarreihe BI-Lectures grosszügig unterstützt und damit den internationalen wissenschaftlichen Austausch im CCR fördert (siehe spezifischer Text).

Sieben Jahre CCR: besser als in der Anfangsphase dieses Zentrums kann man heute abschätzen, was im Hinblick auf die ursprünglichen Ziele bereits gelungen und was noch verbesserungsbedürftig ist. Ablesen lässt sich das auch im Urteil der externen Gutachter: Sie bestätigen den Wert der Interdisziplinarität, mah- nen aber eine höhere Intensität der Zusammenarbeit innerhalb der Arbeitsgruppen des CCR an. Sie aner- kennen das unter erheblichen finanziellen Restriktio- nen bisher Erreichte, wünschen sich aber eine stärkere Förderung durch die Charité sowie mehr Visibilität des Zentrums nach aussen. Sie begrüssen die individuellen wissenschaftlichen Leistungen, sprechen sich aber auch für eine Verstärkung der Vernetzung in grös- seren Forschungsverbünden sowie für Expansion des Zentrums aus, um der kardiovaskulären Forschung an der Charité mehr Gewicht zu verleihen.

Wie wird sich die Zukunft des CCR gestalten?

Gegenwärtig haben sich starke Tendenzen entwickelt, die die Zukunft der Wissenschaft vornehmlich in öffentlich geförderten Forschungsverbünden sehen, wie z.B. den klassischen Sonderforschungsbereichen der DFG oder den neuerdings in Deutschland geför- derten sog. Exzellenzinitiativen, auf die man grosse Hoffnungen setzt. Kritisch ist dazu anzumerken, dass

12 Einführung

schaftliche Netzwerke bergen die Gefahr, diese Entwicklung zu verpassen; auch sie müssen sich weiterentwickeln, um den wissenschaftlichen Fortschritt nicht zu behin- dern.

Das CCR bewegt sich nach wie vor im Spannungsfeld zwischen gewollter inter- disziplinärer Kooperation und individueller Forschungsleistung. Es wird sich, um sein Bestehen zu sichern, intensiv um For- schungsnetzwerke aus seinen eigenen Reihen und im Verbund mit anderen Insti- tutionen kümmern, gleichzeitig jedoch eine noch stärkere wissenschaftliche Fokussie- im Rahmen dieser Entwicklung viele Wissenschaftler rung anstreben und vor allem individuelle Forscher von ihre Forschungsthemen häufig nicht mehr nach indi- hoher Qualität anziehen und integrieren müssen. Dies vidueller Neigung und Eignung, sondern eher nach um so mehr, als die einzelnen Forschungsschwer- ihrer Netzwerktauglichkeit aussuchen. Das muss nicht punkte der Charité gegenwärtig in einem intensiven unbedingt mit einer Qualitätssteigerung einhergehen. Wettbewerb um Wissenschaftler und Ressourcen Während weiterhin nicht von der Hand zu weisen ist, stehen, in dem sich die kardiovaskuläre Forschung dass durch derartige Netzwerke Synergien ausge- gegenüber anderen Disziplinen wie etwa den Neuro- hoben und die oft knappen Ressourcen gemeinsam wissenschaften nur behaupten kann, wenn sie inner- besser genützt werden können, bleibt doch festzuhal- lich geeint auf exzellente wissenschaftliche Qualität ten, dass bis zum heutigen Tag die wirkliche Exzellenz setzt und dies auch nach aussen hin glaubhaft ver- wissenschaftlicher Leistung immer individuell erbracht mitteln kann. worden ist, oft ohne Einbindung in Forschungsver- bünde, ja, dass diese aus verschiedenen Gründen Unter diesen Voraussetzungen sollte das CCR der individuellen Exzellenzentfaltung durchaus auch als Kristallisationspunkt und Integrationszentrum hinderlich sein können. Darüber hinaus gilt es zu kardiovaskulärer Forschung an der Charité auch bedenken, dass auch die wissenschaftlichen Struk- über deren 300-jährigen und seinen eigenen sieb- turen selbst einem kontinuierlichen Wandel unter- ten Geburtstag hinaus in eine erfolgreiche Zukunft worfen sind. Ein Beispiel dafür ist Entwicklung des blicken können. Zusammenspiels von akademischer Forschung und industrieller Entwicklung im Hinblick auf Arzneimit- telentwicklung, auf die in diesem Report ausführlich Prof. Dr. med. Thomas Unger eingegangen wird. Traditionelle akademisch-wissen- Direktor des CCR

13 Introduction

INTRODUCTION

he Center for Cardiovascular Research to content and structure within Berlin’s cardiovascular can look back on seven years of success- research institutions, between cardiologists, nephrol- T ful research work. The CCR was founded in ogists and cardiovascular-oriented physiologists and 2003 as an interdisciplinary cardiovascular research pharmacologists, between basic and clinical research- center at the Charité, a renowned, traditional site of ers at different Charité sites can be viewed as a reason medical research that this year celebrates its 300th why in Berlin – except for one “Transregio” network of anniversary. the German Research Foundation (DFG) on the topic of myocarditis – large-scale research networks like a DFG collaborative research center on cardiovascular diseases have not yet been established.

The founding of the CCR was connected with the hope of bridging this gap and of reorganizing the sometimes divergent cardiovascular research activities into joint At the time the CCR was founded, the “old” Charité, project endeavors. So far this has succeeded in part. i.e. the hospital in Berlin-Mitte, which served as the An initiative for a collaborative research center (SFB) medical faculty of the Humboldt University Berlin and on Vascular Stress has been launched, mainly through was the medical flagship of the German Democratic the efforts of members of the CCR. Much restructuring Republic (GDR), had already undergone a merger with respect to utilization of space and facilities as well with the Virchow Hospital in West Berlin. Another as time-consuming preparations for a new medical merger was undertaken in the following decade with curriculum at the Charité, both of which have had a the university hospital “Benjamin Franklin” of the Free great impact on the preclinical program, have delayed University in (West) Berlin. The Charité as an inde- this project. Parallel to this, a number of CCR scientists pendent corporation with nearly 15,000 employees are participating in a project proposal of the Charité thus became the largest university hospital in Europe. for a Germany-wide network project “German Car- Similar developments have taken place in other Euro- diovascular Center (DZHK)” initiated by the German pean capitals, e.g. in London and Paris. These merg- Federal Ministry of Education and Research (BMBF) in ers had a primarily economic background and were collaboration with the Helmholtz Association. The first not necessarily scientific or patient-oriented. proposals for this project have been submitted; selection of applicants will take place at the end of the year. Basic and clinical cardiovascular research was carried out at all Charité sites on a very high level; however, In May 2010 the CCR made an important gain through it was not coordinated to the desired extent. Different the incorporation of the Institute of Vegetative Physiol- backgrounds and traditions of the individual faculties ogy, directed by Professor Pontus Persson, into the and hospitals and insufficient networking with regard Charité. The resettlement of this institute became

14 Introduction necessary because the Charité was in the process The individual research of selling many of its properties outside of its primary areas of the CCR have locations. The scientific advantages of the incorpora- developed differently in the tion and the willingness of both sides to accept space past years: limitations have facilitated the integration of physiology into our already fully occupied research center. Cardiovascular gender Another addition to the CCR is Dr. Michael Schupp, a research has continued its work. The DFG research former doctoral student at the Institute of Pharmacol- group headed by Prof. Regitz-Zagrosek on the topic of ogy in the CCR, who returned to the research center gender-specific mechanisms of myocardial hypertro- in August 2010 after a five-year research stay in the phy, which is primarily comprised of CCR scientists, is U.S as fellow of the Emmy Noether Foundation of the looking forward to a second grant period (see separate German Research Foundation (see specific text). article). The DFG Graduate School 754 “Myocardial These positive developments stand in contrast to Gene Expression and Function” is a great loss: the research group of Professor Patri- being phased out. This graduate cia Ruiz Noppinger left the CCR at the end of 2009 school with its many scholarship because Professor Ruiz Noppinger accepted a posi- holders has enriched the scientific tion with other scientific-administrative tasks. work at CCR since its inception.

The organizational structure of the CCR has not The relatively new research focus Drug Development changed significantly in this reporting period; it is has developed especially fast. The research group led prescribed by the new statute of the CCR which was by PD Heiko Funke-Kaiser has received funds amount- adopted by the Faculty Council of the Charité in 2008. ing to millions of euros provided by the BMBF within The CCR Executive Board is comprised of the follow- the scope of the Go Bio scheme and by the Investi- ing members: tionsbank Berlin (IBB) as part of the Profit program. These funds have been successfully used for CCR ORGANIZATION: a project to develop an inhibitor of the (pro)- Executive Board renin receptor discovered some years ago and • Prof. Thomas Unger (Director) specifically to find molecules with the desired • Prof. Axel R. Pries (Deputy Director) • Prof. Ulrich Kintscher characteristics. The requirement of the BMBF • Prof. Harm Peters (co-opted in January 2010 after the resignation of Professor Ruiz Noppinger) to found a start-up company as spin-off from • Dean of the Charité the Charité with the aid of the Go Bio scheme According to the statute, there is still another important body, the Users’ Council, has been met: The CCR-Pharma GmbH is which is made up of project leaders and technical staff. currently being established by its founders Recently, yet another body has been established, the Conference of Research Group and Project Leaders, which is not provided for in the CCR statute, but which PD Heiko Funke-Kaiser, Dr. Jan Schefe, Prof. has developed into an important information exchange and which meets once a month. Thomas Unger and Dr. Frank Zollmann (see Since 2010 the CCR also has a scientific coordinator, Dr. Karin Effertz. She is in charge of public relations and provides support for cooperative applications and specific text on this subject). projects at the CCR.

15 Introduction

Another project has made great progress in this area: the great importance of this current change for the the development of an angiotensin AT(2) receptor ago- further development of the CCR, this article contains nist into a drug. The research group led by Dr. Ulrike a report by Prof. Garret A. FitzGerald, director of the Steckelings has successfully tested the first repre- Institute for Translational Medicine and Therapeutics at sentative of this substance group, Compound 21, in a the University of Pennsylvania, on the topic of “Drugs, number of in vitro and in vivo experiments. A number Industry and Academia”. of collaborations related to this project have evolved both in Germany and internationally. Following com- The third research focus Vascular Plasticity has pletion of the current toxocological tests, Compound undergone a modification in recent years, mainly due 21 is due to enter Phase I clinical trials at the beginning to the above-mentioned SFB initiative Vascular Stress. of 2011.A project carried out in collaboration with the More than one-third of all staff members of the CCR are company Vicore Pharma in Gothenburg/Sweden and involved in this initiative, which is headed by Prof. Axel Uppsala University/Sweden has just recently received Pries and Prof. Thomas Unger. The original vascular an EU grant projects of the CCR on arteriogenesis, aneurysma and within the scope transplantation vasculopathy have now been enriched of the Eurostars by new research projects from the area of genetics and program (see genetically modified animal models as well as micro- corresponding vascular function, including new approaches from text about this systems biology. Significantly, many cardiovascular project). research groups in Berlin are involved in this initiative, both from the different Charité locations and from the The research group led by Professor Ulrich Kintscher, Max Delbrück Center for Molecular Medicine (MDC) within the scope of its investigations into cellular tran- in Berlin-Buch. scription factors, is studying modulators of the tran- In the field of vascular research at the CCR, the Arte- scription factor PPARγ in order to develop drugs with riogenesis Network (Art.Net) initiated and headed by a metabolic indication, based on already existing drug PD Ivo Buschmann has since become well established structures. internationally. This research group, which runs a joint laboratory with the Drug development, in the past almost exclusively a research group of Fer- domain of the pharmaceutical industry, as can be seen dinand LeNoble at the in the example of the CCR, has now been taken over MDC, has success- in part by the academic community, especially with fully solicited funding respect to the early phases of development. Keywords from the public sector here are the so-called innovation gap in industry but and from industry and also the translational gap in the implementation of is a good example of new substances originating from academic research translational research in further industrial drug development. In the face of from the theoretical

16 Introduction foundations to successful clinical application (see and see whether a CCR-specific research focus will specific text). develop from this; the CCR Board of Directors and the researchers involved in the FG 1368 research group The research focus Metabolism has been further would welcome such a development. strengthened. With the integration of the Department of Endocrinology, Diabetes and Nutrition Medicine Networks and cooperative research projects (Prof. J. Spranger), the CCR has now succeeded in within the CCR and with other Berlin institutions recruiting a DFG Junior Research Group within the inside and outside the Charité are meanwhile taken scope of the Emmy Noether Program to the CCR: for granted. Furthermore, the scientists working at the Dr. Michael Schupp began his work at the CCR in CCR of course also try to enter collaborations with August 2010 after his postdoc stay at the University researchers of other national and international institu- of Pennsylvania in Philadelphia, USA. tions. With its SICA-HF study, the area of applied research on Examples for this cachexia led by Prof. Stefan Anker has succeeded in include proposals to acquiring a grant for a multi-center study funded by the the Fondation Leducq EU on the prevalence, persistence, and phenotyping (Thomas Unger, Ulrike of adipositas, cachexia and type 2 diabetes mellitus Steckelings) together in patients with chronic heart failure, and Professor with the U.S. research group led by Irvin Zucker (Uni- Anker will now direct the study. This has strengthened versity of Nebraska) and a number of other scientists in an important component of metabolic research at the Germany and abroad; the European research network CCR. Ingenious HyperCare (Heiko Funke-Kaiser), the Berlin The integration of the research area Metabolism into excellence cluster Neurocure (Ivo Buschmann) or the thematically related research networks of the Charité scientific coordination of the NIH-WARCEF study and has also been continued. As a result, there is now the above-mentioned SICA-HF study by Stefan Anker. close active cooperation with the DFG-funded clini- More details are provided in the summary CCR Facts cal research groups 218/1 Hormonal Regulation of further below and in the reports of the individual Body Weight Maintenance and 192/2 Skeletal Muscle research groups. Growth and Regulation, and this enables cross-dis- ciplinary applied research in metabolism at the CCR. It is planned to again solicit an EU-funded graduate program for the CCR, headed by Ulrich Kintscher and Researchers from the CCR, especially in the recently PD Kai Kappert as continuation of the very successful established Vegetative Physiology research group led graduate programs (DFG graduate research school by Professor Pontus Persson, are extensively involved 754, and the EU graduate program Cardiovasc / Marie in a nephrophysiological/ clinical-nephrological Curie Early Stage Training Program) and to continue research group of the DFG (FG 1368, Mechanisms the joint graduate and training program in Vascular of acute kidney injury). It was recently evaluated in the Medicine of the Universities of Padua, Gdansk and CCR and has good chances of funding. One must wait

17 Introduction the Charité/CCR. The above-mentioned SFB-initiative Vascular Stress also has a graduate program. CCR – EXTERNAL EVALUATORS 2010

• Prof. Matthias Blüher, Leipzig, Germany On February 15-16, 2010 the second external sci- • Prof. Thomas Eschenhagen, Hamburg, Germany entific evaluation of the CCR was conducted on- • Prof. Jürgen Floege, Aachen, Germany site by the CCR Scientific Advisory Board, a body of • Prof. Xavier Jeunemaitre, Paris, France internationally renowned scientists from Germany and • Prof. Michael Mulvany, Aarhus, Denmark other countries (see box). • Prof. Jeremy Pearson, London, UK • Prof. Thomas Philipp, Essen, Germany • Prof. Eberhard Ritz, Heidelberg, Germany Prof. Rudolf Tauber, Vice Dean of Research at the • Prof. Heikki Ruskoaho, Oulu, Finland Charité, also took part in this evaluation. The evalua- • Prof. Bernward Schölkens, Frankfurt, Germany tion was prepared on the part of the CCR in a retreat of all project leaders in Bad Saarow / Brandenburg in January. An excerpt from the report of the evaluators to the Medical Faculty of the Charité is printed below:

“General Founded in 2003, the Center for Cardiovascular Research (CCR) is an interdisciplinary center with 12 research groups and about 130 projects. These impressive numbers are echoed by the scientific output, multiple graduate and training programs, participation in comprehensive and collaborative scientific projects, the external funding record, the structural organization representing all research groups and the rich stream of research proposals. Overall, there was a unanimous feeling of an excellent centre of cardiovascular research that should be encouraged to continue based on its remarkable performances and possibly reinforce its activities.

Strengths The committee was favourably impressed by the high quality and the diversity of research performed at the CCR, from basic science and experiments to clinical trials and drug development. There was a consensus that this centre conducts research which is really original, innovative and productive.….The activities related to education and training of young scientists and students are also one of the strengths of the centre. Nine students got their Ph.D. through the Marie Curie program in the last 4 years. This program as well as the Graduate course coordinated by Prof Vera Regitz-Zagrosek are important instruments not only for the students, but also to stimulate discussions and collaborations between the groups of the CCR.

18 Introduction

19 Introduction

CCR governance (especially increased networking between groups, increased common budget, better possibilities for space reallocation with the Faculty) should improve the visibility and the performance of this excellent research centre. The CCR should under all circumstances be continued based on the excellent track record, the past performance and future plans”.

This excerpt was taken from the report of the evaluation of the CCR conducted by the external Scientific Advisory Council in February 2010. On behalf of the Executive Board of the CCR I would once again like to take this opportunity to express our appreciation to the evaluators for their work and for the time they devoted to our center.

As complement to the evaluation report, a summary of key statistics is provided in CCR Facts 2010:

Finally, we would once again like to express our appre- Seven years of the CCR: Better than during the ini- ciation to Boehringer Ingelheim, which for the last tial phase of this center, we can assess today how seven years has so generously supported the CCR many of the original objectives have been achieved lecture series BI Lectures and has thus promoted and what still needs improvement. This can also be international scientific exchange in the CCR (see spe- determined from the evaluation of the external experts: cific text). They confirm the value of interdisciplinarity, but advo-

20 Introduction cate a higher intensity of cooperation within research ment of the interplay between academic research and groups of the CCR. They commend what has been industrial development for drug development which is achieved under considerable financial constraints, but discussed in detail in this report. Traditional academic call for greater support by the Charité and greater vis- and scientific networks pose a risk of missing out on ibility outward for our center. They welcome individual this development; they too must evolve in order not to academic achievement, but also advocate a strength- hinder scientific progress. ening of networking in larger research networks and an expansion of the Center in order to give more weight The CCR is still finding its way between the conflict- in the future to cardiovascular research at the Charité. ing priorities of intentional interdisciplinary cooperation and individual research performance. It will, in order to What form will the CCR take in the future? secure its existence, have to maintain intensive research Currently, there is a strong tendency to view the future networks among its members and increase networking of science primarily in publicly funded research net- with other institutions. However, it must also strive to works, such as the classic Collaborative Research achieve a stronger academic focus. First and foremost, Centers of the DFG and, most recently in Germany, the it must attract top-level researchers and integrate them so-called Excellence Initiatives, in which one places into the overall mission of the CCR. All the more so, great hope. One criticism here is that, due to this devel- because the individual research foci of the Charité are opment, many scientists do not choose their research currently in an intense competition for scientists and topics based on their own interests and abilities, but resources in which cardiovascular research can only rather according to how well it would be suitable for a compete with other disciplines such as neuroscience if network. This must not necessarily be accompanied it is internally united in its pursuit of excellent scientific by an increase in quality. While it must be admitted quality and builds on its outward credibility. that such networks enable synergies and can often make better use of the often scarce resources, the Under these conditions, the Center for Cardiovascular fact remains that even today real excellence in scien- Research, as crystallization and integrative focal point tific performance is always accomplished individually, of cardiovascular research at the Charité, can look often without any connection to research networks forward far beyond the Charité’s 300th anniversary which, for various reasons, may actually turn out and its own seven-year birthday to a successful future. to be a hindrance to the individual development of excellence. In addition, it is important to remember that the scientific structures themselves are subject Thomas Unger MD, PhD, FESC, FAHA to continuous change. One example is the develop- CCR Director

Stefan Anker – Applied Cachexia Research

Head of the group

Prof. Dr. Dr. med. Stefan D. Anker

Curriculum Vitae: Stefan D. Anker is Professor of Cardiology and Cachexia Research at the Department of Cardiology of Charité Berlin, Campus Virchow-Klini- kum, in Berlin / Germany (since 2002). Dr. Anker studied medicine and obtained his M.D. (1993) from Charité Medical School in Berlin, Germany. He went on to earn a Ph.D. (1998) at the National Heart & Lung Institute, Imperial College London, UK. Dr. Anker has authored more than 350 original papers, reviews, and editorials. His published work on the one hand addresses many issues of the pathophysiology and treatment of acute and chronic heart failure and on the other hand focuses on treatment development to prevent and reverse muscle wasting, sarcopenia and cachexia at the animal and human level. Additionally, Dr. Anker is very active in biomarker, anaemia, metabolic, immunological and nutrition research. Since 2004, Dr. Anker is extending his work on cachexia research beyond heart failure to other chronic illnesses, including cancer, COPD, CKD and aging. For his work, Dr. Anker has received numerous grants and several clinical research awards. Dr. Anker serves on the editorial boards of 5 scientific journals. Dr. Anker is founding Editor-in-Chief of the Journal of Cachexia, Sarcopenia and Muscle (JCSM). Dr. Anker is member of a number of international steering committees (chairing or co-chairing 3), several DSMB’s (chairing 2) as well as several end-point committees of clinical trials in heart failure and in the field of cachexia treatment and prevention. Dr. Anker serves in the board of the Heart Failure Association (HFA) of the European Society of Cardiology (since 2006) and now is President Elect of HFA (since 2010). He was the chairman of the scientific committee of the 2010 Annual Meeting of HFA (Berlin, 29.5.–1.6.2010). In 2010, Dr. Anker became Fellow of the European Society of Cardiology (FESC). Dr. Anker is the founding president of the International Society on Cachexia and Wasting Disorders (SCWD) – for more information see www.cachexia.org.

23 Stefan Anker – Applied Cachexia Research

Members of the group

Scientists Students Springer, Jochen Dr. rer nat., lab leader Baumgarten, Anna PhD-student Barkhudaryan, Anush MD, (guest scientist, Erebouni Fetzer, Katharina DVM-student Medical Center, Yerevan, Armenia) Hilman, Arne PhD-student Yulia Elkina Dipl. Biochem. Peske, Katrin DVM-student Rokutan, Hirofumi MD, (guest scientist, Tokyo Metro- Pötsch, Mareike Diploma-student politan Geriatric Schust, Susanne DVM-student Hospital, Japan) Suckow, Christian MD-student Palus, Sandra Veterinarian Tomcakova, Margareta MD-student von Heahling, Stephan Dr. Dr. med Tschirner, Anika PhD-student

Technicians Kiauka, Sabine Apprentice

24 Stefan Anker – Applied Cachexia Research

Summary

The group is focused on the crippling syndrome of in other therapeutical areas than cardiac cachexia. cachexia. An underlying hypothesis of our work is Currently, several projects are focused on cardio- that all forms of cachexia as seen in COPD, cancer, vascular drugs and novel compounds on survival, infections and old age lead to pathophysiologically heart function and body composition in a rat model relevant cardiovascular alterations, namely chronic of cancer cachexia. Additionally, we are interested heart failure, which is clinically relevant for prognosis. in the effects of chronic use of frusemide on kidney Disturbance of the endothelial function and the regu- function and morphology in a rat model of chronic lation of peripheral perfusion result in ischemia and heart failure on heart function and body composition hypoxia of the peripheral tissue as well as the induc- in a rat myocardial infarction model. tion of reactive oxygen species and inflammation. We have some evidence that all forms of cachexia, Methods utilized in these projects include physi- independently of their underlying chronic disease, ological (e.g. monitoring of body weight, body com- show neurohumoral activation, which is commonly position by NMR, high resolution echocardiography, associated with chronic heart failure. Furthermore, invasive hemodynamics and non-invasive ECG), cachectic patients almost with no exception are short immunological (e.g. ELISA, Luminex-assays), bio- of breath, fatigue easily and very often show edema, chemical (e.g. Western blotting, enzyme kinetics, all of which are hallmarks of chronic heart failure. We electron paramagnetic resonance spectroscopy have shown that several therapies of chronic heart and qRT-PCR) and histopathological (e.g. differen- failure display anti-cachectic properties, e.g. ACE- tial stains, immunohistochemistry and morphomet- inhibitors and beta-blockers. Hence, we are testing ric analysis) techniques. whether these cardiovascular therapies are effective

25 Stefan Anker – Applied Cachexia Research

Zusammenfassung

Der Forschungsschwerpunkt unserer Arbeitsgruppe Wir konnten zeigen, dass viele Herzinsuffizienz-The- liegt im Bereich der Herzinsuffizienz -induzierten rapien anti-kachektisch wirken können (z.B. ACE- Kachexie. Hemmer). Wir wollen testen, ob diese kardiovasku- Eine unserer Arbeit zugrunde liegende Hypothese lären Therapien auch außerhalb der (nur scheinbar) ist, dass bei allen Kachexieformen kardiovaskuläre engen Grenzen der kardiovaskulären Medizin anti- Veränderungen von besonderer pathogenetischer kachektisch wirksam sein können. Bedeutung sind. Wir denken, dass sich bei allen Zur Zeit beschäftigt sich ein Projekt mit den Effekten Kachexien (z.B. auch bei COPD, Tumoren oder von Appetit-Stimulanzien auf das Überleben und die Infektionen bzw. im hohen Alter) Herzinsuffizienz kardiale Funktion im Herzinfarkt-Modell der Ratte, ein entwickelt und klinisch und prognostisch bedeut- weiteres, klinisches Projekt mit den Mechanismen sam ist. des Muskelabbaus und der durch Translokation von Störungen der Endothelfunktion und Durchblu- Endotoxin im Darm ausgelösten chronischen Inflam- tungsregulation verursachen Gewebeischämie und mation bei Herzinsuffizienz. Die genannten Projekte -hypoxie und es kommt zu Radikalproduktion und werden mit physiologischen Methoden (Dokumen- lokaler Inflammation. Wir haben erste Hinweise tation der Gewichtsverläufe, Messung der Körperzu- darauf, dass alle Kachexien (unabhängig von der sammensetzung per MRT, Echokardiographie und zugrunde liegenden chronischen Erkrankung) eine invasive Hämodynamik), immunologischen (ELISA), besonders für die Herzinsuffizienz bekannte neuro- biochemischen (Western Blotting, Enzymkinetik und hormonale Aktivierung aufweisen. Außerdem sind Elektronenspin-Resonanz Spektroskopie) und histo Patienten mit Kachexie fast ausnahmslos kurzatmig, pathologischen Methoden (Differentialfärbungen, leicht ermüdbar, und weisen sehr häufig Ödeme auf Immunhistochemie und morphometrische Untersu- - das sind Kardinalsymptome der Herzinsuffizienz. chungen) bearbeitet.

26 Stefan Anker – Applied Cachexia Research

Research projects

1. Diuretics use and kidney function in a rat model of chronic heart failure

Project leader Jochen Springer Coworkers Christian Suckow Funding self funded External cooperations Prof. Dr. med. Berthold Hocher

The use of loop diuretics like frusemide for the treat- (RAAS) in both patients and animals. The RAAS-acti- ment of fluid overload in chronic heart failure (CHF) vation is associated with poorer prognosis and pro- has been recommended by the American College of gression of CHF. Inhibition of angiotensin-converting Cardiology guidelines, as patients immediately ben- enzyme reduces the formation and the activity of efit by the reduction of fluid overload. On the other RAAS effector hormones, thereby counteracting the hand the use of frusemide has been associated with frusemide-induced neurohumoral activation. The an increased risk of hospitalization for the progres- effect on survival of frusemide vs untreated animals sion of CHF and subsequently increased risk of death was investigated in a rat model of chronic heart failure from progressive CHF, as well as an increased car- over a period of 6 months. A high mortality was seen diovascular and all-cause mortality as compared to in the frusemid group, which was counteracted by the patients receiving no diuretic or a combination therapy use of the ACE-inhibitor ramipril on top of frusemide. (Domanski et al, J Am Coll Cardiol, 2003). Frusemide Here we assessed the effect of chronic diuretic-use on has been shown to induce the neurohumoral activ- kideney function and morphology (fibrosis, glomerulo- ity such as the renin-angiotensin-aldosterone system sclerosis and vascular remodeling).

27 Stefan Anker – Applied Cachexia Research

2. Drug screening in a rat model of cancer cachexia

Project leaders Stefan Anker, Jochen Springer Coworkers Anna Baumgarten, Katharina Fetzer, Arne Hilman, Katrin Peske, Mareike Pötsch, Sandra Palus, Hirofumi Rokutan, Susanne Schust, Anika Tschirner, Margareta Tomcakova Funding self funded, Myotec Therapeutics, ARAIM, Solatarium Dietetics

The rat ascites hepatoma Yoshida AH-130 is a suitable The ascites hepatoma Yoshida AH-130 cells (108) model system to study the mechanisms involved in the are inoculated into 180-200g male Wistar rats. Alter- establishment of cachexia. Its growth causes rapid natively animals receive saline injection only (sham). and progressive loss of body weight and tissue waste, The animals are housed in groups of three. The day particularly in skeletal muscle. Acceleration of tissue after inoculation animals are randomized into various protein breakdown accounts for most of the wasting groups for drug treatment. The rats receive treat- in the AH-130 bearers and in particular, skeletal mus- ment with either placebo or compounds in several cle hypercatabolism is mainly due to hyperactivation doses over a period maximal 16 days. The primary of the ATP–ubiquitin-dependent proteolytic system. endpoints of the study include assessment of body High plasma levels of tumour necrosis factor-α (TNF) weight, body composition (via NMR-scanning) and and perturbations in the hormonal homeostasis may survival. Secondary endpoints are locomotor activity play an important role in forcing the metabolic balance and food intake, which serve as indicators for quality towards the catabolic side. In addition to increased of life, cardiac function (high resolution echocardiog- muscle protein degradation during cancer growth, the raphy) and organ weights are assessed at the end of presence of the tumour also induces an increased the study (or after death). The model is used to screen rate of apoptosis in skeletal muscle in rats, which also preclinical compounds as well as registered drugs for seems to be activated by cytokines, TNF in particular. second label use.

28 Stefan Anker – Applied Cachexia Research

3. Progressive loss of cardiac function in cancer cachexia

Project leaders Jochen Springer, Stefan Anker Coworkers Katrin Peske, Mareike Pötsch, Sandra Palus, Susanne Schust, Anika Tschirner Funding self funded External cooperations Dr. med. Elena Kaschina, Prof. Dr. med. Thomas Force, Prof. Dr. Dr. med. Thomas Thum, Prof. Dr. rer.nat. Denise Hilfiker-Kleiner, Prof. Dr. med. Frederic Jaisser

Cachexia is a very common co-morbidity in cancer dramatic impairment of cardiac function (see figure patients which drastically reduces quality of life and below). Cardiac function was not only significantly survival. The exact cause of death in cancer is mostly worse than sham at day 11, but also significantly worse unknown, yet 22% of the patients are thought to die compared to the echocardiographic results obtained from the cachexia itself. However cancer patients pre-inoculation of the tumor. The heart shows a reduc- also develop shortness of breath of unknown reason, tion, i.e. wasting, in weight compared to sham at the which is the hallmark symptom of heart failure. In this end of the study, which is also reflected in the progres- project we assessed the cardiac function in terminally sive loss of left ventricular mass and wall thickness ill rats with cancer. Tumour-bearing animals shown a assessed by echocardiography.

4. Heart failure signs in patients with cancer

Project leader Jochen Springer, Prof. Dr. Dr. med .Wolfram Döhner Coworkers Anush Barkhudaryan Funding self funded, DAAD External cooperations Prof. Dr. med. Manfred Dietel, PD Dr. med. Lars Morawietz

This projects aims to find human correlations for the histology of patients, who died of cancer, is assessed heart failure data that we have gathered in the rat can- and correlated to cachexia vs non-cachexia. cer cachexia model. Cardiac dimensions, weight and

29 Stefan Anker – Applied Cachexia Research

5. Cachexia in stroke

Project leader Jochen Springer, Prof. Dr. Dr. med .Wolfram Döhner Coworkers Katrin Peske, Susanne Schust, Anika Tschirner Funding BMBF External cooperations Prof. Dr. med. Ulrich Dirnagl, PD Dr. med. Andre Rex

The central hypothesis of this project states that body The project addresses this question in a MACO mouse weight and the loss of body weight contributes to the model using 60 min of ischemia to induce large cer- morbidity and mortality of patients with a stroke. The ebral infarctions. The aim of the study is to better loss of body weight is not considered to be an epi- characterize the metabolic changes and to assess the phenomenon or related to co-morbidities, but a result effects of a neuroendocrine activation, which may be of stroke-related metabolic changes. accompanied by inflammatory processes.

6. Effects of xanthine oxidase inhibition in cancer cachexia

Project leader Jochen Springer, Stefan D. Anker Coworkers Dr. rer. nat. Nadine Möller, Dr. vet med. Kai Hartmann, Anika Tschirner Funding self funded External cooperations Prof. Dr. rer. nat. Josep M. Argiles

The xanthine oxidase (XO) has been implicated in considered a “danger signal” for the immune system several major diseases, such as ischemia-reperfusion leading to an enhanced production of pro-inflamma- injury like stroke, chronic heart failure, vascular dis- tory cytokines, which in turn contribute to the wasting eases like hypertension and chronic inflammatory dis- process. Tumor-bearing animals showed a 52-fold orders like gout. Uric acid produced by XO has been increase in XO-generated ROS, assessed by electron shown to have predictive value for mortality in cardiac spin resonance spectroscopy. However, the protein cachexia. The by-product of uric acid production by expression of the enzyme was unchanged. Inhibition of XO are reactive oxygen species (ROS) that not only XO with allopüurinol or oxypurinol did not only reduce alter peripheral blood flow and hence further promote the amount of ROS generated, but also improved sur- oxidative stress, but also induce the expression of pro- vival as well as cardiac function and reduced wasting teins involved in the ubiquitin-proteasome pathway, of fat and lean mass. Furthermore a down-regulation which is considered the most important pathway in of mRNA expression of key proteins of the ubiquitin cachexia-associated loss of protein. Uric acid itself is proteasome pathway was seen in muscle.

30 Stefan Anker – Applied Cachexia Research

Publications 2006 - 2010 Jankowska EA, Biel B, Majda J, Szklarska A, Lopuszanska M, Medras M, Anker SD, Banasiak W, Poole-Wilson PA, Lainscak M, Keber I, Anker SD (2006): Body composition Ponikowski P. (2006): Anabolic deficiency in men with chronic changes in patients with systolic heart failure treated with beta heart failure: prevalence and detrimental impact on survival. blockers: a pilot study. Int J Cardiol 106:319-322 Circulation 114:1829-37

Genth-Zotz S, von Haehling S, Bolger AP, Kalra PR, Wen- Köhler F, Nettlau H, Schweizer T, Waller T, Anker SD (2006): sel R, Coats AJ, Volk HD, Anker SD. (2006): The anti-CD14 The research project of the german federal ministry of eco- antibody IC14 suppresses ex vivo endotoxin stimulated tumor nomics and technology: ‘partnership for the heart’ – a new necrosis factor-alpha in patients with chronic heart failure. approach in telemedicine. Dis Manage Health Outcomes Eur J Heart Fail 8:366-372 14:1-4

Kalantar-Zadeh K, Kuwae N, Wu DY, Shantouf RS, Fouque D, John M, Lange A, Hoernig S, Witt C, Anker SD (2006):The Anker SD, Block G, Kopple JD (2006): Associations of body prevalence of anemia in chronic obstructive pulmonary fat and its changes over time with quality of life and pro- disease: comparison to other chronic diseases. Int J Cardiol spective mortality in hemodialysis patients. Am J Clin Nutr 111:365-370 83:202-210 Krum H, Bailey M, Meyer W, Verkenne P, Dargie H, Lechat P, Levy WC, Mozaffarian D, Linker DT, Sutradhar SC, Anker SD, Anker S (2007): Impact of statin therapy on clinical outcomes Cropp AB, Anand I, Maggioni A, Burton P, Sullivan MD, Pitt in chronic heart failure patients according to beta-blocker B, Poole-Wilson PA, Mann DL, Packer M (2006): The Seattle use: results of CIBIS II. Cardiology 108:28-34 Heart Failure Model: prediction of survival in heart failure. Circulation 113:1424-1433 Koehler F, Doehner W, Hoernig S, Witt C, Anker SD, John M (2007): Anorexia in chronic obstructive pulmonary disease - Komajda M, Anker SD, Charlesworth A, Okonko D, Metra M, Association to cachexia and hormonal derangement. Int J Di Lenarda A, Remme W, Moullet C, Swedberg K, Cleland Cardiol 119:83-9 JG, Poole-Wilson PA (2006): The impact of new onset anaemia on morbidity and mortality in chronic heart failure: results Akashi YJ, Kida K, Suzuki K, Inoue K, Kawasaki K, Yamauchi from COMET. Eur Heart J 27:1440-1446 M, Musha H, Anker SD (2007): The significance of (123) I-BMIPP delayed scintigraphic imaging in cardiac patients. Int Piepoli MF, Kaczmarek A, Francis DP, Davies LC, Rauch- J Cardiol 117:145-51 haus M, Jankowska EA, Anker SD, Capucci A, Banasiak W, Ponikowski P (2006): Reduced peripheral skeletal muscle Habedank D, Ewert R, Hummel M, Wensel R, Hetzer R, Anker mass and abnormal reflex physiology in chronic heart failure. SD (2007): Changes in exercise capacity, ventilation, and Circulation 114:126-134 body weight following heart transplantation. Eur J Heart Fail 9:310-6 Anker SD, Clark AL, Winkler R, Zugck C, Cicoira M, Ponikowski P, Davos CH, Banasiak W, Zardini P, Haass M, Kennedy LM, Anker SD, Dickstein K, Willenheimer R (2007): Senges J, Coats AJ, Poole-Wilson PA, Pitt B (2006): Statin Impact of neurohormonal blockade on association between use and survival in patients with chronic heart failure - results body mass index and mortality. Int J Cardiol 119:33-40 from two observational studies with 5200 patients. Int J Cardiol 112:234-42 von Haehling S, Doehner W, Anker SD (2007): Nutrition, metabolism, and the complex pathophysiology of cachexia in Kennedy LM, Dickstein K, Anker SD, James M, Cook TJ, chronic heart failure. Cardiovasc Res 73:298-309 Kristianson K, Willenheimer R (2006): Weight-change as a prognostic marker in 12550 patients following acute myocardial infarction or with stable coronary artery disease. Eur Heart J 27:2755-62

31 Stefan Anker – Applied Cachexia Research

Ponikowski P*, Anker SD*, Szachniewicz J, Okonko D, Mozaffarian D, Anker SD, Anand I, Linker DT, Sullivan MD, Ledwidge M, Zymlinski R, Ryan E, Wasserman SM, Baker N, Cleland JG, Carson PE, Maggioni AP, Mann DL, Pitt B, Poole- Rosser D, Rosen SD, Poole-Wilson PA, Banasiak W, Coats Wilson PA, Levy WC (2007): Prediction of mode of death in AJ, McDonald K (2007): Effect of darbepoetin alfa on exercise heart failure: the Seattle Heart Failure Model. Circulation tolerance in anemic patients with symptomatic chronic heart 116:392-8 failure: a randomized, double-blind, placebo-controlled trial. J Am Coll Cardiol 49:753-62 Niethammer M, Sieber M, von Haehling S, Anker SD, Munzel T, Horstick G, Genth-Zotz S (2007): Inflammatory pathways in Kohler F, Schieber M, Lucke S, Heinze P, Henke S, Matthe- patients with heart failure and preserved ejection fraction. Int sius G, Pferdt T, Wegertseder D, Stoll M, Anker SD (2007): J Cardiol Jul 19; [Epub ahead of print] [“Partnership for the Heart”--development and testing of a new remote patient monitoring system]. Dtsch Med Jankowska EA, Witkowski T, Ponikowska B, Reczuch K, Wochenschr 32:458-60 Borodulin-Nadzieja L, Anker SD, Piepoli MF, Banasiak W, Ponikowski P (2007): Excessive ventilation during early phase Doehner W, Bunck AC, Rauchhaus M, von Haehling S, of exercise: A new predictor of poor long-term outcome in Brunkhorst FM, Cicoira M, Tschope C, Ponikowski P, Claus patients with chronic heart failure. Eur J Heart Fail 9:1024- RA, Anker SD (2007): Secretory sphingomyelinase is upregu- 31 lated in chronic heart failure: a second messenger system of immune activation relates to body composition, muscular Kempf T, von Haehling S, Peter T, Allhoff T, Cicoira M, functional capacity, and peripheral blood flow. Eur Heart J Doehner W, Ponikowski P, Filippatos GS, Rozentryt P, Drexler 28:821-8 H, Anker SD, Wollert KC (2007): Prognostic utility of growth differentiation factor-15 in patients with chronic heart failure. J Földes G, von Haehling S, Okonko DO, Jankowska EA, Am Coll Cardiol 50:1054-60 Poole-Wilson PA, Anker SD (2007): Fluvastatin reduces increased blood monocyte Toll-like receptor 4 expression in Sandek S, Bauditz J, Swidsinski A, Buhner S, Weber-Eibel whole blood from patients with chronic heart failure. Int J J, von Haehling S, Schroedl W, Karhausen T, Doehner W, Cardiol 124:80-5 Rauchhaus M, Poole-Wilson P, Volk HD, Lochs H, Anker SD (2007): Altered intestinal function in patients with chronic Westermann D, Rutschow S, Jager S, Linderer A, Anker S, heart failure. J Am Coll Cardiol 50:1561-9 Riad A, Unger T, Schultheiss HP, Pauschinger M, Tschope C (2007): Contributions of inflammation and cardiac matrix von Haehling S, Jankowska EA, Morgenthaler NG, Vassanelli metalloproteinase activity to cardiac failure in diabetic cardio- C, Zanolla L, Rozentryt P, Filippatos GS, Doehner W, Koehler myopathy: the role of Angiotensin type 1 receptor antagonism. F, Papassotiriou J, Kremastinos DT, Banasiak W, Struck J, Diabetes 56:641-6 Ponikowski P, Bergmann A, Anker SD (2007): Comparison of mid-regional proANP with N-terminal proBNP in predicting Thum T, Hoeber S, Froese S, Klink I, Stichtenoth DO, Galuppo survival in patients with chronic heart failure. J Am Coll Cardiol P, Jakob M, Tsikas D, Anker SD, Poole-Wilson PA, Borlak J, 50:1973-80 Ertl G, Bauersachs J (2007): Age-dependent impairment of endothelial progenitor cells is corrected by growth-hormone- Springer J, Groneberg DA, Dinh QT, Quarcoo D, Hamelmann mediated increase of insulin-like growth-factor-1. Circ Res E, Braun-Dullaeus RC, Geppetti P, Anker SD, Fischer A 100:434-43 (2007): Neurokinin-1 receptor activation induces reactive oxygen species and epithelial damage in allergic airway inflam- Clark AL, Knosalla C, Birks E, Loebe M, Davos CH, Tsang S, mation. Clin Exp Allergy 37:1788-97 Negassa A, Yacoub M, Hetzer R, Coats AJ, Anker SD (2007): Heart transplantation in heart failure: The prognostic impor- Koehler F, Doehner W, Anker SD, John M (2007): Anorexia tance of body mass index at time of surgery and subsequent in chronic obstructive pulmonary disease - Association to weight changes. Eur J Heart Fail 9:839-44 cachexia and hormonal derangement. Int J Cardiol. [Epub ahead of print]

32 Stefan Anker – Applied Cachexia Research

Metra M, Ponikowski P, Dickstein K, McMurray JJ, Gavazzi A, Belonje AM, Voors AA, van Gilst WH, Anker SD, Slart RH, Bergh CH, Fraser AG, Jaarsma T, Pitsis A, Mohacsi P, Böhm Tio RA, Zijlstra F, van Veldhuisen DJ; HEBE III investigators. M, Anker S, Dargie H, Brutsaert D, Komajda M; Heart Failure (2008): Effects of erythropoietin after an acute myocardial Association of the European Society of Cardiology. (2007): infarction: rationale and study design of a prospective, rand- Advanced chronic heart failure: A position statement from the omized, clinical trial (HEBE III). Am Heart J 155:817-22 Study Group on Advanced Heart Failure of the Heart Failure Association of the European Society of Cardiology. Eur J Strassburg S, Anker SD, Castaneda TR, Burget L, Perez-Tilve Heart Fail 9:684-94 D, Pfluger PT, Nogueiras R, Halem H, Dong JZ, Culler MD, Datta R, Tschop MH (2008): Long-term Effects of Ghrelin and Gazzieri D, Trevisani M, Springer J, Harrison S, Cottrell GS, Ghrelin Receptor Agonists on Energy Balance in Rats. Am J Andre E, Nicoletti P, Massi D, Zecchi S, Nosi D, Santucci M, Physiol Endocrinol Metab 295:E78-E84 Gerard NP, Lucattelli M, Lungarella G, Fischer A, Grady EF, Bunnett NW, Geppetti P (2007): Substance P released by Filippatos GS, Adamopoulos C, Sui X, Love TE, Pullicino PM, TRPV1-expressing neurons produces reactive oxygen spe- Lubsen J, Bakris G, Anker SD, Howard G, Kremastinos DT, cies that mediate ethanol-induced gastric injury. Free Radic Ahmed A (2008): A propensity-matched study of hyperten- Biol Med 15;43(4):581-9 sion and increased stroke-related hospitalization in chronic heart failure. Am J Cardiol 101:1772-6 Okonko DO, Grzeslo A, Witkowski T, Mandal AK, Slater RM, Roughton M, Foldes G, Thum T, Majda J, Banasiak W, Sandek A, Rauchhaus M, Anker SD, von Haehling S (2008): Missouris CG, Poole-Wilson PA, Anker SD*, Ponikowski P* The emerging role of the gut in chronic heart failure. Curr (2008): Effect of intravenous iron sucrose on exercise toler- Opin Clin Nutr Metab Care 11:632-9 ance in anemic and nonanemic patients with symptomatic chronic heart failure and iron deficiency FERRIC-HF: a rand- Alper AB, Campbell RC, Anker SD, Bakris G, Wahle C, Love omized, controlled, observer-blinded trial. J Am Coll Cardiol TE, Hamm LL, Mujib M, Ahmed A (2008): A propensity- 51:103-12 matched study of low serum potassium and mortality in older adults with chronic heart failure. Int J Cardiol Aug 6. [Epub Torre-Amione G, Anker SD, Bourge RC, Colucci WS, ahead of print] Greenberg BH, Hildebrandt P, Keren A, Motro M, Moyé LA, Otterstad JE, Pratt CM, Ponikowski P, Rouleau JL, Sestier F, Evans WJ, Morley JE, Argilés J, Bales C, Baracos V, Guttridge Winkelmann BR, Young JB (2008): Advanced Chronic Heart D, Jatoi A, Kalantar-Zadeh K, Lochs H, Mantovani G, Marks Failure CLinical Assessment of Immune Modulation Therapy D, Mitch WE, Muscaritoli M, Najand A, Ponikowski P, Rossi Investigators. Results of a non-specific immunomodulation Fanelli F, Schambelan M, Schols A, Schuster M, Thomas D, therapy in chronic heart failure (ACCLAIM trial): a placebo- Wolfe R, Anker SD (2008): Cachexia: a new definition. Clin controlled randomised trial. Lancet 371:228-36 Nutr 27:793-9

Habedank D, Ewert R, Hetzer R, Anker SD (2008): Revers- Akashi YJ, Palus S, Datta R, Halem H, Taylor JE, Thoene- ibility of cachexia after bilateral lung transplantation. Int J Reineke C, Dong J, Thum T, Culler MD, Anker SD, Springer Cardiol. [Epub ahead of print] J (2008): No effects of human ghrelin on cardiac function despite profound effects on body composition in a rat model Schumann S, Saggu M, Möller N, Anker SD, Lendzian F, of heart failure. Int J Cardiol Aug 22. [Epub ahead of print] Hildebrandt P, Leimkühler S (2008): The Mechanism of Assembly and Cofactor Insertion into Rhodobacter capsula- Göhler A, Conrads-Frank A, Worrell SS, Geisler BP, Halp- tus Xanthine Dehydrogenase. J Biol Chem 283:16602-11 ern EF, Dietz R, Anker SD, Gazelle GS, Siebert U (2008): Decision-analytic evaluation of the clinical effectiveness and von Haehling S, Schefold JC, Springer J, Anker SD (2008): cost-effectiveness of management programmes in chronic The cholesterol paradox revisited: heart failure, systemic heart failure. Eur J Heart Fail 10:1026-32 inflammation, and beyond. Heart Fail Clin 4:141-51

33 Stefan Anker – Applied Cachexia Research

Doehner W, Gathercole D, Cicoira M, Krack A, Coats AJ, Reinke S, Karhausen T, Doehner W, Taylor W, Hottenrott K, Camici PG, Anker SD (2008): Reduced glucose transporter Duda GN, Reinke P, Volk HD, Anker SD (2009): The influence GLUT4 in skeletal muscle predicts insulin resistance in non- of recovery and training phases on body composition, periph- diabetic chronic heart failure patients independently of body eral vascular function and immune system of professional composition. Int J Cardiol Sep 6. [Epub ahead of print] soccer players. PLoS One 4:e4910

Pocock SJ, McMurray JJ, Dobson J, Yusuf S, Granger CB, Voors AA, von Haehling S, Anker SD, Hillege HL, Struck J, Michelson EL, Ostergren J, Pfeffer MA, Solomon SD, Anker Hartmann O, Bergmann A, Squire I, van Veldhuisen DJ, Dick- SD, Swedberg KB (2008): Weight loss and mortality risk in stein K; OPTIMAAL Investigators (2009): C-terminal provaso- patients with chronic heart failure in the candesartan in heart pressin (copeptin) is a strong prognostic marker in patients failure: assessment of reduction in mortality and morbidity with heart failure after an acute myocardial infarction: results (CHARM) programme. Eur Heart J 29:2641-50 from the OPTIMAAL study. Eur Heart J 30:1187-94

Belonje AM, Westenbrink BD, Voors AA, von Haehling S, Anker SD, Voors A, Okonko D, Clark AL, James MK, von Ponikowski P, Anker SD, van Veldhuisen DJ, Dickstein K Haehling S, Kjekshus J, Ponikowski P, Dickstein K; OPTIMAAL (2009): Erythropoietin levels in heart failure after an acute Investigators. P (2009): Revalence, incidence, and prognostic myocardial infarction: determinants, prognostic value, and the value of anaemia in patients after an acute myocardial infarc- effects of captopril versus losartan. Am Heart J 157:91-6. tion: data from the OPTIMAAL trial. Eur Heart J 30:1331-9

Lainscak M, von Haehling S, Anker SD (2009): Natriuretic von Haehling S, von Bardeleben RS, Kramm T, Thiermann Y, peptides and other biomarkers in chronic heart failure: From Niethammer M, Doehner W, Anker SD, Munzel T, Mayer E, BNP, NT-proBNP, and MR-proANP to routine biochemical Genth-Zotz S (2009): Inflammation in right ventricular dys- markers. Int J Cardiol 32:303-11 function due to thromboembolic pulmonary hypertension. Int J Cardiol May 1. [Epub ahead of print] Morley JE, Anker SD, Evans WJ (2009): Cachexia and aging: an update based on the fourth international cachexia meeting. Jankowska EA, Rozentryt P, Ponikowska B, Hartmann O, J Nutr Health Aging 13:47-55 Kustrzycka-Kratochwil D, Reczuch K, Nowak J, Borodulin- Nadzieja L, Polonski L, Banasiak W, Poole-Wilson PA, Anker Vaz Pérez A, Doehner W, von Haehling S, Schmidt H, Zim- SD, Ponikowski P (2009): Circulating estradiol and mortality in mermann AV, Volk HD, Anker SD, Rauchhaus M (2009): men with systolic chronic heart failure. JAMA 9;301:1892-901 The relationship between tumor necrosis factor-alpha, brain natriuretic peptide and atrial natriuretic peptide in patients Kalra PR, Clague JR, Coats AJ, Anker SD, Poole-Wilson PA, with chronic heart failure. Int J Cardiol Jan 18. [Epub ahead Struthers AD (2009): C type natruiretic peptide production by of print] the human kidney is blunted in chronic heart failure. Clin Sci (Lond) May 18. Ekundayo OJ, Dell’italia LJ, Sanders PW, Arnett D, Aban I, Love TE, Filippatos G, Anker SD, Lloyd-Jones DM, Bakris Jankowska EA, Filippatos G, Ponikowska B, Borodulin-Nadzieja G, Mujib M, Ahmed A (2009): Association between hype- L, Anker SD, Banasiak W, Poole-Wilson PA, Ponikowski P ruricemia and incident heart failure among older adults: A (2009): Reduction in circulating testosterone relates to exercise propensity-matched study. Int J Cardiol Feb 5. [Epub ahead capacity in men with chronic heart failure. J Card Fail 15:442-50 of print] van Veldhuisen DJ, Cohen-Solal A, Böhm M, Anker SD, Babalis Palus S, Akashi Y, von Haehling S, Anker SD, Springer J D, Roughton M, Coats AJ, Poole-Wilson PA, Flather MD; (2009): The influence of age and sex on disease development SENIORS Investigators (2009): Beta-blockade with nebivolol in in a novel animal model of cardiac cachexia. Int J Cardiol elderly heart failure patients with impaired and preserved left ven- 133:388-93 tricular ejection fraction: Data From SENIORS (Study of Effects of Nebivolol Intervention on Outcomes and Rehospitalization in Seniors With Heart Failure). J Am Coll Cardiol 53:2150-8

34 Stefan Anker – Applied Cachexia Research

Anker SD, John M, Laviano A, Filippatos G, Paccagnella Baid-Agrawal S, Frei U, Reinke P, Schindler R, Kopp MA, Mar- A, Ponikowski P, Schols AW (2009): ESPEN Guidelines on tus P, Berg T, Juergensen JS, Anker SD, Doehner W. (2009): Parenteral Nutrition: Cardiology. Clin Nutr Jun 8. [Epub Impaired Insulin Sensitivity as an Underlying Mechanism Link- ahead of print] ing Hepatitis C and Posttransplant Diabetes Mellitus in Kidney Recipients. Am J Transplant Nov 20;9(12):2777-2784 von Haehling S, Hopkinson NS, Polkey MI, Niethammer M, Anker SD, Genth-Zotz S. (2009): Elevated TNFalpha pro- Klapholz M, Abraham WT, Ghali JK, Ponikowski P, Anker SD, duction in whole blood in patients with severe COPD: the Knusel B, Sun Y, Wasserman SM, van Veldhuisen DJ (2009): potential link to disease severity. Wien Klin Wochenschr The safety and tolerability of darbepoetin alfa in patients with 121:303-8 anaemia and symptomatic heart failure. Eur J Heart Fail 11(11):1071-7 Lainscak M, Hodoscek LM, Düngen HD, Rauchhaus M, Doehner W, Anker SD, von Haehling S (2009): The burden of Habedank D, Kung T, Karhausen T, von Haehling S, Doehner chronic obstructive pulmonary disease in patients hospital- W, Schefold JC, Hasper D, Reinke S, Anker SD, Reinke P ized with heart failure. Wien Klin Wochenschr 121:309-13 (2009): Exercise capacity and body composition in living- donor renal transplant recipients over time. Nephrol Dial Ogino K, Kato M, Furuse Y, Kinugasa Y, Ishida K, Osaki Transplant 24(12):3854-60 S, Kinugawa T, Igawa O, Hisatome I, Shigemasa C, Anker SD, Doehner W (2009): Uric Acid Lowering Treatment with Szabo T, von Haehling S, Habedank D, Rauchhaus M, Lains- Benzbromarone in Patients with Heart Failure: a Double-blind cak M, Sandek A, Schefold J, Anker SD, Doehner W (2009): Placebo-controlled Cross-over Preliminary Study. Circ Heart Usefulness of minimal modelling to assess impaired insulin Fail Nov 20 [Epub ahead of print] sensitivity in patients with chronic heart failure. Int J Cardiol 4. [Epub ahead of print] von Haehling S, Schefold JC, Majc Hodoscek L, Doehner W, Mannaa M, Anker SD, Lainscak M (2009): Anaemia is an Mehrhof F, Löffler M, Gelbrich G, Ozcelik C, Posch M, independent predictor of death in patients hospitalized for Hense HW, Keil U, Scheffold T, Schunkert H, Angermann C, acute heart failure. Clin Res Cardiol Nov 17 [Epub ahead of Ertl G, Jahns R, Pieske B, Wachter R, Edelmann F, Wollert print] KC, Maisch B, Pankuweit S, Erbel R, Neumann T, Herzog W, Katus H, Müller-Tasch T, Zugck C, Düngen HD, Regitz- Anker SD, Comin Colet J, Filippatos G, Willenheimer R, Zagrosek V, Lehmkuhl E, Störk S, Siebert U, Wasem J, Neu- Dickstein K, Drexler H, Lüscher TF, Bart B, Banasiak W, mann A, Göhler A, Anker SD, Köhler F, Möckel M, Osterziel Niegowska J, Kirwan BA, Mori C, von Eisenhart Rothe B, KJ, Dietz R, Rauchhaus M; on Behalf of the Competence Net- Pocock SJ, Poole-Wilson PA, Ponikowski P; the FAIR-HF work Heart Failure (2009): A network against failing hearts- Trial Investigators. (2009): Ferric Carboxymaltose in Patients Introducing the German “Competence Network Heart Failure” with Heart Failure and Iron Deficiency. N Engl J Med Int J Cardiol Aug 11. [Epub ahead of print] 361(25):2436-2448 von Haehling S, Schefold JC, Jankowska E, Doehner W, Springer Anker SD, Colet JC, Filippatos G, Willenheimer R, Dickstein K, J, Strohschein K, Genth-Zotz S, Volk HD, Poole-Wilson P, Anker Drexler H, Lüscher TF, Mori C, von Eisenhart Rothe B, Pocock S, SD (2009): Leukocyte redistribution: effects of beta blockers in Poole-Wilson PA, Ponikowski P; FAIR-HF committees and inves- patients with chronic heart failure. PLoS One 4(7):e6411 tigators. (2009) Rationale and design of Ferinject assessment in patients with IRon deficiency and chronic Heart Failure (FAIR-HF) McMurray JJ, Anand IS, Diaz R, Maggioni AP, O’Connor study: a randomized, placebo-controlled study of intravenous C, Pfeffer MA, Polu KR, Solomon SD, Sun Y, Swedberg K, iron supplementation in patients with and without anaemia. Eur J Tendera M, van Veldhuisen DJ, Wasserman SM, Young JB; Heart Fail 11(11):10 RED-HF Committees and Investigators. (2009): Design of the Reduction of Events with Darbepoetin alfa in Heart Failure (RED-HF): a Phase III, anaemia correction, morbidity-mortality trial. Eur J Heart Fail 11(8):795-801

35 Stefan Anker – Applied Cachexia Research

Spintzik J, Springer J, Westermann B (2009): Morphological Poldermans D, Bax JJ, Boersma E, De Hert S, Eeckhout E, and histological organization of the pyriform appendage of the Fowkes G, Gorenek B, Hennerici MG, Iung B, Kelm M, Kjeld- tetrabranchiate Nautilus pompilius (Cephalopoda, Mollusca). sen KP, Kristensen SD, Lopez-Sendon J, Pelosi P, Philippe F, J Morphol 270(4):459-68 Pierard L, Ponikowski P, Schmid JP, Sellevold OF, Sicari R, Van den Berghe G, Vermassen F, Hoeks SE, Vanhorebeek I, Gratze P, Boschmann M, Dechend R, Qadri F, Malchow J, Vahanian A, Auricchio A, Bax JJ, Ceconi C, Dean V, Filip- Graeske S, Engeli S, Janke J, Springer J, Contrepas A, Plehm patos G, Funck-Brentano C, Hobbs R, Kearn P, McDonag T, R, Klaus S, Nguyen G, Luft FC, Muller DN (2009): Energy McGregor K, Popescu BA, Reiner Z, Sechtem U, Sirnes PA, metabolism in human renin-gene transgenic rats: does renin Tendera M, Vardas P, Widimsky P, De Caterina R, Agewall contribute to obesity? Hypertension 53(3):516-23 S, Al Attar N, Andreotti F, Anker SD, Baron-Esquivias G, Berkenboom G, Chapoutot L, Cifkova R, Faggiano P, Gibbs von Haehling S, Schefold JC, Hodoscek LM, Doehner W, S, Hansen HS, Iserin L, Israel CW, Kornowski R, Eizagaechev- Mannaa M, Anker SD, Lainscak M (2010) Anaemia is an inde- arria NM, Pepi M, Piepoli M, Priebe HJ, Scherer M, Stepinska pendent predictor of death in patients hospitalized for acute J, Taggart D, Tubaro M; Task Force for Preoperative Cardiac heart failure. Clin Res Cardiol 99(2):107-13 Risk Assessment and Perioperative Cardiac Management in Non-cardiac Surgery of European Society of Cardiology Ronco C, McCullough P, Anker SD, Anand I, Aspromonte N, (ESC); European Society of Anaesthesiology (ESA). (2010): Bagshaw SM, Bellomo R, Berl T, Bobek I, Cruz DN, Daliento Guidelines for pre-operative cardiac risk assessment and L, Davenport A, Haapio M, Hillege H, House AA, Katz N, Mai- perioperative cardiac management in non-cardiac surgery: sel A, Mankad S, Zanco P, Mebazaa A, Palazzuoli A, Ronco the Task Force for Preoperative Cardiac Risk Assessment and F, Shaw A, Sheinfeld G, Soni S, Vescovo G, Zamperetti N, Perioperative Cardiac Management in Non-cardiac Surgery Ponikowski P; (2010) Acute Dialysis Quality Initiative (ADQI) of the European Society of Cardiology (ESC) and endorsed consensus group. Cardio-renal syndromes: report from the by the European Society of Anaesthesiology (ESA). Eur J consensus conference of the acute dialysis quality initiative. Anaesthesiol. 27(2):92-137. Eur Heart J 31(6):703-11 Bagshaw SM, Cruz DN, Aspromonte N, Daliento L, Ronco F, Muscaritoli M, Anker SD, Argilés J, Aversa Z, Bauer JM, Biolo Sheinfeld G, Anker SD, Anand I, Bellomo R, Berl T, Bobek I, G, Boirie Y, Bosaeus I, Cederholm T, Costelli P, Fearon KC, Davenport A, Haapio M, Hillege H, House A, Katz N, Maisel Laviano A, Maggio M, Rossi Fanelli F, Schneider SM, Schols A, Mankad S, McCullough P, Mebazaa A, Palazzuoli A, A, Sieber CC. (2010): Consensus definition of sarcopenia, Ponikowski P, Shaw A, Soni S, Vescovo G, Zamperetti N, cachexia and pre-cachexia: joint document elaborated by Zanco P, Ronco C (2010): Acute Dialysis Quality Initiative Special Interest Groups (SIG) “cachexia-anorexia in chronic Consensus Group.Epidemiology of cardio-renal syndromes: wasting diseases” and “nutrition in geriatrics”. Clin Nutr workgroup statements from the 7th ADQI Consensus Confer- 29(2):154-9 ence. Nephrol Dial Transplant 25(5):1406-16. Epub 2010 Feb 25

von Haehling S, Filippatos GS, Papassotiriou J, Cicoira M, Jankowska EA, Doehner W, Rozentryt P, Vassanelli C, Struck J, Banasiak W, Ponikowski P, Kremastinos D, Bergmann A, Morgenthaler NG, Anker SD (2010): Mid-regional pro- adrenomedullin as a novel predictor of mortality in patients with chronic heart failure. Eur J Heart Fail 12(5):484-91

36 Stefan Anker – Applied Cachexia Research

House AA, Anand I, Bellomo R, Cruz D, Bobek I, Anker SD, Ronco C, McCullough PA, Anker SD, Anand I, Aspromonte N, Aspromonte N, Bagshaw S, Berl T, Daliento L, Davenport Bagshaw SM, Bellomo R, Berl T, Bobek I, Cruz DN, Daliento A, Haapio M, Hillege H, McCullough P, Katz N, Maisel A, L, Davenport A, Haapio M, Hillege H, House A, Katz NM, Mai- Mankad S, Zanco P, Mebazaa A, Palazzuoli A, Ronco F, Shaw sel A, Mankad S, Zanco P, Mebazaa A, Palazzuoli A, Ronco A, Sheinfeld G, Soni S, Vescovo G, Zamperetti N, Ponikowski F, Shaw A, Sheinfeld G, Soni S, Vescovo G, Zamperetti N, P, Ronco C; Acute Dialysis Quality Initiative Consensus Ponikowski P; Acute Dialysis Quality Initiative (ADQI) consen- Group. (2010): Definition and classification of Cardio-Renal sus group. (2010): Cardiorenal syndromes: an executive sum- Syndromes: workgroup statements from the 7th ADQI Con- mary from the consensus conference of the Acute Dialysis sensus Conference. Nephrol Dial Transplant 25(5):1416-20 Quality Initiative (ADQI). Contrib Nephrol 65:54-67.

Strassburg S, Pfluger PT, Chaudhary N, Tso P, Tschöp MH, Maisel A, Mueller C, Nowak R, Peacock WF, Landsberg Anker SD, Nogueiras R, Perez-Tilve D (2010): Action Profile JW, Ponikowski P, Mockel M, Hogan C, Wu AH, Richards of the Antiobesity Drug Candidate Oleoyl-Estrone in Rats. M, Clopton P, Filippatos GS, Di Somma S, Anand I, Ng L, Obesity (Silver Spring). Mar 25. Daniels LB, Neath SX, Christenson R, Potocki M, McCord J, Terracciano G, Kremastinos D, Hartmann O, von Haehling S, Gheorghiade M, Follath F, Ponikowski P, Barsuk JH, Blair JE, Bergmann A, Morgenthaler NG, Anker SD (2010): Mid-region Cleland JG, Dickstein K, Drazner MH, Fonarow GC, Jaarsma pro-hormone markers for diagnosis and prognosis in acute T, Jondeau G, Sendon JL, Mebazaa A, Metra M, Nieminen dyspnea: results from the BACH (Biomarkers in Acute Heart M, Pang PS, Seferovic P, Stevenson LW, van Veldhuisen DJ, Failure) trial. J Am Coll Cardiol 11;55(19):2062-76 Zannad F, Anker SD, Rhodes A, McMurray JJ, Filippatos G (2010): Assessing and grading congestion in acute heart Jankowska EA, Rozentryt P, Witkowska A, Nowak J, Hart- failure: a scientific statement from the acute heart failure com- mann O, Ponikowska B, Borodulin-Nadzieja L, Banasiak W, mittee of the heart failure association of the European society Polonski L, Filippatos G, McMurray JJ, Anker SD, Ponikowski of cardiology and endorsed by the European society of inten- P (2010): Iron deficiency: an ominous sign in patients with sive care medicine. Eur J Heart Fail 12(5):423-33. systolic chronic heart failure. Eur Heart J 31(15):1872-80

Wu AH, Pitt B, Anker SD, Vincent J, Mujib M, Ahmed A de Boer RA, Doehner W, van der Horst IC, Anker SD, Babalis (2010): Association of obesity and survival in systolic heart D, Roughton M, Coats AJ, Flather MD, van Veldhuisen DJ; failure after acute myocardial infarction: potential confounding SENIORS Investigators. (2010): Influence of diabetes mellitus by age. Eur J Heart Fail 12(6):566-73 and hyperglycemia on prognosis in patients > or =70 years old with heart failure and effects of nebivolol (data from the McCullough PA, Haapio M, Mankad S, Zamperetti N, Massie Study of Effects of Nebivolol Intervention on Outcomes and B, Bellomo R, Berl T, Anker SD, Anand I, Aspromonte N, Bag- Rehospitalization in seniors with heart failure [SENIORS]). Am shaw SM, Bobek I, Cruz DN, Daliento L, Davenport A, Hillege J Cardiol 1;106(1):78-86.e1 H, House AA, Katz N, Maisel A, Mebazaa A, Palazzuoli A, Ponikowski P, Ronco F, Shaw A, Sheinfeld G, Soni S, Vescovo Morley JE, Argiles JM, Evans WJ, Bhasin S, Cella D, Deutz G, Zanco P, Ronco C, Berl T; Acute Dialysis Quality Initiative NE, Doehner W, Fearon KC, Ferrucci L, Hellerstein MK, (ADQI) Consensus Group. (2010): Prevention of cardio-renal Kalantar-Zadeh K, Lochs H, MacDonald N, Mulligan K, syndromes: workgroup statements from the 7th ADQI Con- Muscaritoli M, Ponikowski P, Posthauer ME, Rossi Fanelli F, sensus Conference. Nephrol Dial Transplant 25(6):1777-84 Schambelan M, Schols AM, Schuster MW, Anker SD; Society for Sarcopenia, Cachexia, and Wasting Disease. (2010): Nutri- tional recommendations for the management of sarcopenia. J Am Med Dir Assoc 11(6):391-6

37 Stefan Anker – Applied Cachexia Research

Habedank D, Ewert R, Hummel M, Dandel M, Habedank F, Lainscak M, Podbregar M, Anker SD (2007): How does Knosalla C, Lehmkuhl HB, Anker SD, Hetzer R (2010): The cachexia influence survival in cancer, heart failure and effects of bilateral lung transplantation on ventilatory effi- other chronic diseases? Curr Opin Support Palliat Care ciency, oxygen uptake and the right heart: a two-yr follow-up. 2007;1:299-305 Clin Transplant 18 [Epub ahead of print] Kalantar-Zadeh K, Horwich TB, Oreopoulos A, Kovesdy CP, Younessi H, Anker SD, Morley JE (2007): Risk factor para- Reviews & book chapters (2006-2010) dox in wasting diseases. Curr Opin Clin Nutr Metab Care 10:433-42 Strassburg S, Anker SD (2006): Metabolic and immunologic derangements in cardiac cachexia: where to from here? Földes G, von Haehling S, Jankowska EA, Anker SD (2007): Heart Fail Rev 11:57-64 Targeting the toll-system in cardiovascular sciences. Recent Pat Inflamm Allergy Drug Discov 1(1):57-67 Foldes G, von Haehling S, Anker SD (2006): Toll-like receptor modulation in cardiovascular disease: a target for interven- von Haehling S, Doehner W, Anker SD (2007): Nutrition, tion? Expert Opin Investig Drugs 15:857-871 metabolism, and the complex pathophysiology of cachexia in chronic heart failure. Cardiovasc Res 73:298-309 Springer J, von Haehling S, Anker SD. (2006): The need for a standardized definition for cachexia in chronic illness. Nat Lainscak M, von Haehling S, Springer J, Anker SD (2007): Clin Pract Endocrinol Metab 2:416-7 Biomarkers for chronic heart failure. Heart Fail Monit 5:77-82 von Haehling S, Doehner W, Anker SD (2006): Obesity and Akashi YJ, Springer J, Lainscak M, Anker SD (2007): Atrial natriuretic the heart a weighty issue. J Am Coll Cardiol 6;47(11):2274-6 peptide and related peptides. Clin Chem Lab Med 45:1259-67

Anker SD, John M, Pedersen PU, Raguso C, Cicoira M, Schünemann M, Anker SD, Rauchhaus M (2008): Cancer fatigue Dardai E, Laviano A, Ponikowski P, Schols AM; DGEM (Ger- syndrome reflects clinically non-overt heart failure: an approach man Society for Nutritional Medicine); Becker HF, Bohm M, towards onco-cardiology. Nat Clin Pract Oncol 5(11):632-3 Brunkhorst FM, Vogelmeier C (2006): ESPEN guidelines on enteral nutrition: cardiology and pulmonology. Clin Nutr Maisel A, Mueller C, Adams K Jr, Anker SD, Aspromonte 25:311-318 N, Cleland JG, Cohen-Solal A, Dahlstrom U, DeMaria A, Di Somma S, Filippatos GS, Fonarow GC, Jourdain P, Komajda Springer J, Filippatos G, Akashi YJ, Anker SD (2006): Progno- M, Liu PP, McDonagh T, McDonald K, Mebazaa A, Nieminen sis and therapy approaches of cardiac cachexia. Curr Opin MS, Peacock WF, Tubaro M, Valle R, Vanderhyden M, Yancy Cardiol 21:229-233 CW, Zannad F, Braunwald E (2008): State of the art: using natriuretic peptide levels in clinical practice. Eur J Heart Fail Kalantar-Zadeh K, Abbott KC, Kronenberg F, Anker SD, 10:824-39 Horwich TB, Fonarow GC (2006): Epidemiology of dialysis patients and heart failure patients. Semin Nephrol 26:118- Sandek A, Rauchhaus M, Anker SD, von Haehling S (2008): 133 The emerging role of the gut in chronic heart failure. Curr Opin Clin Nutr Metab Care 11:632-9. Jankowska EA, Ponikowski P, Piepoli MF, Banasiak W, Anker SD, Poole-Wilson PA (2006): Autonomic imbalance and Kalantar-Zadeh K, Anker SD, Horwich TB, Fonarow GC. immune activation in chronic heart failure - pathophysiological (2008): Nutritional and anti-inflammatory interventions in links. Cardiovasc Res 70:434-445 chronic heart failure. Am J Cardiol 101:89E-103E von Haehling S, Doehner W, Anker SD (2006): Ernst Scher Lainscak M, Dagres N, Filippatos GS, Anker SD, Kremastinos ing Res Found Workshop 55:155-168 DT (2008): Atrial fibrillation in chronic non-cardiac disease: Where do we stand? Int J Cardiol 128:311-5

38 Stefan Anker – Applied Cachexia Research

Farmakis D, Filippatos G, Lainscak M, Parissis JT, Anker SD, Szabó T, Felger D, von Haehling S, Lainscak M, Anker SD, Kremastinos DT (2008): Anticoagulants, antiplatelets, and Doehner W (2009): Overview of emerging pharmacotherapy statins in heart failure. Cardiol Clin 26:49-58, vi in chronic heart failure. Expert Opin Pharmacother 10(13):2055-74 von Haehling S, Lainscak M, Springer J, Anker SD (2008): Cardiac cachexia: A systematic overview. Pharmacol Ther 121:227-52 von Haehling S, Schefold JC, Lainscak M, Doehner W, Anker SD (2009): Inflammatory biomarkers in heart failure revis- Doehner W, von Haehling S, Anker SD, Lainscak M (2009): ited: much more than innocent bystanders. Heart Fail Clin Neurohormonal activation and inflammation in chronic car- 5(4):549-60 diopulmonary disease: a brief systematic review. Wien Klin Wochenschr 121:293-6 Soukoulis V, Dihu JB, Sole M, Anker SD, Cleland J, Fonarow GC, Metra M, Pasini E, Strzelczyk T, Taegtmeyer H, Gheorghi- Jaarsma T, Beattie JM, Ryder M, Rutten FH, McDonagh T, Mohacsi ade M (2009): Micronutrient deficiencies an unmet need in P, Murray SA, Grodzicki T, Bergh I, Metra M, Ekman I, Angermann heart failure. J Am Coll Cardiol 54(18):1660-73 C, Leventhal M, Pitsis A, Anker SD, Gavazzi A, Ponikowski P, Dick- stein K, Delacretaz E, Blue L, Strasser F, McMurray J;Advanced Riegel B, Moser DK, Anker SD, Appel LJ, Dunbar SB, Grady Heart Failure Study Group of the HFA of the ESC. (2009): Palliative KL, Gurvitz MZ, Havranek EP, Lee CS, Lindenfeld J, Peterson care in heart failure: a position statement from the palliative care PN, Pressler SJ, Schocken DD, Whellan DJ; American Heart workshop of the Heart Failure Association of the European Society Association Council on Cardiovascular Nursing; American of Cardiology. Eur J Heart Fail 11:433-43 Heart Association Council on Cardiovascular Nursing; Ameri- can Heart Association Council on Clinical Cardiology; Ameri- Lainscak M, Anker SD (2009): Prognostic factors in chronic can Heart Association Council on Nutrition, Physical Activity, heart failure. A review of serum biomarkers, metabolic and Metabolism; American Heart Association Interdisciplinary changes, symptoms, and scoring systems. Herz 34:141-7 Council on Quality of Care and Outcomes Research. (2009): State of the science: promoting self-care in persons with Heymans S, Hirsch E, Anker SD, Aukrust P, Balligand JL, heart failure: a scientific statement from the American Heart Cohen-Tervaert JW, Drexler H, Filippatos G, Felix SB, Gulles- Association. Circulation 120(12):1141-63 tad L, Hilfiker-Kleiner D, Janssens S, Latini R, Neubauer G, Paulus WJ, Pieske B, Ponikowski P, Schroen B, Schultheiss Kung T, Springer J, Doehner W, Anker SD, von Haehling S HP, Tschöpe C, Van Bilsen M, Zannad F, McMurray J, Shah (2010): Novel treatment approaches to cachexia and sarco- AM (2009): Inflammation as a therapeutic target in heart penia: highlights from the 5th Cachexia Conference. Expert failure? A scientific statement from the Translational Research Opin Investig Drugs 19(4):579-85 Committee of the Heart Failure Association of the European Society of Cardiology. Eur J Heart Fail 11:119-29 von Haehling S, Stepney R, Anker SD (2010): Advances in understanding and treating cardiac cachexia: Highlights from Sandek A, Doehner W, Anker SD, von Haehling S (2009): the 5th Cachexia Conference. Int J Cardiol Jun 18. [Epub Nutrition in heart failure: an update. Curr Opin Clin Nutr ahead of print] Metab Care 12:384-91

Patents (2006-2010) WO002008106938A3 (2008) DE102004047955A1 (2006) EP000001818061A1 (2007) EP000001937360A1 (2008) US020090023639A1 (2009) WO002006058748A1 (2006) US020070197485A1 (2007) US000007417038B1 (2008) WO002009056256A1 (2009) EP000001669074A1 (2006) EP000001863492A1 (2007) DE102007010834A1 (2008) WO002009071405A1 (2009) EP000001749531A1 (2006) EP000001915159A1 (2008) WO002008106938A2 (2008) US020090197851A1 (2009) WO002007014586A1 (2007) US020080139521A1 (2008) US020080269179A1 (2008) EP000002099474A1 (2009) DE102005046539A1 (2007) WO002008067833A1 (2008) WO002008135571A1 (2008) US020090248101A1 (2009) WO002007039263A2 (2007) WO002008067831A2 (2008) DE102007022367A1 (2008) EP000002122363A2 (2009)

39 Stefan Anker – Applied Cachexia Research

General information Third party funding (2006-2009)

Project leader Project title Sponsor Period Anker S.D. WARCEF study coordination – Germany NIH USA 2004-2011 & Poland Anker S.D. Warcef study coordination – Nether- NIH USA 2005-2011 lands Springer J. Heart failure cachexia therapy Ipsen Pharma, France 2005-2007 Anker S.D. Anker S.D. Scholarship for biomarker research BRAHMS AG 2006-2007 Springer J. Developing a heart failure in old animals Sonnenfeld-Stiftung Berlin 2006-2007 Anker S.D. Springer J. Development of an in-vitro myocardial Erna-Graff- Stiftung 2006-2007 in faction model based on vital heart sections Springer J. To study the effects of a GLP-1 agonist Curatis Pharma GmbH 2007 on heart in rats with chronic heart failure Döhner W. Investigations on insulin sensitivity in DFG 2007-2010 Anker S.D. patients with heart failure and with COPD with/without cachexia Anker S.D. Scholarship for biomarker research BRAHMS AG 2008-2009 Springer J. Cancer cachexia therapy Myotec Therapeutics 2008-2009 Anker S.D. Döhner W. Metabolic research on mechanisms of DFG – BMBF 2008-2013 Anker S.D. cachexia in stroke and CHF – Substudy of the IFB Stroke Center Berlin Anker S.D. Studies investigating comorbidities EU FP 7 2009-2013 von Haehling S. aggravating heart failure (SICA-HF): Döhner W. EU co-ordination (SDA) and 4 sub- projects Anker S.D. Biomarker research in the Biostat-CHF EU FP 7 2009-2014 von Haehling S. project Döhner W. Springer J. Investigations on weight loss and meta- BMBF 2009-2010 Döhner W. bolic changes in a stroke animal model

40 IVO BUSCHMANN – EXPERIMENTAL AND CLINICAL ANGIOLOGY / ARTERIOGENESIS

Head of the group

Priv.-Doz. Dr. med. Ivo Buschmann, MD, F.E.S.C.

Curriculum Vitae: Ivo Buschmann studied medicine at the University of Hamburg. After 2 years of clinical medicine in cardiology he received a stipend by the Max- Planck-Society and joined the group of Wolfgang Schaper at the Max-Planck-Institute for Physiological & Clinical Research Bad Nauheim, which focused on the biology of the collateral circulation.

In the following years Ivo Buschmann held the position of group leader and published several original papers in the field of therapeutic Arteriogenesis. One of the experimental trials was the first to show that Arteriogenesis may be stimulated therapeutically in the brain (PNAS 2004).

In 2000, Ivo Buschmann was granted a VolkswagenStiftung Excellency Programme-Funding for altogether 6 years and had to change – due to the program - location to the University of Freiburg, Dept. of Internal Medicine (Cardiology). In 2004, Ivo Buschmann and his research group moved to the Charité Berlin, where they are now located at the Center for Cardiovascular Research at the Campus Mitte, the Campus Virchow and the Vascular Center Berlin. Ivo Buschmann currently holds the position of a clinical consultant in vascular medicine (Angiology).

In 2004, Ivo Buschmann received his habilitation in the field of Vascular Medicine. The main focus of the Rich- ard Thoma Laboratories are the experimental and clinical basics of Arteriogenesis. A special emphasis is the translational approach, i.e. on promoting the transfer of experimental data into the clinic scenario.

By the end of 2010, CardioAccel will be a first spin-off company from the Richard Thoma Laboratories. This project is initially funded by the German Federal Ministry of Economy and Technology through the EXIST-For- schungstransfer Programme. The Company develops personalized training programmes for vascular diseases (Herzhose).

Moreover, Ivo Buschmann is a Member of the Board of the Deutsches Angiologie Zentrum Berlin –DAZB- (German Angiology Center Berlin), a foundation to promote research and patient care in the field of peripheral vascular medicine.

41 IVO BUSCHMANN – EXPERIMENTAL AND CLINICAL ANGIOLOGY / ARTERIOGENESIS

Members of the group Spin-Off Company:

Office Wilke, Thorsten

Experimental Arteriogenesis (CCR): Coworkers (sponsored by EXIST-Forschungstransfer Programme of German Federal Ministry of Economy and Technology) Dülsner, André DVM, Dr. med. vet. Krackhardt, Florian MD, Dr. med. Coworkers Hillmeister, Philipp Dr. rer. nat. Buschmann, Eva-Elina Dr. med., MD Pagonas, Nikolaos MD Funke, Katharina Dipl.-Kffr., MBA Carrão, Catarina Dr. rer. nat. Paeschke, Robert Dipl.-Ing. Gatzke, Nora DVM Harnoß, Jonathan med. & PhD student ECRC collaboration (Berlin-Buch): Lee, Eun-Ji med. & PhD student Coworkers Li, Meijing MD, PhD student Le Noble, Ferdinand Prof. Dr. rer. nat. (Co-Director Ma, Chenming MD, PhD student Richard Thoma Laboratories) Nagorka, Stephanie DVM, PhD student Shi, Yu PhD Wang, Haitao MD, PhD student Collaboration with Vascular Center Berlin Scholz, Katja Technician Prof. K.-L. Schulte Director Zimmer, Anja Technician Langhoff, Ralf Deputy-Director Lindhorst, Ruhdja Dr. med., MD

42 IVO BUSCHMANN – EXPERIMENTAL AND CLINICAL ANGIOLOGY / ARTERIOGENESIS

Summary

Stimulation of biological bypass formation (Arte- mechanisms (fluid shear stress, application of para- riogenesis) is an attractive therapeutic alternative crine factors), taking into account pathophysiologi- for patients with diffuse occlusive vessel disease. cal principles (potential pro-arteriogenic effects of Extensive in vivo and in vitro studies have revealed atherosclerosis through undifferentiated stem cells, the basic mechanisms of Arteriogenesis during the acceleration of mitogenous processes etc.). Current past decades. Early non-invasive induction of col- data indicate that a single pharmacon acting on a lateral proliferation in patients at risk is a major aim specific receptor might not be available to sufficiently of current studies. Cytokine-induced Arteriogenesis induce arteriogensis. Especially in patients suffering is a potential novel conservative strategy. Clinical from chronic stenoses, an additive induction of fluid studies have so far been limited by their unifactorial shear stress may be essential to (re-)activate collat- approach and non-specific end points. An additional eral growth and, thus, to allow for an additional ther- induction of the natural trigger of Arteriogenesis, apeutic benefit of any pro-artiogenic strategy. This i.e. an enhanced fluid shear stress acting on the concept ist supported by current experimental data endothelium, can be achieved through active and proving that collateral growth without enhanced fluid “passive” physical training. Clinical studies demon- shear stress does not lead to a functional improve- strate therapeutic safety and promising preliminary ment oft tissue perfusion. The Richard Thoma Labo- results. Arteriogenesis is presumably the most effec- ratories explore the field of Arteriogenesis both with tive endogenous mechanism of compensation for respect to its experimental basics of as well as with arterial stenosis or occlusion. Therapeutic strategies respect to transforming the resulting findings into should therefore support preexistent physiological everyday clinical and ambulant processes.

43 IVO BUSCHMANN – EXPERIMENTAL AND CLINICAL ANGIOLOGY / ARTERIOGENESIS

Zusammenfassung:

Die Stimulation des biologischen Bypasses (Arte- tion der Atherosklerose z. B. durch undifferenzierte riogenese) stellt eine attraktive alternative Behand- Stammzellen, Beschleunigung mitogener Prozesse lungsmethode v. a. für Patienten mit hochgradigen im Körper etc.). Ein weiterer wesentlicher Punkt diffusen stenosierenden Gefäßerkrankungen dar. betrifft die bisher erfolgreich angewandte Strategie Basismechanismen der Arteriogenese konnten in der singulären Pharmakonentwicklung und -therapie ausgedehnten Untersuchungen der letzten Jahr- mittels eines funktionell wirksamen pharmakologi- zehnte grundlegend verstanden werden. Eine schen Wirkmoleküls (z. B. Angiotensinrezeptorblo- rechtzeitige und möglichst wenig invasive Induktion cker mit spezifischem Effekt auf selektiven Rezeptor kollateraler Proliferation bei Risikopatienten ist das und respektive Blutdruck): Die vorliegenden Daten Ziel aktueller Studien. Zytokininduzierte Arterioge- deuten bei der Arteriogenese darauf hin, dass nese ist eine potenzielle medikamentös-konservative womöglich dieses singuläre pharmakologische Prin- Strategie. Klinische Studien waren bislang durch zip bei der Arteriogenese nicht wirksam sein könnte. den unifaktoriellen Ansatz sowie z. T. unspezifische Gerade bei Patienten, bei denen Gefäßstenosen Endpunkte limitiert. Eine zusätzliche Induktion des schon länger bestehen, kann eine additive Erhöhung natürlichen Auslösers der Arteriogenese, der endo- der Schubspannung (durch aktives oder passives thelialen Schubspannung, kann durch aktives und Training) notwendig sein, um kollaterales Wachstum passives körperliches Training erfolgen. Klinische erneut „anzuschalten“. Erst diese Aktivierung des Anwendungen zeigen Therapiesicherheit und erste Endothels ermöglicht dann additive therapeutische vielversprechende Ergebnisse. Zusammenfassend Verbesserungen durch proarteriogene Therapien. gilt, dass die Arteriogenese vermutlich den wich- Diese konzeptionelle Überlegung wird von den der- tigsten Mechanismus darstellt, um Stenosen und zeitigen experimentellen Befunden unterstrichen, Okklusionen im Körper endogen zu kompensieren. die eindeutig belegen, dass kollaterales Wachstum Therapeutische Strategien sollten daher eine Unter- ohne „Shear-Stress-Erhöhung“ zu keinen funktionel- stützung bereits vorhandener nebenwirkungsarmer len Verbesserungen der Gewebsperfusion führt. Die biologischer Mechanismen anstreben (Induktion Richard Thoma Laboratorien forschen im Bereich von Schubspannung, Benefit spezifischer parakriner der experimentellen Grundlagen der Arteriogenese Faktoren), ohne dabei pathophysiologische Prinzi- sowie der Umsetzung in den ambulanten als auch pien aus dem Auge zu verlieren (mögliche Induk- klinischen Alltag.

44 IVO BUSCHMANN – EXPERIMENTAL AND CLINICAL ANGIOLOGY / ARTERIOGENESIS

Research projects

1. Effects of Aspirine on Adaptive Arteriogenesis

Project leader André Dülsner Coworkers Nora Gatzke, Dr. med. vet. Johanna Glaser, Philipp Hillmeister, Meijing Li, Eun-Ji Lee, Dr. rer. nat. Kerstin Lehmann, Stephanie Nagorka DVM, Vesna Furundzija MD, Heike Meyborg BSc, Philipp Stawowy MD, Dr. Andreas Busjahn, Ivo Buschmann Funding Deutsche Forschungsgemeinschaft (Grant DFG BU1141/4-2), VolkswagenStiftung

Background and Purpose – Aspirin and CClopidogrel but not Clopidogrel. Furthermore, it is still significantly are well-used in the prevention of vascular diseases. decreased after treatment with ASA combined with In virtue of its anti-inflammatory effects aspirin inhibits G-CSF compared to G-CSF only. The PCA diameters, peripheral Arteriogenesis. In this study we investigated which are not influenced by Clopidogrel, do not dif- the effects of acetylsalicylic acid (ASA) and Clopidogrel fer after ASA combined with G-CSF-treated rats from on cerebral Arteriogenesis. those, which are treated with ASA alone, and they Methods – Rats underwent the 3-VO method and were are still smaller compared to the G-CSF single treat- treated with either drinking water as control, ASA or ment. The enhanced monocyte migration after treat- Clopidogrel for seven or 21 days. They also received ment with G-CSF, GM-CSF and M-CSF is significantly G-CSF every other day for one week to evaluate their reduced by pretreatment with ASA. effects on therapeutically induced Arteriogenesis. Conclusions – In this study it is shown that ASA, but Cerebrovascular reactivity was examined as well as not Clopidogrel inhibits G-CSF improved cerebral post mortem latex angiography. Arteriogenesis.

Results – Reserve capacity is completely abolished after 3-VO and is still abrogated after 21 days by ASA,

45 IVO BUSCHMANN – EXPERIMENTAL AND CLINICAL ANGIOLOGY / ARTERIOGENESIS

2. Stimulation of coronary collateral growth by granulocyte stimulating factor: role of reactive oxygen species.

Project leader Ana Catarina R. Carrão Coworkers William M. Chilian, June Yun, Christopher Kolz, Petra Rocic, Lehmann Kerstin, Jeroen P.H.M. van den Wijngaard, Pepijn van Horssen, Jos A. E. Spaan, Vahagn Ohanyan, Yuh Fen Pung, Ivo Buschmann Funding EU-Grant Collaborations Department of Integrative Medical Sciences, NEOUCOM, Northeastern Ohio University College of Medicine, Rootstown, Ohio; Department of Biochemistry and Molecular Biology, University of South Alabama, Mobile; Department of Biomedical Engineering and Physics, Academic Medical Centre, Amsterdam, Netherlands.

Objective - The purpose of this study was to determine tion, 60 microg/kg, during two episodes of ischemia). whether G-CSF promotes coronary collateral growth DHE fluorescence was double in G-CSF+RI versus (CCG) and decipher the mechanism for this stimula- vehicle+RI (P<0.01), and even higher in G-CSF with- tion. out RI (P<0.01). Interestingly, the DHE signal did not Methods and results - In a rat model of repetitive epi- colocalize with myeloperoxidase (immunostaining, sodic myocardial ischemia (RI, 40 seconds LAD occlu- neutrophil marker) but appeared in cardiac myocytes. sion every 20 minutes for 2 hours and 20 minutes, 3 The study of isolated cardiac myocytes revealed the times/d for 5 days) CCG was deduced from collateral- cytokine stimulates ROS which elicit production of dependent flow (flow to LAD region during occlusion). angiogenic factors. Apocynin inhibited G-CSF effects After RI, G-CSF (100 microg/kg/d) increased CCG both in vivo and in vitro. (P<0.01) (0.47+/-0.15) versus vehicle (0.14+/-0.06). Conclusions - G-CSF stimulates ROS production Surprisingly, G-CSF treatment without RI increased directly in cardiomyocytes, which plays a pivotal role in CCG (0.57+/-0.18) equal to G-CSF+RI. We evaluated triggering adaptations of the heart to ischemia includ- ROS by dihydroethidine (DHE) fluorescence (LV injec- ing growth of the coronary collaterals.

46 IVO BUSCHMANN – EXPERIMENTAL AND CLINICAL ANGIOLOGY / ARTERIOGENESIS

3. Molecular Mechanisms of the Bradykinin- Pathway for Arteriogenesis

Project leader Philipp Hillmeister Coworkers Kerstin E Lehmann, Anja Bondke, Henning Witt, André Duelsner, Clemens Gruber, Hans-Jörg Busch, Joachim Jankowski, Patricia Ruiz-Noppinger, Konstantin-Alexander Hossmann, Ivo Buschmann Funding Deutsche Forschungsgemeinschaft, (Grant DFG BU 1141/4-2), Volkswagen Foundation, Germany Collaborations Charité University Medicine Berlin: Center for Preclinical Studies (CC2), Institute of Physiology, Max-Planck-Institute for Molecular Genetics, Department of Anatomy, Institute of Integrative Neuroanatomy; Medical Clinic IV,; Department of Internal Medicine (Cardiology), University of Freiburg i. Br.; Max-Planck-Institute for Neurological Research, Cologne;

Cerebral Arteriogenesis constitutes a promising thera- and migration. By using scanning electron micros- peutic concept for cerebrovascular ischaemia; how- copy, morphologic activation of the PCA endothelium ever, transcriptional profiles important for therapeutic was detected. Furthermore, the PCA showed induced target identification have not yet been investigated. proliferation (PCNA staining) and CD68+ macrophage This study aims at a comprehensive characterization staining 24 h after 3-VO, resulting in a significant of transcriptional and morphologic activation during increase in diameter within 7 days after 3-VO, confirm- early-phase collateral vessel growth in a rat model ing the arteriogenic phenotype. Analysis of molecular of adaptive cerebral Arteriogenesis. Arteriogenesis annotations and networks associated with differentially was induced using a three-vessel occlusion (3-VO) expressed genes revealed that early-phase cerebral rat model of nonischaemic cerebral hypoperfusion. Arteriogenesis is characterised by the expression of Collateral tissue from growing posterior cerebral artery protease inhibitors. These results were confirmed by (PCA) and posterior communicating artery (Pcom) was quantitative real-time reverse transcription-PCR, and selectively isolated avoiding contamination with adja- in situ hybridisation localised the expression of tissue cent tissue. We detected differential gene expression inhibitor of metalloproteinase-1 (TIMP-1) and kinino- 24 h after 3-VO with 164 genes significantly deregu- gen to collateral arteries, showing that TIMP-1 and lated. Expression patterns contained gene transcripts kininogen might be molecular markers for early-phase predominantly involved in proliferation, inflammation, cerebral Arteriogenesis.

47 IVO BUSCHMANN – EXPERIMENTAL AND CLINICAL ANGIOLOGY / ARTERIOGENESIS

4. Improvement of fractional flow reserve and collateral flow by treatment with external counterpulsation (Art.Net.-2 Trial)

Project leader Eva-Elina Buschmann Coworkers Utz W, Pagonas N, Schulz-Menger J, Busjahn A, Monti J, Maerz W, le Noble F, Thierfelder L, Dietz R, Klauss V, Gross M, Buschmann IR Funding Principal Investigator funded Collaborations Franz-Volhard-Klinik, Department for Cardiology, Helios-Klinikum Berlin-Buch, HealthTwiSt GmbH, Berlin – Buch, Synlab laboratory services, Eppelheim, Max-Delbrueck-Center for Molecular Medicine, Angiogenesis and Cardiovascular Pathology, Berlin, Medizinische Poliklinik der Universität München; Campus Innenstadt, Dept. for Cardiology, München, Department for Internal Medicine (Cardiology), University Clinic Freiburg/ Germany.

Background - Arteriogenesis (collateral artery growth) pressure-derived collateral flow index (CFIp, primary is nature’s most efficient rescue mechanism to over- endpoint) and fractional flow reserve (FFR). come the fatal consequences of arterial occlusion or Results - In the ECP group, the CFIp (from 0.08 +/- stenosis. The goal of this trial was to investigate the 0.01 to 0.15 +/- 0.02; P < 0.001) and FFR (from 0.68 effect of external counterpulsation (ECP) on coronary +/- 0.03 to 0.79 +/- 0.03; P = 0.001) improved sig- collateral artery growth. nificantly, while in the control group no change was Materials and Methods - A total of 23 patients (age 61 observed. Only the ECP group showed a reduction +/- 2.5 years) with stable coronary artery disease and of the Canadian Cardiovascular Society (CCS, P = at least one haemodynamic significant stenosis eligi- 0.008) and New York Heart Association (NYHA, P < ble for percutaneous coronary intervention were pro- 0.001) classification. spectively recruited into the two study groups in a 2 : 1 Conclusion - In this study, we provide direct functional manner (ECP : control). One group (ECP group, n = 16) evidence for the stimulation of coronary Arteriogenesis underwent 35 1-h sessions of ECP in 7 weeks. In the via ECP in patients with stable coronary artery disease. control group (n = 7), the natural course of collateral These data might open a novel noninvasive and pre- circulation over 7 weeks was evaluated. All patients ventive treatment avenue for patients with non-acute underwent a cardiac catheterization at baseline and vascular stenotic disease. after 7 weeks, with invasive measurements of the

48 IVO BUSCHMANN – EXPERIMENTAL AND CLINICAL ANGIOLOGY / ARTERIOGENESIS

Publications 2006-2010 Hillmeister P, Lehmann KE, Bondke A, Witt H, Duelsner A, Gruber C, Busch HJ, Jankowski J, Ruiz-Noppinger P, Jungehuelsing GJ, Liman TG, Brunecker P, Ebel A, Endres Hossmann KA, Buschmann IR (2008): Induction of cerebral M, Buschmann I, Pagonas N, Buschmann EE. (2010): Does Arteriogenesis leads to early-phase expression of protease External Counterpulsation Augment Mean Cerebral Blood inhibitors in growing collaterals of the brain. J Cereb Blood Flow in the Healthy Brain? Effects of External Counterpulsa- Flow Metab Nov;28(11):1811-23 tion on Middle Cerebral Artery Flow Velocity and Cerebrovas- cular Regulatory Response in Healthy Subjects. Cerebrov Bondke A, Buschmann IR (2007):Visualization of therapeutic asc Dis Oct 15;30(6):612-617 Arteriogenesis by MR angiography: functional measurements are superior to surrogate parameters. J Magn Reson Imag Buschmann I, Pries A, Styp-Rekowska B, Hillmeister P, ing Jul;26(1):215; author reply 216 Loufrani L, Henrion D, Shi Y, Duelsner A, Hoefer I, Gatzke N, Wang H, Lehmann K, Ulm L, Ritter Z, Hauff P, Hlushchuk R, Buschmann IR, Bondke A, Elgeti T, Kühnle Y, Dietz R, Möckel Djonov V, van Veen T, le Noble F. (2010): Pulsatile shear and M (2007): Positive cardiac troponin I and T and chest pain in a Gja5 modulate arterial identity and remodeling events during patient with iatrogenic hypothyroidism and no coronary artery flow-driven Arteriogenesis. Development Jul;137(13):2187-96 disease. Int J Cardiol Feb 7;115(2):e83-5 von Knobelsdorff-Brenkenhoff F, Buschmann EE, Pilz B, Schefe JH, Lehmann KE, Buschmann IR, Unger T, Funke- Schulz-Menger J (2010): Persistent Cabrol shunt causing Kaiser H (2006): Quantitative real-time RT-PCR data analysis: severe right heart failure. Ann Thorac Surg. Jul;90(1):312 current concepts and the novel “gene expression’s CT difference” formula. J Mol Med Nov;84(11):901-10 Carrão AC, Chilian WM, Yun J, Kolz C, Rocic P, Lehmann K, van den Wijngaard JP, van Horssen P, Spaan JA, Ohanyan Bergmann CE, Hoefer IE, Meder B, Roth H, van Royen N, V, Pung YF, Buschmann I (2009): Stimulation of coronary Breit SM, Jost MM, Aharinejad S, Hartmann S, Buschmann IR collateral growth by granulocyte stimulating factor: role of (2006): Arteriogenesis depends on circulating monocytes and reactive oxygen species. Arterioscler Thromb Vasc Biol macrophage accumulation and is severely depressed in op/ Nov;29(11):1817-22 op mice. J Leukoc Biol Jul;80(1):59-65

Buschmann EE, Utz W, Pagonas N, Schulz-Menger J, Busjahn A, Monti J, Maerz W, le Noble F, Thierfelder L, Dietz Reviews & Editorials 2006-2010 R, Klauss V, Gross M, Buschmann IR (2009): Arteriogenesis Network (Art. Net.). Improvement of fractional flow reserve le Noble F, Klein C, Tintu A, Pries A, Buschmann I (2008): and collateral flow by treatment with external counterpulsation Neural guidance molecules, tip cells, and mechanical factors (Art.Net.-2 Trial). Eur J Clin Invest Oct;39(10):866-75 in vascular development. Cardiovasc Res May 1;78(2):232- 41 von Knobelsdorff-Brenkenhoff F, Rudolph A, Wassmuth R, Bohl S, Buschmann EE, Abdel-Aty H, Dietz R, Schulz-Menger Buschmann IR, Lehmann K, Le Noble F; Art.Net. (2008): J (2009): Feasibility of cardiovascular magnetic resonance to Physics meets molecules: is modulation of shear stress the assess the orifice area of aortic bioprostheses. Circ Cardio link to vascular prevention? Circ Res Mar 14;102(5):510-2 vasc Imaging Sep;2(5):397-404, 2 p following 404 Bondke A, Buschmann IR, Bode C, Buschmann EE (2007): Pagonas N, Utz W, Schulz-Menger J, Busjahn A, Monti J, [Inducing collaterals in due time. Arteriogenesis as a preven- Thierfelder L, Dietz R, Klauss V, Gross M, Buschmann IR, tive principle] Haemostaseologie Dec;27(5):363-72 Buschmann EE; on behalf of the Arteriogenesis Network (Art. Net.). (2009): Assessment of the effect of external counter- Bondke A, Hillmeister P, Buschmann IR (2007): Exact assess- pulsation on myocardial adaptive Arteriogenesis by invasive ment of perfusion and collateral vessel proliferation in small functional measurements - design of the Arteriogenesis animal models. Circ Res Apr 27;100(8):e82-3 network trial 2. Int J Cardiol Jun 16

49 IVO BUSCHMANN – EXPERIMENTAL AND CLINICAL ANGIOLOGY / ARTERIOGENESIS

Visualization of therapeutic Arteriogenesis by MR angiog- Donner S: Spaziergänge lassen neue Blutgefäße sprießen, raphy: Functional measurements are superior to surrogate Die Welt – Online, 07.09.2009 parameters. J Magn Reson Imaging 2007 Jul;26(1):215 Donner Susanne, Herzhose sorgt über natürlichen Bypass für Erdo F, Buschmann IR (2007): [Arteriogenesis: a new strategy bessere Durchblutung, VDI-Nachrichten, 25.09.2009 of therapeutic intervention in chronic arterial disorders. Cel- lular mechanism and experimental models] Orv Hetil. Apr N.N.: Blutgefäße können wieder wachsen, Das Goldene 8;148(14):633-42 Blatt, 12.10.2009, 30

Buschmann IR, Bondke A, Elgeti T, Kuhnle Y, Dietz R, Mockel Blech J: Das Blut in Wallung bringen, Der Spiegel M (2007): Positive cardiac troponin I and T and chest pain in a 18.01.2010,102-104. patient with iatrogenic hypothyroidism and no coronary artery disease. Int J Cardiol Feb 7;115(2) Aumiller J: Herzhose bringt das Blut in Wallung, CardioNews, Nr. 4, 2010, 34

Book Chapters 2006-2010 Krause Claudia: Mein Herz schlägt wieder kräftig, Mach Mal Pause, BauerMediaGroup, Nr.33, 1.08.2010, 54 Chapter: Buschmann I, Pagonas N, “Stimulation of Arteriogenesis via External Counterpulsation – State of the Art and Clinical Per- TV- and Radio-broadcasts related to spective” in Schaper, W; Deindl, E, “The Collateral Circulation the Group’s research: – Molecular Regulation, Pathophysiology and Therapeutics”; to be published Autumn 2010 Engler K: Schaufensterkrankheit und „Herzhose“, MDR 1, Radio Sachsen-Anhalt, Vormittagsprogramm vom Boengler K, Buschmann I, Deindl E, Gottwik M, Hoffmeister 21.10.2009 HM, Ito W, Klein H, Mauser M, Nienaber C, Regitz-Zagrosek V, Sack S (2009): On the occasion of Wolfgang Schaper’s Brummerloh D: Biologische Bypässe: Herzhose lässt neue 75th birthday. Basic Res Cardiol Jan;104(1):2-4 Umgehungsarterien wachsen –BR5, 11.07.2010

Chapter: Engler K: Beitrag zu Schaufensterkrankheit und „Herzhose“ Pries A, Buschmann I, Habazettl H, “Key Issues in Vascular im Magazin „Hauptsache Gesund“, MDR Fernsehen, Pathology” in 21.01.2010 Lanzer P. Mastering endovascular techniques: a guide to excellence. Lippincott Williams & Wilkins, 2006 Brettschneider E: Training statt Herz-Bypass: Bewegung lässt neue Blutgefäße am Herzen sprießen, Gesundheitsmagazin „QuiVive“, RBB-Fernsehen ,03.03.2010 Popular Press Publications / Target Customer Publications Rosbach J: Mit der Herzhose zu neuen Arterien, Beitrag im related to the Group’s research: Magazin „Visite“, NDR-Fernsehen, 30.03.2010 (wiederholt im WDR-Fernsehen, Ratgeber Servicezeit Gesundheit, Von Lutterotti N: Kreislaufmassage oder wenn kühne 19.07. & 20.07.2010) Träume platzen, Frankfurter Allgemeine Zeitung, Nr. 179, 05.08.2009, N5

Abel S: Biologische Bypässe, Gesund – Beilage des Springer Verlags zum Hamburger Abendblatt etc., 04.09.2009, 4

50 IVO BUSCHMANN – EXPERIMENTAL AND CLINICAL ANGIOLOGY / ARTERIOGENESIS

General Information Third party funding (2006-2010)

Project leader Project title Sponsor Period Buschmann I. VolkswagenStiftung Arteriogenese VolkswagenStiftung 2003-2006

Buschmann I. Arteriogenese und Risikofaktoren in der Deutsche Forschungsgemein 2003-2007 Zucker Fatty Rat schaft, BU 1151/4-1 Buschmann I. Kompetenznetz Zerebrale Arteriogenese MBWK 2002-2007

Buschmann I. EST Teilprojekt EU 2004-2006 Buschmann I. Porcine Arteriogenesis Medtronic (US) 2006-2007 Buschmann I. Flow driven Arteriogenesis Charité - Experimental and Clini- 2008-2011 Le Noble F. cal Research Center, Berlin-Buch

Lehmann K. Künstliche Arterie BMBF 2007-2010 Lehmann K. NGFN Verbundprojekt NGFN 2008-2010 Buschmann I. Modulation of natural Inhibition as a DFG – German Research 2008-2011 Kappert K. therapeutic principle: Protein-Tyrosine- Community Phosphatases as interventional target structures of Arteriogenesis

Buschmann I. CardioAccel Federal Ministry for Economy 2010-2011 and Technology / EXIST For schungstransfer Hillmeister P. Cerebral Arteriogenesis – A Concept for Creutzfeld Jacob Stipend 2008-2011 Prevention from Charité-Center for Stroke Research Berlin (CSB)

51 IVO BUSCHMANN – EXPERIMENTAL AND CLINICAL ANGIOLOGY / ARTERIOGENESIS

Awards

2010 15. Dreiländertagung der Schweizerischen, Deutschen und Österreichischen Gesellschaft für Angiologie und 39. Jahrestagung der Deutschen Gesellschaft für Angiologie und Gefäßmedizin e.V., Congress Center Basel, Basel, Switzerland , 12.-15.September

1st Poster Prize: “Pioglitazone inhibits Induction of the Biological Bypass“ André Dülsner

1st Poster Prize:, „Induction of the Biological Bypass in the Circle of Willis“ Kongress of the German Society for Vascular Surgery, Berlin, 9.9.10: Nora Gatzke

2010 For the best Presentation held in scope of the Campaign “Young Vascular Medical Scientists” granted a travel grant Jonathan Harnoß

2010 Charité Promotion Stipend granted to Stephanie Nagorka

2009-2010 Charité Promotion Stipend granted to Nora Gatzke

52 Duska Dragun – NEPHROLOGY AND CARDIOVASCULAR IMMUNOLOGY

Head of the group

Prof. Dr. med. Duska Dragun

Curriculum Vitae: 1988-1993 Medical School: University of Zagreb, Croatia 1993-1995 Intern at university hospital "Rebro", Zagreb, Croatia 1995 License to practise medicine from Ministry of Health Croatia 1995-1997 Doctoral thesis at "Max Delbrück Centrum für molekulare Medizin Berlin-Buch", Prof. Dr. Hermann Haller, Berlin, Germany 1998 Degree as a "Dr. med." from the "Humboldt Universität", Berlin, Germany, Grade: "summa cum laude" 1998 Research fellow at "Dept. for Immunology and Organ Transplantation" Prof. Dr. Barry Kahan, University of Texas, Houston, USA 1999-2000 Clinical fellow at "Franz Volhard Klinik", Charité Campus Buch, Prof. Dr. Friedrich C. Luft, Berlin, Germany 2002-2007 Assistant Professor at the Medical faculty of the Charité, Berlin 2000-2002 Clinical fellow at Department of Nephrology, Charité Campus Mitte, 2005 Specialist in Internal Medicine, Berlin 2005-2006 Attendant at Department of Nephrology, Charité Campus Mitte, 2006- Attendant at Clinic for Nephrology and Intensive Care Medicien, Charité Campus Virchow Clinic 2008 Specialist in Nephrology, Berlin 2008- Full Professor at the Medical faculty of the Charité, Berlin

Current administrative functions 2002- Member of the Faculty board for career development 2005 Member of executive board of "Junge Charité" 2006- Advisory board of the Charité Mentoring Program for female scientists 2009- Deputy member of the Board of the Medical faculty of Charité 2009- Co-chair of the Faculty Board for career development 2009- Scientific Advisory Board of the German Society of Transplantation 2009- Task force "Kidney transplantation" German Society of Nephrology

53 Duska Dragun – NEPHROLOGY AND CARDIOVASCULAR IMMUNOLOGY

Members of the group Graduate and undergraduate students Baris, Christina Medical student Scientists Böhm, Susanne Bachelor student; Catar, Rusan Ali Dr. rer. nat. 07/2008 – 02/2009 Chaykovska, Lyubov Dr. med. Eimers, Meike PhD student; until 05/2008 Fielitz-Haase, Anja Dr. rer. medic. Gürgen, Dennis PhD student Fuller, Florian PD Dr. med. Janke, Daniel Medical student Haase, Michael PD Dr. med. Karatas, Aysun PhD student; until 01/2010 Hegner, Björn Dr. med. Krause, Sebastian Medical student Hoff, Uwe Dr. med. Kretzschmar, Tobias Medical student Kusch, Angelika Dr. med. Marinez, Julian Medical student Näther, Melanie Dr. rer. nat. Pützer, Jennifer Medical student Philippe, Aurélie Dr. rer. nat. Schaub, Theres PhD student Schmidt, Maria Medical student Urban, Claudia Diploma student; Technicians 12/2007 – 07/2008 Eigen, Marc Wagner, Philine Medical student Gruber, Gertrud Wischnewski, Oskar Medical student Kämper, Barbara

54 Duska Dragun – NEPHROLOGY AND CARDIOVASCULAR IMMUNOLOGY

Summary

Our group studies the pathogenesis and therapy of • Sex-specific mechanisms of cardiovascular co- alloantigen independent renal transplant injuries and moribidity in renal patients the development of new therapeutic modalities in • Disease and stimulus specific mechanisms of an experimental and clinical setting. The focus of differentiation of MSCs our research is to increase the long-term survival of the graft and to minimize cardiovascular morbidity We rely on ‘bedside to bench’ approach that allows in transplanted patients. Moreover, we are involved us to translate newly identified disease entities or in development of novel diagnostic tests and their results of association studies in disease relevant validation in various contexts of renal and cardiovas- animal models for dissection of pathophysiologic cular pathologies. mechanisms. Animal experiments include highly sophisticated age and gender specific kidney trans- Research areas of interest: plant models in rats as well as different models to • Role of agonistic antibodies targeting G-protein study the progression of kidney disease and result- coupled vascular receptors in cardiovascular, ing cardiac hyperthrophy and fibrosis (DOCA-salt autoimmune, and transplant pathologies HTN, Transgenic animals). Tissue analysis is done • Vascular mechanisms and interventions aimed via histology, immunohistology and molecular biol- at modulation of vascular injury during ischemia ogy. Different types of cell cultures are used for the and reperfusion injury in native kidneys and kid- elucidation of signaltransduction pathways. ney transplants • Development of biomarkers to monitor vascular injury during acute renal failure in native kidneys and organ transplants

55 Duska Dragun – NEPHROLOGY AND CARDIOVASCULAR IMMUNOLOGY

Zusammenfassung

Unsere Arbeitsgruppe untersucht die Pathogenese • Geschlechtsspezifische Mechanismen kardio- und Therapiemöglichkeiten der alloantigenunab- vaskulärer Co-Morbidität bei Nierenpatienten hängigen Schädigung von Nierentransplantaten • Krankheits- und stimulusspezifische Mechanis- und entwickelt neuartige Behandlungsstrategien men der Differenzierung von MSCs auf experimenteller und klinischer Ebene. Unser wichtigstes Ziel ist eine Verbesserung des Langzeit- Unsere Forschung basiert auf dem "bedside to Transplantatüberlebens bei gleichzeitiger Minimie- bench"-Ansatz, der uns gestattet, neu identifizierte rung der kardiovaskulären Morbidität transplantier- Krankheitsentitäten und Ergebnisse von Assozia- ter Patienten. Darüberhinaus beschäftigen wir uns tionsstudien in krankheitsrelevanten Tiermodellen mit der Entwicklung neuer Biomarker in der Diag- in Hinblick auf die zugrunde liegenden pathophy- nostik renaler und kardiovaskulärer Erkrankungen. siologischen Mechanismen aufzuschlüsseln. Die Tierversuche umfassen neben spezifischen alters- Unsere Forschungsgebiete sind: und geschlechtsbezogenen Rattenmodellen auch • Rolle agonistischer Antikörper gegen G-Protein unterschiedliche Modelle zur Untersuchung der gekoppelte vaskuläre Rezeptoren in kardiovas- Progression von Nierenerkrankungen und der dar- kulären, autoimmunen und transplantationsas- aus resultierenden Herzhypertrophie und Fibrose soziierten Pathologien (DOCA-Salz induzierte Hypertonie, transgene Tiere), • Vaskuläre Mechanismen und Interventionen zur sowie experimentelle Nieren- und Gefäßtransplanta- Modulation des Gefäßschadens im Rahmen tion. Die Gewebsanalyse erfolgt mittels Histologie, des Ischämie-Reperfusionsschadens in Eigen- Immunhistologie und molekularbiologischen Metho- nieren und Nierentransplantaten den. Zur Aufklärung von Signaltransduktionsme- • Entwicklung von Biomarkern zur Überwachung chanismen werden verschiedene Zellkultursysteme von Gefäßschäden bei akutem Nierenversagen eingesetzt. in Eingennieren und Organtransplantaten

56 Duska Dragun – NEPHROLOGY AND CARDIOVASCULAR IMMUNOLOGY

Research projects

1. Angiotensin II type 1 receptor (AT1R-Abs) and Endothelin-1 type A receptor antibodies (ETAR-Abs) in transplant and autoimmune pathologies

Project leader Duska Dragun Coworkers Aurélie Philippe, Rusan Catar, Melanie Näther, Tobias Kretzschmar, Oskar Wischnewski, Barbara Kämper Cooperations G. Riemekasten, H. Heidecke, C. Schönemann U. Querfeld, N. Hiemann, R. Hetzer, P. Reinke, HD Volk, J. van Laar (Newcastle), A. Ghofrani (Giessen), W. Seeger (Giessen), A. Melk (Hannover), C. Schmidt, B. Tönshoff (Heidelberg), M. Konrad (Münster), B. Nashan (Hamburg), A. Benigni (Bergamo), G. Remuzzi (Bergamo), S. Schaub (Basel), J. Steiger (Basel), J. P. Soulillou (Nantes), H. U. Meier-Kriesche (Gainesville), N. Reinsmoen (Los Angeles), A. Zeevi (Pittsburgh), T. Coates (Adelaide) Funding Bundesministerium für Wirtschaft und Technologie, Deutsche Stiftung Sklerodermie, Actelion Pharmaceuticals Deutschland GmbH, Institutional funding (Universitäre Forschungsförderung Charité)

Our group has identified functional autoantibodies SSc related complications. Both autoantibodies were against the Ang II type 1 receptor as the first non- biologically active as they enhanced pulmonary vas- HLA target responsible for vascular rejection, provid- cular reactivity to endogenous receptor agonists in ing a link between cardiovascular and immune dis- a dose-dependent manner that could be blocked eases. Obliterative vascular lesions in SSc resemble by respective pharmacologic antagonists. Anti-AT1R those in transplant vasculopathy. We reasoned that and anti-ETAR autoantibodies may directly contribute agonistic autoantibodies to vascular receptors might to the pathogenesis of SSc and could represent a also be present in SSc and might serve as a disease- link between the increased vascular responsiveness specific biomarker. We identified functional autoim- and tissue damage. This interpretation would support munity directed not only against AT1, but also directed the "vascular hypothesis" in the pathophysiology of against and ETA receptors in SSc patients. Increased SSc. To better understand the link between the auto- levels of autoantibodies against these receptors were antibodies we detected, and the two receptors, we associated with more severe disease manifesta- are currently working on protein immunoprecipitation tions. Harboring anti-AT1R and anti-ETAR antibodies using rat vascular smooth muscle cell primary cultures revealed patients with higher likelihood for develop- which express endogenously both receptors. Moreo- ment of pulmonary hypertension and mortality from ver, we are investigating the implication of the auto-

57 Duska Dragun – NEPHROLOGY AND CARDIOVASCULAR IMMUNOLOGY antibodies in the cardiovascular disease by studying Clinical screening studies for agonistic the signaling pathways depending on the AT1 and ETA autoantibodies receptors. In cooperation with the DHZB a prospective study on Angiotensin II (Ang II) and Endothelin-1 (ET-1) mediate the role of AT1R-Abs in cardiac transplant recipients functional alterations in resistance arteries in terms is currently performed. Data submitted for publica- of enhanced contractility or impaired relaxation. An tion show that presence of AT1R-Abs correlated with increased peripheral resistance in renal, pulmonary, earlier onset and more frequent development of car- mesenteric and coronary arteries may contribute to diac transplant microvasculopathy as determined in severe vascular pathologies observed in organ trans- protocol biopsies. In cooperation with Departments plant recipients and patients with systemic oblitera- of Pediatric Nephrology at the Charité and at the tive vasculopathy due to systemic sclerosis. We study Medical School Hannover, we prospectively recruit vascular-bed specific effects of autoantibodies target- pediatric renal transplant recipients. Children with ing vascular G-protein coupled receptors and their role history of transplant losses due to vascular rejection as an enhancer of vasoactive responses initiated by and presence of AT1R-Abs are enrolled in preemptive natural ligands, Ang II and ET-1. We also study influ- therapy study employing immunoadsorption, intensi- ence of permissive phenomena such is ischemia on fied immunosuppression and high-dose sartanes. In autoantibody-receptor target interaction. cooperation with the Department of Visceral Surgery and Organ Transplantation at the Charité Campus Vir- chow Clinic, we prospectively evaluate liver-, small- bowel and multivisceral transplant recipients for the presence of AT1R-Abs and initiate rescue protocols in case of antibody related transplant injury. Preliminary results implicate association with bile-duct vanishing lesion in liver transplants and vascular rejection in multivisceral transplants. In cooperation with the Depart- ment of Nephrology and Transplant Center Nantes in France we prospectively study cohort of several hundreds of renal transplant patients for AT1R-Abs in conjuction with immune monitoring procedures for multiple T-cell and B-cell markers, as well as regular Agonistic autoantibodies as allosteric receptor activators surveillance of allograft histology by protocol biopsies.

58 Duska Dragun – NEPHROLOGY AND CARDIOVASCULAR IMMUNOLOGY

2. Endothelial mechanisms in thrombotic microangiopathy induced acute renal failure

Project leader Duska Dragun Coworkers Melanie Näther, Aurélie Philippe, Rusan A. Catar Cooperations U. Rauch Funding Dr. Werner Jackstädt Stiftung Institutional funding (Universitäre Forschungsförderung Charité)

Thrombotic microangiopathy (TMA) is the morphologi- tibodies directed against AT1 and ETA receptors in cal substrate in a variety of diseases associated with patients with systemic sclerosis (SSc) and renal crisis. acute renal failure. Typical histological features of TMA We could show in cultured HMEC-1 cells that stimula- are vessel wall thickening with endothelial swelling and tion with IgG from SSc patients resulted in activation formation of platelet–fibrin hyaline microthrombi that of stress signalling kinase ERK1/2 and upregulation occlude arterioles and capillaries. TMA occurs in a of prothrombotic Tissue Factor (TF). The observed wide range of diseases including haemolytic urae- effects could be inhibited by specific pharmacologic mic syndrome (HUS), thrombotic thrombocytopenic blockade of AT1, and ETA receptors. Based on these purpura (TTP), autoimmune disorders, malignant results we study transcription factor Ets-1 dependent hypertension, and vascular transplant rejection. All activation of TF in HMEC-1 cells and expression of of these diseases have a severe and rapid progres- soluble TF-isoforms in blood samples and renal biop- sion in common with very limited therapeutic options sies of patients with acute transplant vasculopathy, because the underlying mechanisms are unknown. SSc with renal crisis, and atypic HUS / TTP. In previous studies we identified activating autoan-

IgG from patients with systemic sclerosis upregulates the proteinexpression of prothrombotic Tissue Factor in the cytosolic compartment (B) of cultured HMEC-1 cells but not in membrane extracts (A).

59 Duska Dragun – NEPHROLOGY AND CARDIOVASCULAR IMMUNOLOGY

3. Systems Biology towards Novel Chronic Kidney Disease - Diagnosis and Treatment: SysKid

Project leader Duska Dragun Coworkers Björn Hegner, Aurélie Philippe, Theres Schaub Cooperations The SysKid consortium members Funding European Commission FP7

Chronic kidney disease affects up to 10% of the popu- the materials for development and clinical evaluation lation, and besides eventual progression towards end of tools for early stage diagnosis as well as prognosis stage renal disease massively impacting patient’s and treatment monitoring. quality of life, comorbidities including cardiovascular The main goal of our SysKid project is not only to complications and bone metabolism disorders are identify biomarkers for fast progression and worse serious consequences. But still, early stage diagnosis disease outcome but also to elucidate novel patho- and tailored treatment is not adequate on the everyday mechanisms involved in renal disease and related car- clinical level. On the one hand chronic kidney disease diovascular conditions on the molecular, cellular, and might not yet have reached its appropriate emplace- organism level. Here we focus on auto-antibodies that ment in an epidemiological and healthcare perspec- activate vascular receptors and thereby exacerbate tive, but also the pathophysiology of the disease on a renal and vascular damage and circulating progeni- molecular and cellular level is not well enough under- tor cells that might contribute to disease progression stood. The sysKID consortium was installed for pre- when malfunctioning in pathologic contexts. Within cisely addressing these issues: Unravel the molecular the SysKid consortium we screen large well defined and cellular mechanisms of chronic kidney disease patient cohorts for association of auto-antibodies with development, combine this information with clinical specific disease and progression characteristics. Our risk factors, and on this basis delineate chronic kid- biologic models allow for evaluation of a huge variety ney disease biomarkers. These markers will allow us of disease relevant processes to test candidate mol- preclinical studies of novel therapy approaches for ecules identified by us and other SysKid groups for halting disease progression, and will provide us with their impact on renal and vascular damage.

60 Duska Dragun – NEPHROLOGY AND CARDIOVASCULAR IMMUNOLOGY

4. Disease specific mechanisms of smooth muscle and fibroblastic differentiation of mesenchymal stem cells (MSC)

Project leader Björn Hegner, Duska Dragun Coworkers Melanie Näther, Theres Schaub Cooperations G. Riemekasten, C. Lange (Hamburg) Funding Deutsche Stiftung Sklerodermie Institutional funding (Universitäre Forschungsförderung Charité)

Bone marrow derived MSC home to injured vessels deposition and tissue calcification. We focus on signal and contribute to neointima formation during instent transduction and transcriptional control of mobilisa- restenosis and transplant arteriosclerosis. Systemic tion, homing, proliferation, apoptosis, smooth muscle sclerosis (SSc) is an autoimmune form of obliterative differentiation, matrix and calcium deposition. Influ- vasculopathy that eventually leads to systemic fibrosis ence on Angiotensin II and Endothelin-1 and putative affecting skin and various organs. We propose that amplification of aberrant cellular processes via ago- MSC from patients with SSc differ from healthy con- nistic autoantibodies against AT1 and ETA receptors trols in terms of aberrant smooth muscle and fibrob- is an additional focus. lastic differentiation leading to vasculopathy, matrix

L-type Ca channel dependent calcium influx into MSC upon stimulation with 60 mM KCl

61 Duska Dragun – NEPHROLOGY AND CARDIOVASCULAR IMMUNOLOGY

5. Sexual dimorphisms in myocardial hypertrophy and cardiorenal interaction – signal transduction and intervention strategies

Project leader Angelika Kusch, Björn Hegner, Duska Dragun Coworkers Dennis Gürgen, Lyubov Chaykovska, Aysun Karatas, Claudia Urban, Christina Baris Cooperations L. de Windt (Utrecht), F. Luft, U. Kintscher, V. Regitz-Zagrosek, O. Huber Funding DFG, DR 498/1-1 – FOR 1054, TP 2 und GK 752-III, TP 3

We have described sex-specific regulation of the cal- DOCA-salt model of mineralocorticoid excess. In addi- cineurin pathway in response to largely blood pres- tion, transverse aortic constriction and voluntary cage sure-independent mineralocorticoid action in DOCA- wheel running are employed as models for pressure salt induced left ventricular myocardial hypertrophy and exercise induced MH, respectively. (MH) and renal hypertrophy in mice. We now study Sex differences in elements upstream and down- the contribution of individual estrogen receptors (ERα stream from mTOR are studied in vitro in female murine and ERβ) in this setting and expand our research on cardiomyocytes (HL-1) using genomic overexpression diverse signalling pathways implicated in the develop- and depletion approaches. Presence or absence of ment of adaptive and maladaptive MH. A major focus estradiol resembles pre- and postmenopausal hormo- is the Akt/mTOR pathway as a putative master switch nal status to determine the influence of estrogen on between adaptive and maladaptive MH. We use a hypertrophic signal transduction focusing on a linear pharmacologic approach of mTOR blockade in our versus non-linear pathway involvement.

Uni-Nx+salt controls compared with DOCA-salt mice that were treated with hydralazine to remove confounding effects of blood pressure elevation A: RT-PCR for calcineurin Aβ in the heart B: RT-PCR for modulating calcineurin and inhibiting protein 1 (MCIP1) C: Immunoprecipitation and subsequent Western blot for phosphorylated nuclear factor of activated T cells c2 (pNFATc2)

62 Duska Dragun – NEPHROLOGY AND CARDIOVASCULAR IMMUNOLOGY

6. Relevance of auto-antibodies directed against protease activated receptors 1 and 2 (PAR-1 and PAR-2) receptors in immune-mediated renal disease and after renal transplantation

Project leader Duska Dragun Coworkers Rusan Catar, Uwe Hoff, Björn Hegner, Barbara Kämper Cooperations G. Riemekasten, H. Heidecke, R. Schindler, M. Noris (Bergamo), G. Remuzzi (Bergamo), C. Licht (Toronto), S. Coughlin (San Diego) Funding Bundesministerium für Wirtschaft und Technologie

Initiation of thrombin dependent pathways mediated Celltrend an assay for detection of PAR-1 and PAR-2 through activation of PAR-1 and PAR-2 is increasingly autoantibodies. We screen our cohorts of transplant recognized as an important trigger of microthrombo- recipients, patients with thrombotic microangipathies sis in crescentic glomerulonephritis and thrombotic in native kidneys, patients with glomerulonephritis microangiopathies. We propose that agonistic anti- associated thrombotic complications, and patients bodies directed against PAR-1 and PAR-2 mediate with renal involvement of systemic autoimmune dis- sustained receptor activation and thereby actively eases PAR1 and 2 auto-antibodies and correlate contribute to various renal pathologies. Based on our autoantibody concentrations with outcome param- experience in the development of assays for screening eters. Isolated patient-IgG is used to study activation of autoantibodies against G-protein coupled recep- of endothelial cells as well as for the characterization tors we have currently developed in collaboration with of target epitopes.

63 Duska Dragun – NEPHROLOGY AND CARDIOVASCULAR IMMUNOLOGY

7. The use of antiinflammatory agents for the improvement of early graft function in marginal renal transplants

Project leader T. Florian Fuller, Duska Dragun Coworkers Lyubov Chaykovska, Angelika Kusch, Rusan Catar, Jennifer Pützer, Uwe Hoff Cooperations J. Troppmair (Innsbruck) Funding Novartis Pharma Institutional funding (Universitäre Forschungsförderung Charité)

Due to severe organ shortage, marginal kidneys (PKC)- inhibitors have an antiinflammatory effect via from older age donors or with severe preservation- reduction of leukocyte-derived superoxide release in reperfusion injury are increasingly used in human ischemically damaged organs. We employ our syn- renal transplantation. These organs typically have a geneic and allogeneic rat kidney transplant models high incidence of delayed graft function (DGF) asso- to determine protective capacity of antiinflammatory ciated with impaired long-term graft survival. Inflam- (AEB-protein-kinase-C inhibitor). Functional and mor- matory reactions accompanying preservation-reper- phological parameters as well as cellular and molecu- fusion injury in kidney transplants cause significant lar markers of inflammation, proliferation and tubular endothelial and tubular damage. Protein-kinase-C fibrosis serve as endpoints.

Sotrastaurin has renoprotective capacity by local modulation of Erk and p66Shc-activities

64 Duska Dragun – NEPHROLOGY AND CARDIOVASCULAR IMMUNOLOGY

8. Cytochrome P-450-(CYP)-derived eicosanoids in acute kidney injury and acute transplant dysfunction – genetic susceptibilities, mechanisms and interventions

Project leader Uwe Hoff, Duska Dragun, Anja Haase-Fielitz, Michael Haase Coworkers Florian Fuller, Lyubov Chaykovska, Björn Hegner, Sebastian Krause, Jennifer Pützer, Barbara Kämper, Julian Marinez, Katja Klemz, Melanie Näther Cooperations W. H. Schunck, F. C. Luft, M. Gollasch, M. Rother, JR Falck (Dallas), D. N. Müller, R. Bellomo, M. Oppert and SEPNET Funding Bundesministerium für Wirtschaft und Technologie, Alexander von Humboldt-Stiftung, Deutsche Herzstiftung, Else-Kröner Stiftung, Dr. Werner Jackstädt Stiftung, Institutional funding (Universitäre Forschungsförderung Charité)

Recent studies indicate that an impaired balance ing a system for the measurement of CYP-dependent between production of nitric oxide (NO) and CYP- eicosanoids in serum and urine. This novel diagnos- dependent eicosanoids plays a crucial role in the tic tool is intended to be employed as a biomarker development of endothelial dysfunction and subse- of endothelial dysfunction in recipients of solid organ quent tubular injury. In particular, 20-HETE (20-hydrox- yeicosatetraenoic acid), which is excessively released and overproduced during ischemia/reperfusion (I/R) may be responsible for sustained vasoconstriction and aggravation of inflammation. Our studies demonstrate that renal injury can be significantly ameliorated by inhibiting the synthesis (HET 0016) and even more favorably by blocking the action of 20-HETE (20-HEDE) during the acute phase of ischemia and reperfusion. The protective action is attributed to anti- apoptic, anti-vasoconstrictive and anti-inflammatory effects due to 20-HETE inhibition. We now extend our studies on our syngeneic and allogeneic rat kidney transplant models employing marginal donor kidneys by means of extended cold preservation times and increased donor age. In cooperation with the Max-Delbrück-Center for Apoptotic tubular cells after 45 minutes of warm ischemia Molecular Medicine (MDC), Lipidomix GmbH and the with and without inhibition of 20-HETE production or 20-HETE Deutsche Herzzentrum Berlin (DHZB) we are develop- blockade

65 Duska Dragun – NEPHROLOGY AND CARDIOVASCULAR IMMUNOLOGY transplants (heart, liver, kidney, simultaneous kidney- vascular tone. Circulating and local catecholamines pancreas) to detect sequelae of calcineurin inhibitors are primarily catabolized through enzymatic pathways (CNI)-nephrotoxicity. We currently screen serum and involving the enzyme catechol-O-methyltransferase urine of organ transplant recipients for differences in (COMT). We found that cardiac surgery patients who concentrations of 20-HETE and EETs and correlate were homozygous for the low activity COMT L allele their levels with structural and functional determinants (LL patients) more commonly developed prolonged of CNI mediated toxicity. vasodilatory shock, AKI, more severe AKI requiring renal replacement therapy, and prolonged hospital Vasodilatory shock and acute kidney injury (AKI) are stay. These patients also had increased concentra- associated with an increased mortality after cardiac tions of plasma catecholamine and MAO dependent surgery and/or during sepsis, the two most common catecholamine degradation products that correspond contributing factors to AKI. We are focused on predic- well in terms of genotype-phenotype correlations. Our tion of the risk for AKI and improvement in individual- findings provide a conceptual frame for further studies ized patient care by the identification of genetic risk in vasodilatory shock and AKI in sepsis. factors and biomarkers involved in the regulation of the

Publications 2006 - 2010

Fuller TF, Hoff U, Rose F, Linde Y, Freise CE, Dragun D, Feng Fuller TF, Rose F, Singleton KD, Linde Y, Hoff U, Freise CE, S (2006): Effect of mycophenolate mofetil on rat kidney grafts Dragun D, Niemann CU (2007): Glutamine donor pretreatment with prolonged cold preservation. Kidney Int 70:570-577 in rat kidney transplants with severe preservation reperfusion injury. J Surg Res 140:77-83 Haase M, Morgera S, Bamberg C, Halle H, Martini S, Dragun D, Neumayer HH, Budde K (2006): Successful pregnancies in Scornik JC, Guerra G, Schold JD, Srinivas TR, Dragun D, dialysis patients including those suffering from cystinosis and Meier-Kriesche HU (2007): Value of posttransplant antibody familial Mediterranean fever. J Nephrol 19:677-681 tests in the evaluation of patients with renal graft dysfunction. Am J Transplant 7:1808-1814 Muller G, Catar RA, Niemann B, Barton M, Knels L, Wendel M, Morawietz H (2006): Upregulation of endothelin receptor Patecki M, von Schaewen M, Tkachuk S, Jerke U, Dietz R, B in human endothelial cells by low-density lipoproteins. Exp Dumler I, Kusch A (2007): Tyk2 mediates effects of urokinase Biol Med (Maywood); 231:766-71 on human vascular smooth muscle cell growth. Biochem Biophys Res Commun 359:679-84 Stielow C, Catar RA, Muller G, Wingler K, Scheurer P, Schmidt HH, Morawietz H (2006): Novel Nox inhibitor Kiyan J, Kusch A*, Tkachuk S, Krämer J, Haller H, Dietz R, of oxLDL-induced reactive oxygen species formation in Smith G, Dumler I (2007): Rosuvastatin regulates vascu- human endothelial cells. Biochem Biophys Res Commun lar smooth muscle cell phenotypic modulation in vascular 26;344:200-5 remodeling: role for the urokinase receptor. Atherosclerosis 195:254-61 (* first co-author)

66 Duska Dragun – NEPHROLOGY AND CARDIOVASCULAR IMMUNOLOGY

Catar RA, Müller G, Heidler J, Schmitz G, Bornstein SR, Chaykovska L, Deger S, Wille A, Friedersdorff F, Kasper A, Morawietz H (2007): Low-density lipoproteins induce the Dragun D, Liefeldt L, Miller K, Giessing M, Fuller TF (2009): renin-angiotensin system and their receptors in human Kidney transplantation into urinary conduits with ureter- endothelial cells. Horm Metab Res 39:801-5 oureterostomy between transplant and native ureter: single- center experience. Urology 73:380-5 Krug AW, Kopprasch S, Ziegler CG, Dippong S, Catar RA, Bornstein SR, Morawietz H, Gekle M (2007): Aldosterone Hegner B, Lange M, Kusch A, Essin K, Sezer O, Schulze- rapidly induces leukocyte adhesion to endothelial cells: a new Lohoff E, Luft FC, Gollasch M, Dragun D (2009): mTOR link between aldosterone and arteriosclerosis? Hyperten regulates vascular smooth muscle cell differentiation from sion 50:e156-7 human bone marrow-derived mesenchymal progenitors. Arterioscler Thromb Vasc Biol 29:232-8 Haase M, Haase-Fielitz A, Ratnaike S, Reade MC, Bagshaw SM, Morgera S, Dragun D, Bellomo R (2008): N-Acetyl- Haase M, Haase-Fielitz A, Bellomo R, Devarajan P, Story D, cysteine does not artifactually lower plasma creatinine con- Matalanis G, Reade MC, Bagshaw SM, Seevanayagam N, centration. Nephrol Dial Transplant 23:1581-1587 Seevanayagam S, Doolan L, Buxton B, Dragun D (2009): Sodium bicarbonate to prevent increases in serum creatinine Karatas A, Hegner B, de Windt LJ, Luft FC, Schubert C, after cardiac surgery: a pilot double-blind, randomized con- Gross V, Akashi YJ, Gürgen D, Kintscher U, da Costa Gon- trolled trial. Crit Care Med 37:39-47 calves AC, Regitz-Zagrosek V, Dragun D (2008): Deoxycorti- costerone acetate-salt mice exhibit blood pressure-independ- Haase-Fielitz A, Bellomo R, Devarajan P, Story D, Matalanis ent sexual dimorphism. Hypertension 51:1177-1183 G, Dragun D, Haase M (2009): Novel and conventional serum biomarkers predicting acute kidney injury in adult cardiac sur- Kintscher U, Hartge M, Hess K, Foryst-Ludwig A, Clemenz gery -a prospective cohort study. Crit Care Med 37:553-60 M, Wabitsch M, Fischer-Posovszky P, Barth TF, Dragun D, Skurk T, Hauner H, Blüher M, Unger T, Wolf AM, Knippschild Haase-Fielitz A, Haase M, Bellomo R, Lambert G, Mata- U, Hombach V, Marx N (2008): T-lymphocyte infiltration in lanis G, Story D, Doolan L, Buxton B, Gutteridge G, Luft FC, visceral adipose tissue: a primary event in adipose tissue Schunck WH, Dragun D (2009): Decreased catecholamine inflammation and the development of obesity-mediated insulin degradation associates with shock and kidney injury after resistance. Arterioscler Thromb Vasc Biol 28:1304-1310 cardiac surgery. J Am Soc Nephrol 20:1393-403

Riad A, Jäger S, Sobirey M, Escher F, Yaulema-Riss A, West- Haase-Fielitz A, Bellomo R, Devarajan P, Bennett M, Story D, ermann D, Karatas A, Heimesaat MM, Bereswill S, Dragun D, Matalanis G, Frei U, Dragun D, Haase M (2009): The predic- Pauschinger M, Schultheiss HP, Tschöpe C (2008): Toll-like tive performance of plasma neutrophil gelatinase-associated receptor-4 modulates survival by induction of left ventricular lipocalin (NGAL) increases with grade of acute kidney injury. remodeling after myocardial infarction in mice. J Immunol Nephrol Dial Transplant 24:3349-54 180:6954-6961 Haase M, Bellomo R, Devarajan P, Ma Q, Bennett MR, Amico P, Hönger G, Bielmann D, Lutz D, Garzoni D, Steiger Möckel M, Matalanis G, Dragun D, Haase-Fielitz A (2009): J, Mihatsch MJ, Dragun D, Schaub S (2008): Incidence Novel biomarkers early predict the severity of acute kidney and prediction of early antibody-mediated rejection due to injury after cardiac surgery in adults. Ann Thorac Surg non-human leukocyte antigen-antibodies. Transplantation 88:124-30 85:1557-1563 Kinkley S, Staege H, Mohrmann G, Rohaly G, Schaub T, Krämer J, Ruf RG, Schmidt S, Axthelm C, Strasser R, Jans- Kremmer E, Winterpacht A, Will H (2009): SPOC1: a novel sen G, Thieme T, Kusch A, Waigand J, Dietz R, Gross CM PHD-containing protein modulating chromatin structure and (2008): The CAIRP (CAndesartan for In-stent Restenosis mitotic chromosome condensation. J Cell Sci 122:2946-56 Prevention) Trial--a multicenter study of AT1-receptor blocker therapy in coronary stenting. J Invasive Cardiol 20:205-10

67 Duska Dragun – NEPHROLOGY AND CARDIOVASCULAR IMMUNOLOGY

Jerke U, Tkachuk S, Kiyan J, Stepanova V, Kusch A, Hinz M, Reinsmoen NL, Lai CH, Heidecke H, Haas M, Cao K, Ong G, Dietz R, Haller H, Fuhrman B, Dumler I. (2009): Stat1 nuclear Naim M, Wang Q, Mirocha J, Kahwaji J, Vo AA, Jordan SC, translocation by nucleolin upon monocyte differentiation. Dragun D (2010): Anti-Angiotensin Type 1 Receptor Antibod- PLoS One 14;4:e8302 ies Associated with Antibody Mediated Rejection in Donor HLA Antibody Negative Patients. Transplantation in press Kusch A, Tkachuk S, Tkachuk N, Patecki M, Park JK, Dietz R, Haller H, Dumler I (2009): The tight junction protein ZO-2 Riemekasten G, Philippe A, Näther M, Slowinski T, Müller DN, mediates proliferation of vascular smooth muscle cells via Heidecke H, Matucci-Cerinic M, Czirják L, Lukitsch I, Becker regulation of Stat1. Cardiovasc Res 1;83:115-22 M, Kill A, van Laar JM, Catar R, Luft FC, R. Burmester GR, Hegner B, Dragun D (2010): Involvement of functional autoan- Haase-Fielitz A, Bellomo R, Devarajan P, Dragun D, Haase M tibodies against vascular receptors in systemic sclerosis. Ann (2010): The predictive performance of neutrophil gelatinase- Rheum Dis in press associated lipocalin (NGAL) increases with severity of acute kidney injury. Am J Kidney Dis 24:3349-54 Reviews & book chapters (2006-20010) Ksi K, Mikusa-Pietrasik J, Catar R, Dworacki G, Winckie- wicz M, Frydrychowicz M, Dragun D, Ryszard S, Jörres A, Fuller TF, Liefeldt L, Dragun D, Tüllmann M, Loening SA, Witowski J (2010): Oxidative stress-dependent increase in Giessing M (2006): Urological evaluation and follow-up of the ICAM-1 expression promotes adhesion of colorectal and kidney transplant patient. Urologe 45:53-59 pancreatic cancers to the senescent peritoneal mesothelium. International Journal of Cancer 127:293-303 Dragun D (2006): Complementary matching. A definite maybe. Nephrol Dial Transplant 21:3371-3373. Editorial Zebger-Gong H, Kampmann J, Kong L, Roigas J, Sommer K, Hoff U, Krämer S, Peters H, Müller D, Dragun D, Querfeld U Dragun D, Hegner B (2006): "FTY720 - Mechanism of (2010): Decreased transplant arteriosclerosis in endothelial nitric Action of a Novel Immune Modulator: Potential Applica oxide synthase-deficient mice. Transplantation 89:518-26 tion for Autoimmune Diseases", 1. Auflage, Herausgeber: T. Böhler,UNI-MED Verlag AG Bremen, Kapitel: FTY protec- Duerr M, Glander P, Diekmann F, Dragun D, Neumayer HH, tion against ischemia-reperfusion damage Budde K (2010): Increased incidence of angioedema with ACE inhibitors in combination with mTOR inhibitors in kidney Dragun D (2007): The role of angiotensin II type 1 receptor- transplant recipients. Clin J Am Soc Nephrol 5:703-8 activating antibodies in renal allograft vascular rejection. Pediatr Nephrol 22:911-914 Fuller TF, Hoff U, Kong L, Naether M, Wagner P, Nieminen- Kelhä M, Nolting J, Luft FC, Hegner B, Dragun D (2010): Haase-Fielitz A, Haase M, Bellomo R, Dragun D (2007): Genetic Cytoprotective actions of FTY720 modulate severe preserva- polymorphisms in sepsis- and cardiopulmonary bypass-associ- tion reperfusion injury in rat renal transplants. Transplanta ated acute kidney injury. Contrib Nephrol 156:56-91 tion 89:402-8 Dragun D (2007): Agonistic antibody-triggered stimulation Hoff U, Lukitsch I, Chaykovska L, Ladwig M, Arnold C, Mant- of Angiotensin II type 1 receptor and renal allograft vascular hati VL, Fuller TF, Schneider W, Gollasch M, Muller DN, Flem- pathology. Nephrol Dial Transplant 22:1819-22 ming B, Seeliger E, Luft FC, Falck JR, Dragun D, Schunck WH (2010): Inhibition of 20-HETE synthesis and action protects Riemekasten G, Dragun D (2007): Clinical risk-adapted from renal ischemia/reperfusion injury. Kidney Int in press Therapies in systemic sclerosis. Z Rheumatol 66:672-674, 676-678

68 Duska Dragun – NEPHROLOGY AND CARDIOVASCULAR IMMUNOLOGY

Dragun D, Rudolph B (2007): Novelties in diagnostics and Dragun D, Haase-Fielitz A (2009): Low catechol-O-methyl- therapy of antibody mediated rejection. Nephrol Dial Trans transferase and 2-methoxyestradiol in preeclampsia: more plant 22Suppl 8:viii50-viii53 than a unifying hypothesis. Nephrol Dial Transplant 24:31-3

Dragun D (2008): Humoral responses directed against non- Schewior L, Dragun D, Schaeffner E (2009): The challenge HLA antigens in solid-organ transplantation. Transplantation of Wegener’s granulomatosis after kidney transplantation. 86:1019-25 Transpl Int 22:503-5

Dragun D, Scornik J, Meier-Kriesche HU (2008): Kidney- Dragun D, Distler JH, Riemekasten G, Distler O 2009): Stimu- transplant rejection and anti-MICA antibodies. N Engl J Med latory autoantibodies to platelet-derived growth factor recep- 358:196. Letter tors in systemic sclerosis: what functional autoimmunity could learn from receptor biology. Arthritis Rheum 60:907-11 Dragun D (2008): Cellular responses involved in pathogenesis of chronic allograft nephropathy. International Transplanta Dragun D, Philippe A, Catar R, Hegner B (2009): Autoimmune tion Updates, Herausgeber: J. Grinyo, Permanyer Publica- mediated G-protein receptor activation in cardiovascular and tions Barcelona, Kapitel: Late renal allograft dysfunction renal pathologies. Thromb Haemost101:643-8

Dragun D, Hegner B (2008): "Neue Medikamente in der Dragun D, Fielitz-Haase A (2009): Low catechol-O-meth- Transplantationsmedizin", 2. Auflage,Herausgeber: H.-H. ytransferase and 2-methoxyestradiol in preeclampsia: More Neumayer, K. Budde, J. Waiser, UNI-MED Verlag AG Bremen, than a unifying hypothesis. Nephrol Dial Transplant 24:31- 2008 Kapitel: Antikörper vermittelte Allograftabstoßung - 3. Editorial Humorale Rejektion Dragun D (2009): "Management of acute kidney prob Kusch A. (2008): Linking proteolysis to lipids. Thromb Res lems", Herausgeber: A. Jörres, Springer Verlag Berlin-Hei- 123:191-3 delberg, Kapitel: Acute kidney failure during pregnancy and postpartum. Dragun D, Hegner B (2009): Non-HLA antibodies post- transplantation: clinical relevance and treatment in solid organ Dragun D, Philippe A (2010): From mother to child--trans- transplantation. Contrib Nephrol 162:129-39 placental effect of AT1R-AA in preeclampsia. Nephrol Dial Transplant 25:1774-6.

69 Duska Dragun – NEPHROLOGY AND CARDIOVASCULAR IMMUNOLOGY

General information Third party funding (2006-2010)

Project leader Project title Sponsor Period Näther M. Endothelial mecha-nisms in thrombotic Dr. Werner Jackstädt Foundation 2010–2011 Dragun D. microangiopathy induced acute renal failure Dragun D. Autoimmune mediated endothelin Actelion Pharmaceuticals Deut- 2009–2010 Catar R. receptor activation in renal crisis schland GmbH Philippe A. Hegner B. Dragun D. SysKID (Systems biology towards novel EU: Large-scale integrating project 2010–2012 Hegner B. chronic kidney disease diagnosis and (EU)-FP7 Philippe A. treatment) Dragun D. Functional characterization of CXCR3 BMWi (Federal Ministry of Econ- 2009–2011 Catar R. and CXCR4 auto-antibodies omy and Technology) Näther M. Chaykovska L. Dragun D. Sex differences in adaptive and mala- DFG (German Research Foun- 2008–2011 Hegner B. daptive myocardial hypertrophy – role dation) DR 498/1-1 – FG1054 Kusch A. of the Akt/mTOR pathway (Research group 1054) Haase M. Multi center trial of sodium bicarbonate Else Kröner Fresenius Foundation 2008–2010 Haase-Fielitz A. to prevent acute renal failure in patients Dragun D. undergoing cardiopulmonary surgery Haase M. Interventions to prevent acute renal German Heart Foundation 2008–2010 Haase-Fielitz A. failure in patients undergoing cardiopul- Dragun D. monary surgery Dragun D. Effect of the protein-kinase-C-inhibitor Novartis Pharma GmbH 2008–2009 Fuller F. AEB on rat kidney transplants with Chaykovska L. severe preservation-reperfusion injury Kusch A. Hoff U. Hegner B. Mesenchymal stem cells in systemic Deutsche Stiftung Sklerodermie 2008–2009 Dragun D. sclerosis German Scleroderma Foundation Dragun D. CYP-Eicosanoid status after organ BMWi (Federal Ministry of Econ- 2008–2009 Kämper B. transplantation omy and Technology) Hegner B. Hoff U.

70 Duska Dragun – NEPHROLOGY AND CARDIOVASCULAR IMMUNOLOGY

Dragun D. Relevance of autoantibodies against BMWi (Federal Ministry of Econ- 2007–2009 Catar R. PAR-1 and PAR-2 receptors in renal omy and Technology) Hoff U. transplant patients and kidney diseases Näther M. Dragun D. Sex and gender specific mechanisms DFG (German Research Founda- 2006–2010 Hegner B. of myocardial hypertrophy tion) –Graduiertenkolleg 754-3 Gürgen D. (Graduate course 754-3) Dragun D. Kidney involvement in systemic scle- German Scleroderma Foundation 2006–2008 Hegner B. rosis Dragun D. Agonistic Autoantibodies EFRE (European Foundation for 2006–2007 Philippe A. Regional Development) Hegner B. Catar R. Hoff U. Gender aspects in acute kidney injury Dr. Werner Jackstädt Foundation 2006–2007 Dragun D.

Awards

2010 – 2011 Dr. Werner Jackstädt Fellowship Melanie Näther 2009 – 2010 Dr. Werner Jackstädt Fellowship Anja Fielitz-Haase 2009 Bernd-Schönberger-Preis –Stiftung Urologische Forschung Lyubov Chaykovska 2009 Poster award International Society of Gender in Medicine Angelika Kusch 2009 Nils-Alwall-Preis – DAGKN Duska Dragun, Michael Haase 2009 Poster award – DGRh Björn Hegner 2008 Young investigator award – ERA-EDTA Michael Haase 2006 – 2008 Rachel Hirsch Fellowship Angelika Kusch 2006 – 2007 Alexander-von-Humboldt Fellowship Michael Haase 2007 "New key opinion leader" - WTC Duska Dragun 2006 – 2007 Dr. Werner Jackstädt Fellowship Uwe Hoff 2006 Theodor-Frerichs-Preis – DGIM Duska Dragun 2006 Charité Allianz Research Award - European Students Conference Lyubov Chaykovska 2006 Poster of distinction – World transplant congress Björn Hegner 2005 Young Investigator Award – ATC Duska Dragun 2005 Poster of distinction – ATC Uwe Hoff 2005 Apherese Innovationspreis – DAfKN Duska Dragun 2005 Biotest Award Clinical Science – ESOT Björn Hegner

Berthold Hocher – Pharmacology/Nephrology

Head of the Group

Prof. Dr. med. Berthold Hocher

Curriculum Vitae: Prof. Dr. med. Berthold Hocher studied veterinary medicine and medicine at the Free University of Berlin and at the University of Heidelberg.He started his research career at the Department of Molecular Biology and Biochemistry of the Free University of Berlin where he also accomplished his doctoral thesis. In the following years he worked clinically in the field of Internal Medicine with the focus on Nephrology. After clinical posts at the Benjamin Franklin University Hospital of the Free University of Berlin and at the Charité, he became a Consultant Nephrologist and Associated Professor of Internal Medicine at the University Hospital Bern/Switzerland. He is currently Translational Medicine Leader at Roche in Basel/Switzerland, holds a full professorship at the University in Potsdam and is guest scientist at the CCR.His research group in Berlin is associated to the Department of Pharmacology/CCR of the Charité. The main topics of his research group are the nitric oxide/endothelin system in renal and cardiovascular diseases and fetal programming of renal and cardiovascular diseases.

Members of the group

Scientists Alter, Markus Dr. med. Pfab, Thiemo Priv-Doz. Dr. med. Chen, Youpeng Dr. med. Rahnenführer, Jan Biotechnologist Godes, Michael Dr. med. Sharkovska, Yuliya Dr. med. Kalk, Philipp Dr. med. Simon, Alexandra Nutritionist Krause-Relle, Katharina Dr. rer. medic. Websky, Karoline von Veterinarian Ott, Ina Nutritionist

73 Berthold Hocher – Pharmacology/Nephrology

Students Andermann, Tim Medical student Mengeringhausen, Eva Medical student Blancke Jan-Arne Medical student Mersmann, Astrid Medical student Dowuona-Hammond, Ekua Medical student Pursche, Lars Medical student Einem, Gina Franziska von Medical student Reichetzeder, Christoph Physician Gorodetski, Jenny Medical student Runge, Silke Medical student Haumann, Hannah Medical student Stange, Dörte Master student Heunisch, Fabian Medical student Sceplik, Ewelina Medical student Hohmeier, Sophie Medical student Schlemm, Ludwig Medical student Hübner, Sarah Medical student Schwietzer, Marcel Medical student Kempiners, Nina Medical student Sievers, Gerlind Medical student Köder, Ina Medical student Tsuprykov, Oleg PhD student, Physician Krause, Christian Medical student Volochko, Evgeny Medical student Kyeyamwa, Sarah Gynaecologist Weist, Andreas Physician Leonhardt, Claudia Medical student Ziegner, Maja Medical student

74 Berthold Hocher – Pharmacology/Nephrology

Summary ular dialysis – with upward trend. Another important risk Our research is focused on four major topics: factor is hypertension. Thus, we test new drug classes in experiments with rats and mice. Mainly, we use the 1. The impact of the endothelin (ET) system on hyperglycaemia model induced by streptozotocin and the pathogenesis of cardiac and renal disease hypertension models induced either by renal arterial We established and characterized ET-1 transgenic stenosis by surgery or by genetical knock out of the mice and analyzed the impact of the ET system in endothelial NO synthase (eNOS knock out). Of course, cardiovascular disease models. Current projects we combine the models as well to simulate the patho- are focusing on the interaction of the ET – and nitric genesis of diabetic nephropathy as close as possible. oxide system. We thus generated ET-1 transgenic So we can analyse all common renal and cardiovascu- mice lacking the endothelial NOS in order to further lar biomarkers in plasma and urine and round research characterize the complex physiology of the ET-NO- off by histological and immunohistological evaluation System in vivo. Other projects are focussing on the of kidneys, hearts and further vital organs. Results are impact of the ETB receptor on tubular anion trans- compared with already established treatment regimes porters as well as the impact of the ETB receptor for such as AT2 receptor blockers or ACE inhibitors. the pathogenesis of diabetic nephropathy. 4. Detection of biomarkers – German 2. Fetal programming of cardiovascular disease Radiocontrast Media Study There is meanwhile clear evidence indicating that early The increasing incidence of CIN is one of the major life events play an important role in the pathogene- challenges which nephrologists are facing worldwide. sis of cardiovascular diseases in later life. We have Mortality is over one third of the in-hospital acute renal introduced the concept of maternal genes exerting failure after contrast media (CM) administration and unfavorable effects on the offspring into this currently affects especially patients with diabetes mellitus and rapidly growing research field. We were furthermore impaired renal function. Therefore the prevention of able to demonstrate for the first time that insulin resist- CM nephropathy is highly relevant to clinical out- ance may also be present in otherwise healthy human comes. In collaboration with the Clinic for Cardiol- newborns already at birth. Our animal studies dem- ogy at the Charité Campus Mitte we investigate the onstrated that also over-nutrition may exert harmful course and outcome of a huge number of patients effects on the offspring in a gender dependent manner. who undergo coronary angiography and receive CM Current clinical and preclinical studies are designed application. In addition, we collect blood and urine to detect molecular pathways of fetal programming samples during the course. This material is to be ana- including epigenetic DNA modifications. lysed by surface-enhanced laser desorption ionization 3. Diabetes mellitus and diabetic nephropathy time-of-flight mass spectrometry (SELDI-TOF MS) and As the prevalence of diabetes mellitus and its complica- results are to be compared with clinical data. The goal tions, such as diabetic nephropathy, rises in the western with this open, non-hypothesis driven approach is to industrial nations, the severe consequences become detect new biomarkers in blood and urine which are more and more present: Diabetic nephropathy is able to predict the incidence, course and outcome of accountable for one third of all patients who require reg- contrast media induced nephropathy.

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Zusammenfassung nie genannt werden. Wir erproben neue Medikamen- Unsere Arbeitsgruppe beschäftigt sich tenklassen im Ratten- und Mäuse-Tierversuch. Dabei schwerpunktmäßig mit vier Hauptthemen: arbeiten wir hauptsächlich, einzeln oder in Kombina- 1. Der Einfluss des Endothelinsystems auf die tion, mit dem Streptozotocin-induzierten Hypergly- Pathogenese kardialer und renaler Erkrankungen kämiemodell und Hypertoniemodellen, die durch Wir untersuchen den Einfluss des Endothelinsystems operatives Setzen einer Nierenarterienstenose (2K1C) auf kardiovaskuläre Erkrankungen anhand der von uns oder genetischem Knockout der endothelialen NO- etablierten ET-1 transgenen Mauslinie. Um die Interak- Sythase (eNOS) induziert werden. Neben Untersu- tion zwischen Endothelin- und NO-System zu erfor- chungen der gängigen renalen und kardiovaskulären schen, wurde die ET-1 transgene Linie mit einer eNOS Biomarker erfolgt die histologische und immunhisto- (endotheliale NO-Synthase) Knockout Linie gekreuzt. logische Aufarbeitung von Herz, Nieren und weiteren Ein weiterer Schwerpunkt liegt auf der Untersuchung lebenswichtigen Organen. Die Ergebnisse werden mit der tubulären ETB-Rezeptoren in der Niere und ihrer den aktuell gängigen Therapiemöglichkeiten (zumeist Wirkung auf Anionen-Transporter bzw. ihre Rolle in der Sartane und ACE-Hemmer) verglichen. Pathogenese der diabetischen Nephropathie. 4. Ermittlung von Biomarkern – Deutsche 2. Fetale Programmierung kardiovaskulärer Radiokontrastmittel-Studie Erkrankungen Die Inzidenz des kontrastmittelinduzierten Nierenversa- Es gibt klare Hinweise, die belegen, dass bestimmte gens steigt stetig und ist mittlerweile die dritthäufigste Einflüsse im frühen Leben prädispositionierend sind für Ursache des akuten Nierenversagens. Das kontrast- kardiovaskuläre Erkrankungen im weiteren Lebensver- mittelinduzierte Nierenversagen ist insbesondere bei lauf. Wir konnten als Erste zeigen, dass Insulinresistenz Patienten mit Diabetes mellitus und vorbestehender schon bei der Geburt ansonsten gesunder Neugebo- Einschränkung der Nierenfunktion mit einer hohen rener nachweisbar sein kann. Anhand unserer Tierver- Mortalität verbunden. Vor diesem Hintergrund hat die suche konnten wir darlegen, dass eine Überernährung Prävention eines kontrastmittelinduzierten Nierenversa- der Mutter geschlechtsabhängige Auswirkungen auf die gens einen hohen Stellenwert für die Prognose der Pati- Nachkommen hat. Ein Teil unserer laufenden Projekte enten. Wir führen in Zusammenarbeit mit der Klinik für beschäftigt sich damit, die molekularen Signalwege der Kardiologie am Campus Mitte der Charité einen Studie fetale Programmierung aufzudecken. durch, in der wir den klinischen Verlauf von einer hohen 3. Diabetes mellitus und diabetische Nephropathie Zahl von Patienten, die sich zur Koronarangiographie Der Diabetes mellitus und damit auch seine Spät- vorstellen und dabei Radiokontrastmittel appliziert komplikationen wie die diabetische Nephropathie zei- bekommen, verfolgen und dabei Blut- und Urinproben gen eine zunehmende Prävalenz in den westlichen sammeln. Dieses Material soll einer Proteomanalyse Industrienationen mit ernstzunehmenden Folgen: mittels SELDI-TOF-Massenspektrometrie zugeführt Inzwischen ist bei gut einem Drittel aller Dialysepati- und mit den klinischen Daten verglichen werden. Ziel enten der Diabetes mellitus ursächlich für die termi- ist es, neue Biomarker zu ermitteln, die imstande sind, nale Niereninsuffizienz – mit steigender Tendenz. Als das Auftreten und die Prognose eines kontrastmittelin- weiterer entscheidender Risikofaktor soll die Hyperto- duzierten Nierenversagens vorherzusagen.

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Research Projects

1. Nitric oxide-independent stimulation of soluble guanylate cyclase reduces organ damage in experimental low-renin and high-renin models

Project leader Berthold Hocher Coworkers Yuliya Sharkovska, Philipp Kalk, Wellkisch K, Michael Godes, Katharina Krause-Relle Collaborations Prof. Dr. Johannes-Peter Stasch, Dr. Bettina Lawrenz, Linda Sarah Hoffmann, Sandra Geschka, Bayer Schering Pharma AG, Cardiovascular Research, Martin-Luther-University, Halle, Germany

The nitric oxide-soluble guanylate cyclase (sGC)-cGMP as indicated by lower plasma ANP, lower relative left signal transduction pathway is impaired in different car- ventricular weight, lower myocyte diameter and lower diovascular diseases, including pulmonary hypertension, arterial media/lumen ratio, and reduced renal target heart failure and arterial hypertension. Riociguat is a novel organ damage as indicated by improved creatinine stimulator of soluble guanylate cyclase (sGC). However, clearance (Fig. 2) and less renal interstitial fibrosis. little is known about the effects of sGC stimulators in Our study demonstrate for the first time that the novel experimental models of hypertension. We thus investi- sGC stimulator riociguat shows in two independent gated the cardio-renal protective effects of riociguat in models of hypertension a potent protection against low-renin and high-renin rat models of hypertension. cardiac and renal target organ damage. The vasorelaxant effect of riociguat was tested in vitro on isolated saphenous artery rings of normal and nitrate toler- ant rabbits. The cardiovascular in-vivo effects of sGC stimulation were evaluated in hypertensive renin-transgenic rats treated with the nitric oxide-synthase inhibitor N-nitro- L-arginine methyl ester (L-NAME) (high-renin model) and in rats with 5/6 nephrectomy (low-renin model). Glomerulosclerosis in renin-transgenic rats under L-NAME In both animal models, riociguat treatment improved administration (controls) with riociguat treatment at 3 mg/kg survival and normalized blood pressure. Moreover, (riociguat low dose) or 10 mg/kg (riociguat high dose). Values in the L-NAME study part, riociguat reduced cardiac are given as means ± SEM; *: p< 0.01 vs. control. target organ damage as indicated by lower plasma ANP, lower relative left ventricular weight and lower cardiac interstitial fibrosis, and reduced renal target Creatinine clearance during the 5/6 nephrectomy (NX) organ damage as indicated by lower plasma creati- study was calculated from nine and urea, less glomerulosclerosis (Fig. 1) and less blood and urine creatinine. Values are given as means ± renal interstitial fibrosis. In the 5/6 nephrectomy study SEM; ** : p< 0.001 vs. Sham- part, riociguat reduced cardiac target organ damage OP, † : p< 0.05 vs. 5/6 NX.

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2. New evidence for the fetal insulin hypothesis: fetal angiotensinogen M235T polymophism is aasociated with birth weight and elevated fetal total glycated hemoglobin at birth

Project leader Berthold Hocher Coworkers Ludwig Schlemm, Hannah M. Haumann, Maja Ziegner, Bulza Stirnberg, Andreas Sohn, Markus Alter, Thiemo Pfab Collaboration PD Dr. med. Florian Guthmann, Clinic for Neonatology, Charité, Berlin, Germany PD Dr. med. Karim Kalache, Department of Obstetrics and Gynecology, Charité, Berlin

Low birth weight is associated with an increased risk polymorphism. The AGT M235T TT polymorphism of cardiovascular events in later life. Insulin resistance is associated with reduced birth weight (TT: 3288 g is a key finding in adult patients with cardiovascular versus TM+MM: 3435 g, P<0.05). Furthermore, new- diseases. The neonatal phenotype of an individual with borns with a high percentage of fTGH (>6.5%) are more insulin resistance might be low birth weight, as insulin likely to have the TT genotype than those with normal influences fetal growth. The renin–angiotensin–aldos- fTGH (<=6.5%, P<0.05). With higher cutoffs for fTGH, terone system has been associated with cardiovascular the significance increases to P less than 0.005. No disease and insulin resistance. We analyzed whether association was seen between these parameters and fetal polymorphisms of the angiotensinogen (AGT) the fetal angiotensin-converting enzyme insertion/dele- and angiotensin-converting enzyme genes influence tion phenotype. The fetal AGT M235T polymorphism birth weight and/or fetal total glycated hemoglobin is associated with low birth weight and elevated fetal (fTGH), a surrogate parameter of fetal insulin resistance fTGH at birth. Previous findings show that elevated fetal at birth. In 1132 white women delivering singletons, fTGH correlates with low birth weight and that higher neonatal umbilical blood samples and clinical data of activity of the renin–angiotensin–aldosterone system is the mothers and newborns were obtained. Newborns an independent risk factor for the development of dia- were genotyped with respect to the AGT M235T and betes mellitus and coronary artery disease. Therefore, angiotensin-converting enzyme insertion/deletion our data are supportive of the fetal insulin hypothesis.

Newborns carrying the TT genotype are more likely to be SGA and less likely to be LGA. TT-homozygosity is most often found in babies SGA, and least likely in those who are LGA (Padj =0.01). AGA, appropriate for gestational age; AGT, angiotensinogen; LGA, large for gestational age; SGA, small for gestational age.

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3. Fetal sex determines the impact of maternal progins and PPARgamma2 Pro12Ala polymorphism on maternal physiology during pregnancy

Project leader Berthold Hocher, Thiemo Pfab Coworkers Ludwig Schlemm, Hannah Haumann, C. Poralla, Aline Burdack, Michael Godes Collaboration Dr. Youpeng Chen, Univeristy of Goughzou, China, Prof. Dr. med. Horst Halle, Dr. med. Christian Bamberg, Prof. PD Dr. med. Karim Kalache, Department of Obstetrics and Gynecology, Charité, Berlin; PD Dr. med. Florian Guthmann, Clinic for Neonatology, Charité, Berlin, Germany

It was suggested that fetal sex may substantially sus mothers carrying the same alleles but delivering affect maternal glycemic control during pregnancy boys (5.8+/-0.6%; p<0.05; see Figure). Comparing in genetically susceptible mothers. We tested the mothers with the GG genotype delivering girls with hypothesis that fetal sex as a major genetic variant mothers with CC or CG genotypes also delivering of the fetal genome may affect maternal physiology girls (6.3+/-0.7%) revealed a significantly higher during pregnancy in genetically susceptible preg- maternal total glycated hemoglobin at delivery in nant women. the former group (p<0.01, see Figure). The peroxisome proliferator-activated receptor We also analyzed the impact of fetal sex on mater- gamma2 (PPARgamma2) Pro12Ala polymorphism nal physiology during pregnancy in relationship with is known to affect glycemic control and may act in the maternal PROGINS progesterone receptor gene a sex-specific manner. We analyzed the impact of polymorphism. The maternal PROGINS progester- fetal sex on maternal glycemic control during preg- one receptor polymorphism on its own had no effect nancy in relation to the maternal PPARgamma2 on blood pressure, new onset of proteinuria, and Pro12Ala polymorphism. Over 2000 Caucasian total glycated hemoglobin at delivery. However, by women without preexisting diabetes and preexisting considering the offspring’s sex, the AA variant of hypertension with singleton pregnancies delivering the PROGINS progesterone receptor polymorphism consecutively at the Charité obstetrics department was associated with profound cardiovascular/meta- were genotyped. Glycemic control was analyzed by bolic effects; mothers carrying both A alleles (AA measuring total glycated hemoglobin at birth. The genotype) delivering a boy had significantly lower maternal PPARgamma2 Pro12Ala polymorphism systolic blood pressure during the first trimester without consideration of fetal sex had no effect on of pregnancy versus AA mothers delivering girls blood pressure, new onset of proteinuria and total (107.9+/-10.2 vs. 116.6+/-15.1 mmHg; p<0.05). glycated hemoglobin at delivery. Mothers carrying Diastolic blood pressure was similarly lower during both G alleles (GG genotype) delivering a girl had a the first trimester of pregnant AA women delivering higher total glycated hemoglobin (6.8+/-0.5%) ver- boys in comparison with AA women delivering girls

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(63.4+/-5.7 vs. 68.2+/-10.9 mmHg; p<0.05). Total glycated hemoglobin at delivery was significantly higher in AA mothers delivering boys (6.6+/-0.7%) versus AA mothers delivering girls (5.9+/-0.6%; p<0.01). Our studies indicate that fetal sex may substantially affect maternal blood pressure as well as glycemic control during pregnancy in genetically susceptible mothers. Low birth weight, a risk factor for cardiovascular diseases in later life, is already associated with elevated fetal glycosylated hemoglobin at birth.

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4. Impact of maternal body mass index on neonatal outcome

Project leader Berthold Hocher, Philipp Kalk Coworkers Katharina Relle, Kathrin Krause, Michael Godes Collaboration Gabriele Gossing, Horst Halle, Department of Gynaecology and Obstetrics, Charité, Campus Mitte, Florian Guthmann, Roland Wauer, Department of Neonatology, Charité, Campus Mitte

Maternal body mass index (BMI) has an impact on macrosomia and increased admission of the newborn maternal and fetal pregnancy outcome. An increased to a neonatal care unit, whereas decreased BMI was maternal BMI is known to be associated with more associated with preterm birth and lower birthweight frequent admission of the newborn to a neonatal care in the complete study population. In the subgroup of unit. The reasons and impact of this admission on fetal children admitted to a neonatal ward children from outcome, however, remain to be elucidated. obese mothers were characterized by hypoglycaemia. Thus, the aim of our study was to investigate the They need less oxygen, and exhibit a shorter stay on impact of maternal BMI on maternal and fetal preg- the neonatal ward compared to children from normal nancy outcome with special focus on the subgroup of weight mothers, whereas children from underweight children admitted to a specialized neonatal care unit. mothers are characterized by lower umbilical blood A cohort of 2049 non-diabetic mothers giving birth in pH and increased incidence of death corresponding the Charite university hospital was prospectively stud- to increased prevalence of preterm birth. ied. Mothers and children were grouped according to Taken our results together we conclude that preg- maternal BMI. The impact of maternal BMI on mater- nancy outcome is worst in babies from mothers with nal and fetal outcome parameters was tested using low body mass index as compared to healthy weight multivariate regression analysis. Outcome of children mothers with respect to increased incidence of pre- admitted to a specialized neonatal ward (n = 505) was term birth, lower birth weight and increased neonate analysed using chi² and t-test while the children were mortality on the neonatal ward. We demonstrate that grouped according to maternal BMI. the increased risk for neonatal admission in children We found that increased maternal BMI was associ- from obese mothers does not necessarily indicate ated with an increased risk for hypertensive compli- severe fetal impairment as in our population it is mostly cations, peripheral edema, caesarean section, fetal due to surveillance because of hypoglycaemia.

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5. Cell-type specific interaction of endothelin and the nitric oxide system: pattern of prepro-ET-1 expression in kidneys of L-NAME treated prepro-ET-1 promoter-lacZ-transgenic mice.

Project leaders Berthold Hocher, Philipp Kalk, Franz Theuring Coworkers Maren Christian. Katharina Relle, Michael Godes Collaboration Heiko Funke-Kaiser, AG Unger, CCR Torsten Slowinski, Hans-H. Neumayer, Department of Nephrology, Charité, Campus Mitte; Christian Bauer, Department of Biochemistry, Free University, Berlin Fred Schmager, SCHERING AG, now BAYER

Nitric oxide (NO) and endothelin-1 (ET-1) are known to In order to investigate the interaction of ET-1 and NO play a major role in renal and vascular pathophysiology in the kidney in vivo, transgenic mice at the age of 3-4 and exhibit a close interaction with ET-1 stimulating NO months were treated with a single dose of the NO production and NO in turn inhibiting ET-1 expression. synthase inhibitor L-NAME (25 mg (kg bw)(-1) i.p.) 12 h Our objectives were (1) to establish a novel transgenic before kidney removal. Bluo-Gal staining of kidney sec- mouse model facilitating ET-1 expression assessment tions revealed intracellular blue particles as indicators in vivo, (2) to validate this model by assessing prepro- of prepro-ET-1 promoter activity in tubular and vascu- ET-1 promoter activity in mice embryos by means of lar endothelium and glomerular cells. Particle count our novel model and comparing expression sites to was closely correlated to kidney tissue ET-1 content well-established data on ET-1 in fetal development and (R=0.918, P<0.001). Comparison of counts revealed (3) to investigate renal ET-NO interaction by assessing an increase by 135+/-53% in L-NAME treated (n=12) prepro-ET-1 promoter activity in different structures of compared to non-treated mice (n=10, P=0.001). Cell- the renal cortex in the setting of blocked NO synthases type specific evaluation revealed an increase of 136+/- via L-NAME administration. 51% in tubular (P=0.001) and 105+/-41% in glomerular We established transgenic mice carrying a lacZ cells (P=0.046), but no significant increase in vascular reporter gene under control of the human prepro-ET-1 endothelium. gene promoter sequence (8 kb of 5’ sequences). Bluo- We thus conclude that we indeed a close interaction Gal staining of tissue sections revealed intracellular of renal endothelin and renal NO system is present, blue particles as indicators of prepro-ET-1 promoter but the interaction is modulated in a cell-type specific activity. In mouse embryos, we detected high prepro- manner. Furthermore, our new transgenic model pro- ET-1 promoter activity in the craniofacial region, as well vides a unique opportunity to analyse regulation of the as in bone and cartilage consistent with the literature. ET system on a cellular level in vivo.

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6. Low birth weight – a risk for cardiovascular diseases in later life – is associated with elevated fetal glycated hemoglobin already at birth

Project leader Berthold Hocher, Thiemo Pfab Coworkers Torsten Slowinski, Michael Godes Collaboration Friedrich Priem, Institute of Laboratory Medicine, Charité, Berlin Horst Halle, Department of Obstetrics and Gynecology, Charité, Berlin

Several epidemiological studies have confirmed the increase in TGH by 1% in the child was significantly intriguing finding that low birth weight is an independ- associated with a mean birth weight reduction of 135g ent risk factor for glucose intolerance and type 2 (p<0.0001), whereas the same increase in the mother diabetes mellitus, as well as cardiovascular disease. was associated with a mean birth weight increase of Insulin resistance probably plays an important role; 88g (p<0.0001). The ratio of fetal/maternal TGH sug- because it is one of the major underlying causes of glu- gests that lighter newborns have a higher percentage cose intolerance, and it is multifaceted consequences of TGH than would be expected from maternal TGH. include dyslipidemia, hypertension, hypercoagulation, The study demonstrates for the first time in a large and impaired fibrinolysis. population that there is an inverse association between We quantified total glycosylated hemoglobin (TGH) at TGH of a newborn and its birth weight. This might be delivery in 1295 mother/child pairs serving as a sur- due to increased insulin resistance in newborns with rogate of maternal and fetal glycemia. Multivariable lower birth weight. Our data suggest that the patho- regression analyses considering gestational age al physiological mechanism linking prenatal growth and delivery, the child´s sex, maternal body mass index, postnatal sensitivity to insulin are present as early as and smoking during pregnancy revealed that an before birth.

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Birth weight (in kilograms) according to TGH of child, mother, and its ratio. A through C, Scatterplots with regression line. D through F, Six groups of equal size (≈200 cases per group) were formed for TGH of child, mother, and its ratio with low to high values (1– 6) from left to right. Data are given as mean ± SEM. Correlation coefficients and probability values are given.

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7. Novel Approches for the treatment of diabetic end-organ damage (diabetic nephropathy/ diabetic heart disease)

Project leader Berthold Hocher Coworkers Markus Alter, Ina Ott, Karoline von Websky, Alexandra Simon, Katharina Krause-Relle Collaboration Prof. Dr. Florian Schweigert, PD Dr. med. vet. Jens Raila, Dr. rer. nat. Alexandra Henze, Institute of Nutritional Science, University of Potsdam

As the prevalence of diabetes mellitus and hypertension betic mice. BI1356 in combination with Telmisartan was rises in the western industrial nations, their complications able to significantly reduce albuminuria compared to dia- such as diabetic nephropathy become more and more betic, vehicle treated animals (3.07±0.67 µg/[g*24h] vs. present. The model of diabetic and eNOS deficient mice 7.27±1.50 µg/[g*24h]; mean±SEM; n= 12-13; p<0.05). comes very close to the situation in humans and estab- No significant differences were measured between dia- lished treatments such as medication with AT2 receptor betic animals treated solely with BI1356 (4.92±1.60 blockers still lack satisfactory efficacy. Dipeptidylpepti- µg/[g*24h]) or Telmisartan (4.53±1.19 µg/[g*24h]; n= dase-IV inhibition (BI1356) is reported to have beneficial 12-14), respectively, and vehicle (7.27±1.50 µg/[g*24h]). effects on glucose tolerance and to act antifibrotically. The Plasma Cystatin C levels were not significantly altered. aim of the study was to investigate the renal and cardio- This is due to the short hyperglycaemic and treatment vascular effects of treatment with BI1356 on diabetic and period resulting in hyperfiltrating, stage 1 renal disease. eNOS deficient mice and to compare the results with the We demonstrate for the first time, that treatment with the established treatment with Telmisartan. novel dipeptidylpeptidase-IV inhibitor BI1356 in combi- 65 male eNOS knock out C57BL/6J mice were ran- nation with Telmisartan reduces albuminuria in an early domly divided into four groups 1,5 week after they had stage hypertensive diabetic nephropathy model and intraperitonally received streptozotocin (each 100 mg/ might enhance the effects of established treatments. kg body weight on 2 consecutive days): Telmisartan (1 mg/ kg), BI1356 (3 mg/ kg), BI 1356+ Telmisartan (3+1 mg/ kg), and vehicle (n=14-18 per group). Another 14 mice received vehicle after they had been administered citrate puffer instead of streptozotocin (non-diabetic controls). All substances were given once daily by oral gavage with equal volumes per body weight. After 11 weeks of treatment, experiments with metabolic cages were performed, urine and blood were obtained and blood pressure was measured. None of the substances had an influence on survival. Unlike BI1356, Telmisartan reduced systolic blood pressure compared to vehicle treated, diabetic and non-dia-

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8. The combined endothelin-converting enzyme / neutral endopeptidase inhibitor SLV338 prevents myocardial remodelling in rats with renovascular hypertension in a blood pressure independent manner

Project leader Berthold Hocher Coworkers Philipp Kalk, Yuliya Sharkovska, Katharina Krause-Relle, Thiemo Pfab Collaboration Prof. Dr. Yvan Fischer, Dr. Katrin Hoffmann, Abbott Products GmbH, Hannover, Germany; Philippe Guillaume, Daniel Provost, Porsolt and partners Pharmacology, Le Genest-Saint-Isle, France; Prof. Dr. Sebastian Bachmann, Dr. Kerim Mutig, Dr. Alexander Paliege, Institut für Vegetative Anatomie, Charité, Campus Mitte: Dr. Elena Kaschina, Center for Cardiovascular Research/ Institut für Pharmakologie, Charité, Campus Mitte

Hypertensive heart disease is a major contributor to Treatment duration was 12 weeks (starting 2 weeks cardiovascular morbidity and mortality. Endothelin is a after clipping). Blood pressure was assessed every potent vasoconstrictive, pro-inflammatory, profibrotic four weeks. At study end hearts were taken for histol- and proliferative mediator produced by the endothelin ogy/computer-aided histomorphometry. Basic phar- converting enzyme(s) (ECE), whereas natriuretic pep- macological properties of SLV338 are also described. tides – degraded by the neutral endopeptidase (NEP) SLV338 is a dual ECE/NEP inhibitor as demonstrated — have diuretic, vasodilatory, anti-inflammatory, anti- both in vitro and in vivo in functional assays. In the fibrotic, and anti-proliferative properties. Thus com- 2K1C study losartan lowered blood pressure by up bined ECE/NEP inhibition may represent an option to 46 mmHg, whereas both dosages of SLV338 had to halt hypertensive cardiac remodelling. The present no effect. However, SLV338 (both dosages) com- study examined the effects of SLV338, a novel ECE/ pletely normalized cardiac interstitial fibrosis (Figure NEP inhibitor, on cardiac tissue protection in a rat 1), perivascular fibrosis, myocyte diameter and media/ model of renovascular hypertension (2-kidney-1-clip; lumen-ratio of cardiac arteries, as did losartan. 2K1C). These observations show that the dual ECE/NEP Male rats were allocated to 5 groups: Sham-oper- inhibitor SLV338 prevents cardiac remodelling to ated rats, untreated animals with 2K1C, 2K1C animals the same extent as losartan, but in a blood pressure treated with oral SLV338 (30 and 100 mg/kg/d), and independent manner, in a rat model of renovascular 2K1C animals treated with oral losartan (20mg/kg/d). hypertension.

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Cardiac interstitial fibrosis in the 2K1C study. Results were calculated as percentage of Sirius-Red- positive area in randomly chosen pictures from cardiac sections; all values are given as mean ± SEM; ; *: p < 0.05 vs Sham-group; †††/† - p < 0.001/0.05 vs 2K1C.

Publications 2006-2010

Quaschning T, Hocher B, Ruhl S, Kraemer-Guth A, Tilgner J, Pfab T, Thöne-Reineke C, Theilig F, Lange I, Witt H, Maser- Wanner C, Galle J (2006): Vasopeptidase inhibition normal- Gluth C, Bader M, Stasch JP, Ruiz P, Bachmann S, Yanagi- izes blood pressure and restores endothelial function in sawa M, Hocher B (2006): Diabetic endothelin B receptor- renovascular hypertension. Kidney Blood Press Res 29 (6): deficient rats develop severe hypertension and progressive 351-9 renal failure. J Am Soc Nephrol 17 (4): 1082-9

Langnickel D, Enghard P, Klein C, Undeutsch R, Hocher B, Chen YP, Pfab T, Slowinski T, Richter CM, Godes M, Hocher Manz R, Burmester GR, Riemekasten G (2006): Induction of B (2006): Impact of genetic variation of tumor necrosis factor- pathogenic anti-dsDNA antibodies is controlled on the level of alpha on gestational hypertension. Chin Med J (Engl) 119 (9): B cells in a non-lupus prone mouse strain. J Clin Immunol 719-24 26 (1): 86-95 Wagner FD, Buz S, Zais H, Stasch JP, Hetzer R, Hocher B Pfab T, Franz U, Herfeld F, Lun A, Armbruster F, Hocher B (2006): Humoral and hemodynamic responses after left ven- (2006): Rapid immunochromatographic strip test for the tricular assist device implantation and heart transplantation. detection of albuminuria and brief literature review on albu- Exp Biol Med (Maywood) 231 (6): 861-4 minuria screening. Eur J Med Res 11 (1): 3-6 Kalk P, Godes M, Relle K, Rothkegel C, Hucke A, Stasch Regitz-Zagrosek V, Hocher B, Bettmann M, Brede M, Had- JP, Hocher B (2006): NO-independent activation of soluble amek K, Gerstner C, Lehmkuhl HB, Hetzer R, Hein L (2006): guanylate cyclase prevents disease progression in rats with Alpha2C-adrenoceptor polymorphism is associated with 5/6 nephrectomy. Br J Pharmacol 148 (6): 853-9 improved event-free survival in patients with dilated cardiomyopathy. Eur Heart J 27 (4): 454-9

87 Berthold Hocher – Pharmacology/Nephrology

Thone-Reineke C, Kalk P, Dorn M, Klaus S, Simon K, Pfab T, Kalk P, Eggert B, Relle K, Godes M, Heiden S, Sharkovska Godes M, Persson P, Unger T, Hocher B (2006): High-protein Y, Fischer Y, Ziegler D, Bielenberg GW, Hocher B (2007): The nutrition during pregnancy and lactation programs blood adenosine A1 receptor antagonist SLV320 reduces myocar- pressure, food efficiency, and body weight of the offspring in dial fibrosis in rats with 5/6 nephrectomy without affecting a sex-dependent manner. Am J Physiol Regul Integr Comp blood pressure. Br J Pharmacol 151 (7): 1025-32 Physiol 291 (4): R1025-30 Quaschning T, Voss F, Relle K, Kalk P, Vignon-Zellweger N, Pfab T, Slowinski T, Godes M, Halle H, Priem F, Hocher B Pfab T, Bauer C, Theilig F, Bachmann S, Kraemer-Guth A, (2006): Low birth weight, a risk factor for cardiovascular Wanner C, Theuring F, Galle J, Hocher B (2007): Lack of diseases in later life, is already associated with elevated fetal endothelial nitric oxide synthase promotes endothelin-induced glycosylated hemoglobin at birth. Circulation 114 (16): 1687- hypertension: lessons from endothelin-1 transgenic/endothe- 92 lial nitric oxide synthase knockout mice. J Am Soc Nephrol 18 (3): 730-40. Pfab T, Poralla C, Richter CM, Godes M, Slowinski T, Priem F, Halle H, Hocher B (2006): Fetal and maternal peroxisome Wiessner R, Hannemann-Pohl K, Ziebig R, Grubitzsch H, proliferator-activated receptor gamma2 Pro12Ala does not Hocher B, Vargas-Hein O, Lun A, Schimke I, Liefeldt L (2008): influence birth weight. Obesity 14 (11): 1880-5 Impact of kidney function on plasma troponin concentrations after coronary artery bypass grafting. Nephrol Dial Trans Bamberg C, Diekmann F, Haase M, Budde K, Hocher B, Halle plant 23 (1): 231-8 H, Hartung J (2007): Pregnancy on intensified hemodialysis: fetal surveillance and perinatal outcome. Fetal Diagn Ther Quaschning T, Voss F, Herzfeld S, Relle K, Kalk P, Godes 22 (4): 289-93 M, Pfab T, Kraemer-Guth A, Bonz AW, Theuring F, Galle J, Hocher B (2008): Lack of iNOS impairs endothelial function in Hocher B, Liefeldt L, Quaschning T, Kalk P, Ziebig R, Godes endothelin-1 transgenic mice. Kidney Blood Press Res 31 M, Relle K, Asmus G, Stasch JP (2007): Soluble CD154 is a (2): 127-34 unique predictor of nonfatal and fatal atherothrombotic events in patients who have end-stage renal disease and are on Hocher B, Kalk P, Godes M, Liefeldt L, Ziebig R, Stasch JP, hemodialysis. J Am Soc Nephrol 18 (4): 1323-30 Quaschning T, Pfab T (2008): Gender-dependent impact of risk factors for cardiovascular and non-cardiovascular mortal- Seeliger E, Flemming B, Wronski T, Ladwig M, Arakelyan K, ity in end-stage renal disease patients on haemodialysis. Godes M, Möckel M, Persson PB (2007): Viscosity of contrast Kidney Blood Press Res 31 (5): 360-6 media perturbs renal hemodynamics. J Am Soc Nephrol 18 (11) :2912-20 Kalk P, Westermann D, Herzfeld S, Relle K, Pfab T, Bauer C, Tschöpe C, Stasch JP, Hocher B (2008): Additional lack of Pfab T, Stirnberg B, Sohn A, Krause K, Slowinski T, Godes iNOS attenuates diastolic dysfunction in aged ET-1 transgenic M, Guthmann F, Wauer R, Halle H, Hocher B (2007): Impact mice. Can J Physiol Pharmacol 86 (6): 353-7 of maternal angiotensinogen M235T polymorphism and angiotensin-converting enzyme insertion/deletion polymor- Heiden S, Pfab T, von Websky K, Vignon-Zellweger N, Godes phism on blood pressure, protein excretion and fetal outcome M, Relle K, Kalk P, Theuring F, Zidek W, Hocher B (2008): in pregnancy. J Hypertens 25 (6): 1255-61 Tissue specific activation of the endothelin system in severe acute liver failure. Eur J Med Res 13 (7): 327-9 Slowinski T, Kalk P, Christian M, Schmager F, Relle K, Godes M, Funke-Kaiser H, Neumayer HH, Bauer C, Theuring F, Kalk P, Senf P, Deja M, Petersen B, Busch T, Bauer C, Hocher B (2007): Cell-type specific interaction of endothelin Boemke W, Kaisers U, Hocher B (2008): Inhalation of an and the nitric oxide system: pattern of prepro-ET-1 expression endothelin receptor A antagonist attenuates pulmonary in kidneys of L-NAME treated prepro-ET-1 promoter-lacZ- inflammation in experimental acute lung injury. Can J Physiol transgenic mice. J Physiol 581 (Pt 3): 1173-81 Pharmacol 86 (8): 511-5

88 Berthold Hocher – Pharmacology/Nephrology

Kalk P, Mach A, Thone-Reineke C, Godes M, Heiden S, Seeliger E, Wronski T, Ladwig M, Dobrowolski L, Vogel Sharkovska Y, von Websky K, Relle K, Hocher B (2008): T, Godes M, Persson PB, Flemming B (2009): The renin- Pulmonary fibrosis in L-NAME-treated mice is dependent on angiotensin system and the third mechanism of renal blood an activated endothelin system. Can J Physiol Pharmacol flow autoregulation. Am J Physiol Renal Physiol 296 (6): 86 (8): 541-5 F1334-45

Raila J, Kalk P, Pfab T, Thöne-Reineke C, Godes M, Yana- Mitrovic V, Seferovic P, Dodic S, Krotin M, Neskovic A, Dick- gisawa M, Schweigert FJ, Hocher B (2008): Urinary protein stein K, de Voogd H, Böcker C, Ziegler D, Godes M, Nakov R, profiling with surface-enhanced laser desorption/ionization Essers H, Verboom C, Hocher B (2009): Cardio-renal effects time-of-flight mass sxpectrometry in ETB receptor-deficient of the A1 adenosine receptor antagonist SLV320 in patients rats. Can J Physiol Pharmacol 86 (8): 566-70 with heart failure. Circ Heart Fail 6: 523-31

Hocher B, Chen YP, Hügle S, Repey J, Krause K, Slowinski Kalk P, Rückert M, Godes M, von Websky K, Relle K, Neu- T, Godes M, Schaeffeler E, Guthmann F, Wauer R, Halle H, mayer HH, Hocher B, Morgera S (2009): Does endothelin Gossing G, Pfab T (2008): Impact of maternal endothelial B receptor deficiency ameliorate the induction of peritoneal nitric oxide synthase gene polymorphisms on blood pressure, fibrosis in experimental peritoneal dialysis? Nephrol Dial protein excretion and fetal outcome in pregnancy. J Hum Transplant 25:1474-1478 Hypertens 22 (9): 641-7 Hocher B, Schlemm L, Haumann H, Poralla C, Chen YP, Li J, Aufdenblatten M, Baumann M, Raio L, Dick B, Frey BM, Guthmann F, Bamberg C, Kalache KD, Pfab T (2009): Interac- Schneider H, Surbek D, Hocher B, Mohaupt MG (2009): Pre- tion of maternal peroxisome proliferator-activated receptor maturity is related to high placental cortisol in preeclampsia. gamma2 Pro12Ala polymorphism with fetal sex affects mater- Pediatr Res 65 (2): 198-202 nal glycemic control during pregnancy. Pharmacogenet Genomics 20:139-142 Kleeberg L, Morgera S, Jakob C, Hocher B, Schneider M, Peters H, Rötzer S, Müller C, Kaiser M, Fleissner C, Heider Sharkovska Y, Kalk P, Lawrenz B, Godes M, Hoffmann LS, U, Neumayer HH, Sezer O (2009): Novel renal replacement Wellkisch K, Geschka S, Relle K, Hocher B, Stasch JP (2010): strategies for the elimination of serum free light chains in NO-independent Stimulation of Soluble Guanylate Cyclase patients with kappa light chain nephropathy. Eur J Med Res Reduces Target Organ Damage in Low- and High-Renin 14 (2): 47-54 Models of Hypertension. J Hypertens 28 (8):1666-1675

Kalk P, Thöne-Reineke C, Schwarz A, Godes M, Bauer C, Schlemm L, Haumann HM, Ziegner M, Stirnberg B, Sohn A, Pfab T, Hocher B (2009): Renal Phenotype of ET-1 Transgenic Alter M, Pfab T, Kalache KD, Guthmann F, Hocher B (2010): Mice is Modulated by Androgens. Eur J Med Res 14 (2): New evidence for the fetal insulin hypothesis: fetal angi- 55-8 otensinogen M235T polymorphism is associated with birth weight and elevated fetal total glycated hemoglobin at birth. J Kalk P, Guthmann F, Krause K, Relle K, Godes M, Gossing G, Hypertens 28 (4) :732-739 Halle H, Wauer R, Hocher B (2009): Impact of maternal body mass index on neonatal outcome. Eur J Med Res 14 (5): Vinon-Zellweger N, Relle K, Kienlen E, Rahnenführer J, 216-22. Sharkovska Y, Heiden S, Kalk P, Schwab K, Neuman B, Scheler C, Albrecht-Küpper B, Stasch JP, Theuring F, Hocher Hocher B, Chen YP, Schlemm L, Burdack A, Li J, Halle H, B (2010): Endothelin-1 overexpression restores cardiac func- Pfab T, Kalk P, Lang F, Godes M (2009): Fetal sex determines tion in eNOS knockout mice. submitted the impact of maternal PROGINS progesterone receptor polymorphism on maternal physiology during pregnancy. Pharmacogenet Genomics 9: 710

89 Berthold Hocher – Pharmacology/Nephrology

Wengenmayer C, Krikov M, Müller S, Lucht K, Villringer A, Zebger-Gong H, Müller D, Diercke M, Haffner D, Hocher B, Unger T, Hocher B, Thöne-Reineke C (2010): Novel therapy Verberckmoes S, Schmidt S, D’Haese PC, and Querfeld U approach in primary stroke prevention: Simultaneous inhibi- (2010): 1,25-Dihydroxyvitamin D3 - induced Aortic Calcifica- tion of endothelin converting enzyme and neutral endopepti- tions in Experimental Uremia: Upregulation of Osteoblast dase in spontaneously hypertensive, stroke–prone rats Markers, Calcium-transporting Proteins and Osterix. J improves survival. Neurological Research in press Hypertens in press

Föller M, Mahmud H, Qadri SM, Gu S, Braun M, Bobbala D, Hocher B, Heimerl D, Slowinski T, Godes M, Halle H, Priem F, Hocher B, Lang F (2010): Endothelin B receptor stimulation Pfab T (2010): Birthweight and fetal glycosylated hemoglobin inhibits suicidal erythrocyte death. FASEB J 24 (9):3351-9 at birth in newborns carrying the GLUT1 XbaI gene polymorphism. Clin Lab accepted Alter M, Pfab T, Guthmann F, Burdack A, Kempiners N, Kalk P, Hocher B (2010): Maternal and Fetal PROGINS Progester- one Receptor Polymorphism reduces the Risk for Transient Reviews & book chapters (2006-2010) Tachypnea of the Newborn. Clin Lab in press Hocher B (2006): The cost-effectiveness analysis (CEA) is Hocher B, Schlemm L, Haumann H, Rahnenführer J, Li J, dependent on the quality of the NEPHRIC study. Am Heart Guthmann F, Bamberg C, Kalk P, Pfab T, Chen YP (2010): J 151 (1) Offspring sex determines the impact of the maternal ACE I/D polymorphism on maternal glycemic control during the last Hocher B (2007): Fetal programming of cardiovascular dis- weeks of pregnancy. J Renin Angiotensin Aldosterone eases in later life - mechanisms beyond maternal undernutri- Syst in press. tion. J Physiol 579 (Pt 2): 287-8

Schmerbach K, Kalk P, Wengenmayer C, Lucht K, Unger T, Li J, Doerffel Y, Hocher B, Unger T (2007): Inflammation in Hocher B, Thoene-Reineke C (2010): Renal Outcome in Equi- the genesis of hypertension and its complications--the role of potent Antihypertensive Treatment with Telmisartan, Ramipril angiotensin II. Nephrol Dial Transplant 22 (11): 3107-9 and Combination in SHR-SP Rats. submitted Hocher B (2009): Neonatal handling: a potential new underly- Kalk P, Sharkovska Y, Relle K, Pfab T, Guillaume P, Provost ing reason for programming of renal diseases in later life. D, Hoffmann K, Fischer Y, Hocher B (2010): The Combined Kidney Blood Press Res 32 (4): 284-5 Endothelin-Converting Enzyme / Neutral Endopeptidase Inhibitor SLV338 Prevents Myocardial Remodelling in Rats von Websky K, Heiden S, Pfab T, Hocher B (2009): Patho- with Renovascular Hypertension in a Blood Pressure Inde- physiology of the endothelin system - lessons from genetically pendent Manner. Hypertension under review manipulated animal models. Eur J Med Res 14 (1): 1-6

Fischer SS, Kempe DS, Leibrock CB, Rexhepaj R, Siraskar B, Hocher B (2010): Adenosine A1 Receptor Antagonists in Clini- Boini KM, Ackermann TF, Föller M, Hocher B, Rosenblatt KP, cal Research and Development. Kidney Int 78 (5): 438-45 Kuro-O M, Lang F (2010): Hyperaldosteronism in Klotho-deficient mice. Am J Physiol Renal Physiol Epub ahead print

Schildroth J, Rettig-Zimmermann J, Kalk P, Steege A, Fähling M, Sendeski M, Paliege A, Lai EY, Bachmann S, Persson PB, Hocher B, Patzak A (2010): Endothelin type A and B receptors in the control of afferent and efferent arterioles in mice. Nephrol Dial Transplant Epub ahead print

90 Berthold Hocher – Pharmacology/Nephrology

General information Third party funding

Project leader Project title Sponsor Period Hocher B. Generation of cross-bred mice of ET DFG 2005-2007 transgenic and eNOS knockout mice Hocher B. Impact of NO-independent activation of BayerHealthcare 2006 sGC on cardiac and renal target organ damage in rats with 5/6 nephrectomy Hocher B. Impact of adenosine A1 receptor Solvay Pharmaceuticals 2007 antagonist SLV320 on cardiac and renal target organ damage in rats with 5/6 nephrectomy Hocher,B. Role of endothelin B receptor in dia- EU-Comission-Marie-Curie early 2006-2009 betic cardiomyopathy stage training programme Hocher B. Role of ETB receptor in acute renal Dr. Werner-Jackstädt-Stiftung 2005-2006 failure Hocher B. Therapeutic potential of selective Solvay Pharmaceuticals 2006-2008 adenosine A1 receptor blockade in hepatorenal syndrome in rats Kalk P. sGC Activation as potential therapeutic Bayer Schering Pharma AG 2010-2011 approach in diabetic nephropathy Hocher B. Cathepsin S in uremic animal models Roche 2010-2013

Ulrich Kintscher – Pharmacology/Obesity Research

Head of the group

Prof. Dr. med. Ulrich Kintscher

Curriculum Vitae: Prof. Dr. Ulrich Kintscher studied medicine in Giessen and Ham- burg, where he completed his MD thesis in 1997. He then worked as a clinical fellow at the German Heart Centre/Humboldt University in the Dept. of Cardiology in Berlin till 2002, interrupted by a three year postdoctoral research fellowship at the Univer- sity of California in Los Angeles in the of Division of Endocrinology, Diabetes and Hypertension. In 2002 he joined the CCR/ Institute for Pharmacology at the Charite Universitary Medicine in Berlin, where he passed his habilitation for Experimental Pharmacology in 2004. In 2006 he became a W2-Professor for Pharmacology/ Obesity Research at the Charité, where he obtained his board certification in Internal Medicine in 2007.

Members of the group

Scientists Students Anna Foryst-Ludwig Dr. rer. nat. (Laboratory Head) Bähr, Ilse Nirmala PhD student Mandy Bloch Dipl. Biol. Benz, Verena PhD student Böhm, Christian PhD student Giersch, Katja PhD student Technicians Herbst, Lena PhD student Sprang, Christiane Wardat, Sami PhD student Thalke, Beata Winkler, Robin PhD student

93 Ulrich Kintscher – Pharmacology/Obesity Research

Summary

The focus of the Kintscher group is the characteriza- cofactors (qPCR) and their binding on target gene tion of mechanisms which mediate cardiovascular promoters (ChIP) in human samples from fat tissue disease in obesity, insulin resistance and diabetes. biopsies and monocyte/ macrophages to character- Based on these findings we are aiming on the iden- ize mechanisms of selective PPARgamma modula- tification of new pharmacological targets for cardio- tion during disease progression. vascular disease prevention. This work has been recently expanded to the characterization of metabolic functions of nuclear coacti- A centrepiece of our research is a group of nuclear vator- and corepressor complexes which have been hormone receptors named peroxisome prolifer- identified as dysregulated cofactors in metabolic tis- ator-activated receptors (PPAR). The PPAR-fam- sues (e.g. adipose, skeletal muscle, liver) during the ily includes the isoforms PPARalpha, delta, and development of diet-induced obesity/ insulin resist- gamma. Activated by specific ligands these recep- ance. We have identified candidate cofactors which tors function as transcriptional regulators of genes are prominently dysregulated during disease pro- involved in lipid- and glucose metabolism. In addition gression. Functional characterization of candidate to their role in metabolic diseases, these receptors genes are currently ongoing. exert direct cardio-vasculo protective effects. Lig- PPARgamma is activated by synthetic ligands, glita- and-mediated activation of PPARs requires a coor- zones, which are used as insulin-sensitizers in anti- dinated binding and release of nuclear cofactors, a diabetic therapy. However, the clinical usage of gli- process also called PPAR-modulation. tazones is limited by its propensity to induce weight gain, fluid retention and edema. Therefore, we are Our work is focussing on the characterization of currently in the process to investigate substances mechanism involved in selective PPARgamma-mod- which are called selective PPARgamma modulators ulation during the development of insulin resistance, (SPPARMs), which regulate a distinct set of PPAR- diabetes mellitus and associated cardiovascular dis- gamma-target genes, thereby improving insulin sen- ease by using a translational approach ranging from sitivity without the induction of weight gain. We have in-vitro molecular studies in cell culture to animals to recently identified a subgroup of angiotensin type molecular studies in patients. Hereby, the expression 1 receptor blockers (ARB) as SPPARMs. Following levels of the different components of PPARgamma- this line, we are currently analyzing new bi-modal transcriptional complexes, in-vivo binding of PPAR- ARB/ SPPARM compounds (synthesized in collabo- gamma-cofactor complexes on target gene promot- ration with the Institute of Pharmacy, Free University, ers and their functional analysis play an important Berlin, Germany) as potential new treatment options role. In our preclinical studies we use a broad array for the therapy of patients suffering from both high of molecular techniques, and genetically-modified blood pressure and impaired insulin- and glucose in-vivo mouse models. In clinical studies we investi- metabolism. gate the expression levels of defined PPARgamma-

94 Ulrich Kintscher – Pharmacology/Obesity Research

Along the line of nuclear hormone receptors as impact of ER-regulated adipose tissue metabolism major pharmacological targets for cardiovascular during exercise, and its interaction with the myocar- prevention, we are actively investigating the role of dium via defined systemic substrate disposal. Fur- estrogen receptors (ER) in sex differences in myo- thermore, the impact of ER-pathways on adipose cardial hypertrophy and their role in body weight loss tissue lipolysis and lipogenesis is investigated dur- and maintenance of reduced body weight. These ing weight reduction and weight regain in mice. For projects are focussed on tissue crosstalk, in particu- this adipose-tissue specific ER-deficient mice will lar adipose tissue – heart, during the development of be utilized. myocardial hypertrophy. We want to understand the

Zusammenfassung

Der Schwerpunkt der Arbeitsgruppe Kintscher liegt während der Pathogenese von Insulinresistenz/ in der Charakterisierung von molekularen Mechanis- Diabetes Mellitus und begleitender kardiovaskulä- men kardiovaskulärer Erkrankungen bei Adipositas, rer Erkrankungen konzentriert. Hierbei folgen wir Insulinresistenz und Diabetes mellitus. Das Ziel ist einem strikt translationalen Ansatz und versuchen die Identfizierung neuer pharmakologischer Zielmo- molekularbiologische in-vitro Studien mit Tierstu- leküle für die Prävention kardiovaskulärer Erkrankun- dien und klinischen Studien zu kombinieren. Wir gen. charakterisieren die Expression und Funktion von beteiligten Komponenten des PPARgamma-Tran- Im Mittelpunkt steht eine Gruppe nukleärer Hor- skriptionskomplexes in verschiedenen Geweben in monrezeptoren, die Peroxisome Proliferator-Acti- unterschiedlichen Stadien der Krankheitsentwick- vated Receptors (PPARs) welche aus 3 Isoformen lung. In unseren präklinischen Studien kommen eine besteht PPARalpha, PPARdelta und PPARgamma. Vielzahl an molekularbiologischen Techniken zur PPARs werden durch Liganden aktiviert und regu- Anwendung, begeleitet durch Studien in Knock-out lieren als Transkritionsfaktoren wichtige Gene im Mausmodellen. Im Rahmen klinischer Studien ver- Lipid- und Glukosestoffwechsel. Zusätzlich zu ihrer suchen wir unsere molekularbiologische Expertise metabolischen Funktion vermitteln diese Rezeptoren auf humane Proben anzuwenden und charakteri- kardiovaskulär-protektive Wirkungen. Die liganden- sieren PPARgamma-Transkriptionskomplexe mittels vermittelte Aktivierung von PPARs setzt die koor- Geweb-ChIP und weiterführenden Methoden in iso- dinierte Bindung und Abspaltung sog. nukleärer lierten Monozyten und Fettgewebsbiopsien. Kofaktoren voraus, ein Prozess der als selektive Diese Arbeiten wurden kürzlich ergänzt durch die PPAR-Modulation bezeichnet wird. Analyse der metabolischen und kardiovaskulären Funktionen von nukleären Koaktivatoren und – Unsere Arbeit ist auf die molekulare Charakteri- repressoren in unterschiedlichen Geweben (z.B. sierung der selektiven PPARgamma-Modulation Skeletmuskel, Fett, Monozyten/ Makrophagen),

95 Ulrich Kintscher – Pharmacology/Obesity Research

welche während der Entstehung der diät-induzier- gleichzeitig an einer Störung des Insulin- und Gluko- ten Insulinresistennz als dysreguliert charakteri- sestoffwechsel leiden. siert wurden. In noch laufenden Studien konnten Ein weiterer Schwerpunkt des Bereiches: Nukleäre wir Kandidatengene aus dieser Gruppe identifizie- Hormonrezeptoren als Zielmoleküle für neue phar- ren, welcher einer ausgeprägten diät- und/ oder makologische Ansätze zur kardiovaskulären Prä- gewichtsabhängigen Regulation unterliegen. vention, liegt auf der Analyse von Estrogenrezep- PPARgamma wird durch synthetische Liganden aus toren (ERs) als zentrale Mediatoren von Geschlech- der Gruppe der Glitazone aktiviert, welche als Insu- terunterschieden bei Myokardhypertrophie, sowie linsensitizer in der oralen antidiabetischen Therapie auf Untersuchungen zur Bedeutung von ERs bei etabliert sind. Der klinische Einsatz von Glitazonen der Körpergewichtsreduktion und dem Gewichts- ist wesentlich durch das Nebenwirkungsspektrum erhalt nach Reduktion. Diese Studien untersuchen dieser Substanzen limitiert einschließlich Körperge- die Interaktionen zwischen Fettgewebe und Myo- wichtszunahme, Flüssigkeitsretention und Ödemen. kard im Rahmen der trainings-induzierten kardia- Aus diesem Grunde entwickeln wir in einem wei- len Hypertrophieentstehung. Die Bedeutung der teren Projektbereich derzeitig sogenannte selektive Estrogenrezeptor-vermittelten Regulation der Fett- PPARgamma Modulatoren (SPPARMs), die bei gewebslipolyse als myokardiale Energiesubstrat- verbesserter Wirksamkeit (anti-diabetisch, anti- quelle während Training und deren Einfluss auf die atheroskerlotisch) weniger Nebenwirkungen auf- kardiomyozytäre Hypertrophie werden untersucht. weisen sollen. In diesem Zusammenhang konnten Im Weiteren wird die Bedeutung der Fettgewebs- wir eine Subgruppe von Angiotensin Typ1 – Rezep- ERs im Rahmen der geschlechtsspezifischen Kör- torblockern (ARB) als SPPARMs charakterisieren. pergewichtsregulation untersucht. Hierbei wird die In Kollaboration mit dem Institut für Pharmazie der Relevanz der ERs bei der Fettgewebslipolyse und Freien Universität Berlin (Prof. R. Gust) analysieren –lipogenese im Verlauf einer Körpergewichtsre- wir derzeitig neue bi-modale ARB/ SPPARM Subs- duktion und erneuter Zunahme („Weight Cycling“) tanzen als eine zukünftige Therapieoption für Pati- analysiert. enten, welche bei bestehender arterieller Hypertonie

96 Ulrich Kintscher – Pharmacology/Obesity Research

Research projects

1. Molecular Characterisation of New Selective Modulators for the Peroxisome Proliferator-Activated Receptor gamma

Project leader Ulrich Kintscher Coworkers Anna Foryst-Ludwig, Lena Herbst, Beata Thalke Funding - DFG (KI 712/ 3-2) External cooperations Prof. Ronald Gust, Institute of Pharmacy, University Innsbruck, Austria

Aim of this project is the development of detailed 2010). To further characterize these compounds, we structure-activity relationships for the design of new are currently performing cofactor-PPARgamma LBD selective PPARgamma modulators (SPPARMs) based binding studies using FRET analysis. These studies on our knowledge of the PPARgamma-modulating will be complemented by Microarray analysis for gene properties of the angiotensin type 1 receptor blocker expression profiling under distinct SPPARM stimula- telmisartan. In our previous work, we characterized tion. Finally, we want to connect cofactor binding with major telmisartan components regarding their rel- the regulation of gene expression, and will perform evance for PPARgamma activation. (Goebel M et al. ChIP on identified target gene promoters. ChemMedChem 2009/1+2, and BioorgMedChem

Results of FRET (Fluores- cence-Resonance Energy Transfer) analysis for binding of DRIP205 to PPARg-LBD. A specific signal is generated when ligand-induced cofactor - PPARg-LBD interaction occurs. The number represent newly synthesized SPPARMs; Pio: Pioglitazon.

97 Ulrich Kintscher – Pharmacology/Obesity Research

2. The role of sex specific lipolysis in exercise-induced cardiac hypertrophy

Project leader Anna Foryst-Ludwig Coworkers Beata Thalke, Christiane Sprang, Christian Böhm Funding - DFG-FG 1054 (TP6) External cooperations Dr. med. Michael Kreissl, Universität Würzburg Erin Kershaw, PhD, University of Pittsburgh, USA

Adipose tissue undergoes profound molecular mediate sexual dimorphisms in the development of changes during exercise such as increased lipolysis physiological cardiac hypertrophy (see figure) as a result of an activation of hormone-sensitive lipase The aim of the present project is the identification and (HSL) and possibly additional adipose tissue lipases, characterization of molecular and biochemical lipolytic such as adipose tissue triglyceride lipase (ATGL). targets such as HSL/ ATGL in adipose tissue, which Women show higher adipose tissue lipolysis than men mediate sexual dimorphism in exercise-induced car- during exercise. Importantly, HSL is regulated in a sex- diac hypertrophy with a focus on estrogen receptor- dependent manner that might be mediated through dependent gene regulation in adipose tissue. Male direct estrogen actions. Sex-specific modulation of and female mice will be challenged with active tread- adipose fatty-acid metabolism during exercise may mill running and comprehensive metabolic, cardiac, result in alteration of circulating fatty acid levels leading and molecular phenotyping will be performed. Finally, to changes in cardiac substrate utilization. Dysregula- HSL and ATGL gene regulation by estrogen receptors tion of cardiac fatty oxidation plays an important role will be studied in-vitro. in the development of cardiac hypertrophy, and may

98 Ulrich Kintscher – Pharmacology/Obesity Research

3. Adipose tissue estrogen receptors and body weight loss/ maintenance of reduced weight

Project leader Anna Foryst-Ludwig and Mandy Bloch Coworkers Verena Benz, Beata Thalke, Christiane Sprang Funding - DFG-KFO 218 (TP1) External cooperations Dr. rer. nat. Petra Wiedmer, DIFE, Potsdam Jan-Åke Gustafsson, Karolinska Institute, Stockholm, Sweden

99 Ulrich Kintscher – Pharmacology/Obesity Research

4. Function of HDAC6 for glucocorticoid receptor-mediated impairment of glucose metabolism

Project leader Ulrich Kintscher Coworkers Robin Winkler Funding - German Diabetes Foundation External cooperations Prof. Patrick Matthias, Friedrich Miescher Institute, Basel, Switzerland

The glucocorticoid receptor (GR) is an important regu- dex-mediated hyperinsulinemia in wt mice did not lator of insulin sensitivity and glucose tolerance. Since occur in HDACko mice and GR-mediated insulin the histone deacetylase 6 (HDAC6) deacetylates heat resistance in these mice was ameliorated ascertained shock protein 90, a GR chaperone, we investigated by ITT. After dex stimulation glucose production was the effect of HDAC6 on GR function in a metabolic augmented by in wt hepatocytes whereas the increase context in vivo and in vitro. in HDAC6ko hepatocytes was diminished. Wildtype (wt) and HDAC6-deficient (HDAC6ko) mice The present study identifies HDAC6 as an important were subjected to dexamethasone (dex) treatment. In regulator of metabolic GR-function preventing ligand- wt-mice dex injection resulted in a marked glucose dependent induction of gluconeogenesis. Modulation intolerance. In HDACko mice dex-induced glucose of GR-function by HDAC6 appears to be gene-spe- intolerance was completely abolished. Furthermore, cific which may provide an opportunity for therapeutic intervention.

100 Ulrich Kintscher – Pharmacology/Obesity Research

Publications 2006 - 2010 Kintscher U, Hartge M, Hess K, Foryst-Ludwig A, Clemenz M, Wabitsch M, Fischer-Posovszky P, Barth TF, Dragun D, Skurk Schupp M, Kintscher U, Fielitz J, Thomas J, Pregla R, Hetzer T, Hauner H, Bluher M, Unger T, Wolf AM, Knippschild U, R, Unger T, Regitz-Zagrosek V. 2006 Cardiac PPARalpha Hombach V, Marx N. 2008 T-lymphocyte infiltration in visceral expression in patients with dilated cardiomyopathy. Eur J adipose tissue: a primary event in adipose tissue inflammation Heart Fail 8:290-4 and the development of obesity-mediated insulin resistance. Arterioscler Thromb Vasc Biol 28(7):1304-1310 Schupp M, Lee LD, Frost N, Umbreen S, Schmidt B, Unger T, Kintscher U. 2006 Regulation of peroxisome proliferator- Schmerbach K, Schefe JH, Krikov M, Muller S, Villringer A, activated receptor gamma activity by losartan metabolites. Kintscher U, Unger T, Thoene-Reineke C. 2008 Comparison Hypertension 47:586-9 between single and combined treatment with candesartan and pioglitazone following transient focal ischemia in rat brain. Kintscher U, Bramlage P, Paar WD, Thoenes M, Unger T. Brain Res 1208:225-233 Irbesartan for the treatment of hypertension in patients with the metabolic syndrome: A sub analysis of the Treat to Foryst-Ludwig A, Clemenz M, Hohmann S, Hartge M, Sprang Target post authorization survey. Prospective observational, C, Frost N, Krikov M, Bhanot S, Barros R, Morani A, Gus- two armed study in 14,200 patients. Cardiovasc Diabetol tafsson JA, Unger T, Kintscher U. 2008 Metabolic actions of 2007;6(1):12 estrogen receptor beta (ERbeta) are mediated by a negative cross-talk with PPARgamma. PLoS Genet 4(6):e1000108 Scholze J, Grimm E, Herrmann D, Unger T, Kintscher U. 2007 Optimal treatment of obesity-related hypertension: the Kaschina E, Grzesiak A, Li J, Foryst-Ludwig A, Timm M, Hypertension-Obesity-Sibutramine (HOS) study. Circulation Rompe F, Sommerfeld M, Kemnitz UR, Curato C, Namsolleck 115(15):1991-1998 P, Tschope C, Hallberg A, Alterman M, Hucko T, Paetsch I, Dietrich T, Schnackenburg B, Graf K, Dahlof B, Kintscher U, Kappert K, Meyborg H, Clemenz M, Graf K, Fleck E, Kint- Unger T, Steckelings UM. 2008 Angiotensin II Type 2 Recep- scher U, Stawowy P. 2007 Insulin facilitates monocyte tor Stimulation. A Novel Option of Therapeutic Interference migration: A possible link to tissue inflammation in insulin- With the Renin-Angiotensin System in Myocardial Infarction? resistance. Biochem Biophys Res Commun 19:19 Circulation 24:24

Walcher D, Hess K, Heinz P, Petscher K, Vasic D, Kintscher Goebel M, Clemenz M, Staels B, Unger T, Kintscher U, Gust U, Clemenz M, Hartge M, Raps K, Hombach V, Marx N. R. 2009 Characterization of new PPARgamma agonists: 2008 Telmisartan inhibits CD4-positive lymphocyte migration analysis of telmisartan‘s structural components. ChemMed independent of the angiotensin type 1 receptor via peroxi- Chem 4(3):445-456 some proliferator-activated receptor-gamma. Hypertension 51:259-266 Brinckmann M, Kaschina E, Altarche-Xifro W, Curato C, Timm M, Grzesiak A, Dong J, Kappert K, Kintscher U, Unger T, Li Clemenz M, Frost N, Schupp M, Caron S, Foryst-Ludwig A, J. 2009 Estrogen receptor alpha supports cardiomyocytes Bohm C, Hartge M, Gust R, Staels B, Unger T, Kintscher U. indirectly through post-infarct cardiac c-kit+ cells. J Mol Cell 2008 Liver-specific peroxisome proliferator-activated receptor Cardiol 47(1):66-75 alpha target gene regulation by the angiotensin type 1 receptor blocker telmisartan. Diabetes 57(5):1405-1413 Mai K, Andres J, Biedasek K, Weicht J, Bobbert T, Sabath M, Meinus S, Reinecke F, Mohlig M, Weickert MO, Clemenz M, Karatas A, Hegner B, de Windt LJ, Luft FC, Schubert C, Pfeiffer AF, Kintscher U, Spuler S, Spranger J. 2009 Free fatty Gross V, Akashi YJ, Gurgen D, Kintscher U, da Costa Gon- acids link metabolism and regulation of the insulin-sensitizing calves AC, Regitz-Zagrosek V, Dragun D. 2008 Deoxycorti- fibroblast growth factor-21. Diabetes 58(7):1532-1538 costerone acetate-salt mice exhibit blood pressure-independent sexual dimorphism. Hypertension 51(4):1177-1183

101 Ulrich Kintscher – Pharmacology/Obesity Research

Goebel M, Staels B, Unger T, Kintscher U, Gust R. 2009 Reviews & book chapters (2006-2010) Characterization of new PPARgamma agonists: benzimida- zole derivatives - the importance of position 2. ChemMed Clemenz M, Kintscher U, Unger T (2006): The metabolic Chem 4(7):1136-1142 syndrome: cluster with a self-fulfilling loop? J Hypertens 24:257-8 Kappert K, Tsuprykov O, Kaufmann J, Fritzsche J, Ott I, Goebel M, Bahr IN, Hassle PL, Gust R, Fleck E, Unger T, Sta- Hartge MM, Kintscher U, Unger T (2006): Endothelial dys- wowy P, Kintscher U. 2009 Chronic Treatment With Losartan function and its role in diabetic vascular disease. Endocrinol Results in Sufficient Serum Levels of the Metabolite EXP3179 Metab Clin North Am 35:551-60, viii-ix for PPAR{gamma} Activation. Hypertension 54(4):738-743 Kintscher U (2007): Does adiponectin resistance exist in Kilter H, Werner M, Roggia C, Reil JC, Schafers HJ, Kintscher chronic heart failure? 2007 Eur Heart J 28(14):1676-1677 U, Böhm M. 2009 The PPAR-gamma agonist rosiglitazone facilitates Akt rephosphorylation and inhibits apoptosis in car- Kappert K, Unger T, Kintscher U (2008): [Aliskiren hemifuma- diomyocytes during hypoxia/reoxygenation. Diabetes Obes rat]. Dtsch Med Wochenschr 133(24):1308-1312 Metab 11:1060-1067 Kintscher U (2008): The cardiometabolic drug rimonabant: Van Linthout S, Foryst-Ludwig A, Spillmann F, Peng J, Feng Y, after 2 years of RIO-Europe and STRADIVARIUS. Eur Heart Meloni M, Van Craeyveld E, Kintscher U, Schultheiss HP, De J 13:13 Geest B, Tschope C. 2010 Impact of HDL on adipose tissue metabolism and adiponectin expression. Atherosclerosis Kintscher U (2008): Pharmacological Differences of Glita- 210(2):438-444 zones - Does PPARa Activation Make the Difference? J Am Coll Cardiol 52(10):882-884 Fliegner D, Schubert C, Penkalla A, Witt H, Kararigas G, Dworatzek E, Staub E, Martus P, Ruiz Noppinger P, Kintscher Kintscher U, Foryst-Ludwig A, Unger T (2008): Inhibiting Angi- U, Gustafsson JA, Regitz-Zagrosek V. 2010 Female sex and otensin Type 1 Receptors as a Target for Diabetes. Expert estrogen receptor-beta attenuate cardiac remodeling and Opinion on Therapeutic Targets 12(10):1257-1263 apoptosis in pressure overload. Am J Physiol Regul Integr Comp Physiol 298(6):R1597-1606 Kintscher U, Goebel M (2009): INT-131, a PPARgamma agonist for the treatment of type 2 diabetes. Curr Opin Investig Kintscher U, Marx N, Martus P, Stoppelhaar M, Schimkus J, Drugs 10(4):381-387 Schneider A, Walcher D, Kummel A, Winkler R, Kappert K, Dorffel Y, Scholze J, Unger T. Effect of high-dose valsartan Kintscher U (2009): ONTARGET, TRANSCEND, and PRo- on inflammatory and lipid parameters in patients with Type 2 FESS: new-onset diabetes, atrial fibrillation, and left ventricu- diabetes and hypertension. 2010 Diabetes Res Clin Pract lar hypertrophy. J Hypertens 27 Suppl 2(2):S36-39. 89(3):209-215 Foryst-Ludwig A, Kintscher U (2010): Metabolic impact of Goebel M, Wolber G, Markt P, Staels B, Unger T, Kintscher U, estrogen signalling through ERalpha and ERbeta. J Steroid Gust R. Characterization of new PPARgamma agonists: ben- Biochem Mol Biol 122(1-3):74-81. zimidazole derivatives-importance of positions 5 and 6, and computational studies on the binding mode. 2010 Bioorg Kintscher U (Herausgeber) und Marx N (Ko-Herausgeber) Med Chem 18(16):5885-5895 2008 Antihypertensiva bei Metabolischem Syndrom und Diabetes Mellitus. Unimed Buch-Verlag

102 Ulrich Kintscher – Pharmacology/Obesity Research

General information Third party funding (2006-2010)

Project leader Project title Sponsor Period Kintscher U. Der Einfluß von Fettgewebe auf die DFG – GRK 754-III Geschlech- 2006-2010 Myokardhypertrophie: Bedeutung von tsspezifische Mechanismen bei PPARs und Estrogenrezeptoren Myokardhypertrophie Kintscher U. Molekulare Charakterisierung neuer DFG-KI 712/ 3-1 2006-2008 selektiver Modulatoren des Peroxisome Proliferator-Activated Receptor (PPAR) Kintscher U. Molekulare Charakterisierung neuer DFG-KI 712/ 3-2 2010-2012 selektiver Modulatoren des Peroxisome Proliferator-Activated Receptor (PPAR) Kintscher U. The role of sex-specific lipolysis in DFG-FG 1054/1 2008-2011 exercise-induced cardiac hypertrophy TP6 – KI 712/ 5-1 Sex-specific mechanisms in myocardial hypertrophy Kintscher U. The role of adipose tissue estrogen DFG-KFG 218/1 2009-2012 Foryst-Ludwig, A. receptors during body weight loss and TP6 – KI 712/ 6-1 the maintenance of reduced weight Hormonal regulation of body weight maintenance Kintscher U. The role of HDAC6 in GR-mediated Deutsche Diabetes Stiftung 2010-2011 impairment of glucose metabolism Boschmann M. Caveolopathies - insulin resistance, DFG-KFG 192/2 2010-2013 Kintscher U. lipid metabolism TP9 – Skeletal muscle growth regulation and dysregulation Kintscher U. Regulation of tissue-specific endothelial Nippon Boehringer 2006-2008 Unger T. dysfunction by telmisartan: The role of PPAR activation Kintscher U. PPAR activation by Losartan in Hyper- Merck, Sharp & Dohme 2006-2008 Fleck E. tensive Patients: The Importance of Gust R. Losartan-Metabolites

103 Ulrich Kintscher – Pharmacology/Obesity Research

Kintscher U. Einfluss von Telmisartan auf Parameter Bayer Vital 2007-2009 der Inflammation bei hypertensiven Patienten (METATEL-STUDIE) Kintscher U. Regulation of Muscular and Hepatic Boehringer Ingelheim 2005-2007 Unger T. Insulin Signaling by Telmisartan Kintscher U. Regulation of histone deacetylases by Boehringer Ingelheim 2008-2009 telmisartan Kintscher U. Metabolic actions of telmisartan in Boehringer Ingelheim 2009-2010 adipose-specific PPAR-deficient mice

Awards

2008 Best Oral Abstract Presentation; 13th Annual Meeting of the European Council for Cardiovascular Research Mandy Bloch

104 Harm Peters – NEPHROLOGY / MOLECULAR FIBROSIS AND HYPOXIA RESEARCH

Head of the group

Prof. Dr. Harm Peters

Curriculum Vitae: Prof. Peters studied medicine at the Free University Berlin and started his scientific career as a MD candidate at the Department for Endocrinology at the Benjamin Franklin Hospital in Steglitz, today Charité Campus Benjamin Franklin. In 1992 he earned his degree with the thesis entitled “Impact of maternal TSH receptor-blocking antibodies in cases of genetic hypothyroidism”. In 1993 he changed to the Department of Nephrology at the Benjamin Franklin Hospital, followed by a 2-year postdoctoral research period in the lab of Prof. Nancy Noble and Prof. Wayne Border, University of Utah, Salt Lake City, USA. Since 1999 Prof. Peters works as a assistant medical director at the Medical Clinic / Focus Nephrology, Charité Campus Mitte. In 2000 he passed his habilitation on the topic: ”Impact of the L-Arginin-/ NO metabolism in cases of acute and chronic glomerulosclerosis”.

Members of the group

Scientists Technicians Rosenberger, Christian PD Dr. med. Mika, Alice Brand, Daniel Veterinarian Gaedeke, Jens Dr. med. Students Grosz, Bianca Physician Grüger, Jens Medical student Halleck, Fabian Physician Horoszynska, Lillianna Medical student Khadzhynov, Dmytro Physician Scheidl, Julia Medical student Krämer, Stephanie Dr. vet. med. Loof, Tanja Dipl. Biochem. Martini, Sebastian Dr. med. Wang-Rosenke, Yingrui Dr. med.

105 Harm Peters – NEPHROLOGY / MOLECULAR FIBROSIS AND HYPOXIA RESEARCH

Summary

The main research of the group „molecular fibro- tion, as well as potential therapeutic use of hypoxia sis and hypoxia research“ concentrates on the adaptation in renal and cardiac diseases. The one hand on new aspects in the pathogenesis and emphasis lies on acute kidney injury, chronic renal treatment of acute and chronic fibrotic diseases fibrosis, diabetic nephropathy, renal transplanta- of the kidney and their consequences for the car- tion and myocardial infarction. Particular attention diovascular system. With regard to the kidney, the is given to the clinical relevance of injury models, research group compares immune-mediated (acute as well to subtle analysis of morphological tissue anti-Thy1 glomerulonephritis, lupus nephritis) with injury. Our group is dedicated to in vivo transcrip- not primarily inflammatory disease models (5/6 tional response to hypoxia, which is complex and nephrectomy, diabetic nephropathy). With regard still poorly understood. It is widely accepted that to the vascular consequences of impaired renal hypoxia-inducible transcription factors (HIFs) play a function, the research focus is on changes of the pivotal role in this setting. Theoretically, both benefi- myocard, coronary arteries and large vessels. In cial and detrimental effects can arise from HIF acti- order to unravel the underlying mechanism of patho- vation depending on the HIF target genes involved. logical changes, the group concentrates on cellular Our group investigates the impact of HIF up-reg- mediators such as mast cells, lymphocytes (S1P ulation achieved with different hypoxic stimuli, modulation by FTY720) und platelets (inhibition by cell types and co-transcription factors. HIF target clopidogrel) and several extra- and intracellular path- genes like erythropoietin, heme oxygenase-1, glu- ways. These include transforming growth factor beta cose transporter-1 are located within tissues with (small molecule inhibition), PDGF (receptor block- help of high amplification immunohistochemistry, ade with imatinib), mammalian target of rapamycin RT-PCR, or in situ hybridisation. Therapeutical HIF (mTOR-inhibitor sirolimus), NO/cGMP signal transactivation is achieved by pharmaceutical blockade duction (stimulator of soluble guanylate cyclase BAY of HIF degradation or using inducible transgenic 41-2272) and the renin-angiotensin-systems (maxi- technology. Human biopsies help to test for the mizing RAS inhibition and AT2 receptor stimulation). clinical relevance of our findings in animal mod- On the other hand, the research focus Hypoxia els. In the following we provide an overview of our includes detection of hypoxia and hypoxia adapta- projects from 2008 until present.

106 Harm Peters – NEPHROLOGY / MOLECULAR FIBROSIS AND HYPOXIA RESEARCH

Zusammenfassung

Die Forschungsschwerpunkte der Arbeitsgruppe Nachweis von Hypoxie bzw. Hypoxieanpassung, „Molekulare Fibrose- und Hypoxieforschung“ kon- sowie die mögliche therapeutische Nutzung der zentrieren sich auf neue Aspekte in der Pathoge- Hypoxieanpassung bei Nieren- und Herzerkran- nese und Therapie von akuten und chronischen kungen. Ein besonderes Augenmerk liegt auf aku- fibrotischen Nierenerkrankungen sowie ihrer Aus- tes Nierenversagen, chronische Nierenfibrose, dia- wirkungen auf das Herzkreislaufsystem. In Bezug betische Nephropathie, Nierentransplantation und auf Erkrankungen der Nieren werden insbesondere Herzinfarkt. Zum Leitbild der Arbeitsgruppe gehört immun-vermittelte (akute Anti-Thy1-Glomeruloneph- eine sorgfältige Bewertung experimenteller Modelle ritis, Lupusnephritis) mit nicht primär entzündlichen auf ihre klinische Übertragbarkeit, sowie eine sorg- Krankheitsmodellen (5/6 Nephrektomie, diabetische fältige anatomisch-pathologische Aufarbeitung von Nephropathie) verglichen. Bei den kardiovaskulären Organschäden. Die komplexe transkriptionelle Ant- Auswirkungen renaler Funktionseinschränkung ste- wort auf Hypoxie wird in vivo untersucht. Dabei spie- hen Veränderungen des Myokards, der Koronarien len sogenannte Hypoxie-induzierbare Faktoren (HIF) und der großen Gefäße im Vordergrund. Bei der eine zentrale Bedeutung. Deren Aktivierung kann Aufarbeitung der den pathologischen Veränderun- theoretisch positive oder negative Auswirkungen gen zugrunde liegenden Mechanismen fokussieren auf den Krankheitsverlauf haben. Unsere Gruppe sich die Untersuchungen der Arbeitsgruppe auf erforscht die HIF-Wirkung in Abhängigkeit verschie- zelluläre Mediatoren, wie Mastzellen, Lymphozyten dener Schädigungsreize, Zelltypen und Ko-Tran- (S1P-Modulation mit FTY720) und Thrombozyten skriptionsfaktoren. HIF-Zielgene wie Erythropoietin, (Inhibition mit Clopidogrel) sowie auf verschiedene Hämoxygenase-1, Glukose-Transporter-1 werden extra- und intrazellulare Pathways. Hierzu gehören innerhalb von Organen mittels Immunhistochemie, Transforming Growth Factor-beta (Hemmung mit- RT-PCR und/oder In-situ-Hybridisierung nachge- tels small molecules), PDGF (Rezeptorinaktivierung wiesen. HIF-Aktivierung erfolgt über verschiedene mitles Imatinib), mammalian Target of Rapamycine Hypoxietypen, medikamentös, oder durch den Ein- (mTOR-lnhibitor Sirolimus), NO/cGMP Signaltrans- satz transgener Tiere. Die klinische Relevanz des duktion (Stimulator der löslichen Guanylatcyclase Systems wird anhand humaner Biopsien überprüft. BAY 41-2272) und des Renin-Angiotensin-Systems Im folgenden finden Sie einen Überblick über die (Optimierung der RAS-Hemmung, Stimulation des 2008 bis aktuell bearbeiteten Projekte und Frage- AT2-Rezeptors). Auf der anderen Seite umfasst der stellungen. Forschungsschwerpunkt „Hypoxieforschung“ den

107 Harm Peters – NEPHROLOGY / MOLECULAR FIBROSIS AND HYPOXIA RESEARCH

Research projects

1. Dysfunction in epithelial sodium transport under proteinuric conditions

Project leader Harm Peters and Franziska Theilig, Institute of Anatomy Coworkers Henriette J. Kaminski, medical student, Institute of Anatomy Tanja Loof Christian Kastner, doctoral fellow, Institute of Anatomy Sebastian Bachmann, Institute of Anatomy Funding DFG FOR 667, Epithelial mechanisms in renal volume regulation, TP8

Changes of tubular sodium reabsorption play an in expression and localisation of epithelial transporters important role in the maintenance of volume home- and channels, concentration of systemic parameters ostasis under physiological conditions. In proteinuric of the renin-angiotensin-system and vasopressin will states however, defective sodium balance likely con- be evaluated. GBM-GN induced in megalin transgenic tributes to accompanied clinically evident sequelaes mice will clarify the role of increased proximal tubu- like edema and hypertension. The underlying mecha- lar protein endocytosis on expression and activity of nisms, as well as the nephron segments and trans- proximal tubular transporters and channels. Mecha- port mechanism involved need to be clarified. We nisms of activation and deactivation of transporters test the hypothesis that proteinuria leads to changes and channels, participating cell compartment specific in epithelial transporters and channels which favour proteins, and the role of lipid composition under pro- volume retention. We aspire to identify possible varia- teinuria will be characterized. The anticipated results tions in sorting and expression of tubular membrane will provide a comprehensive analysis of the cell bio- proteins. Therefore, proteinuric rat models of anti- logical basis underlying the clinically important conse- Thy1-glomerulonephritis (Thy1-GN), and puromycin- quences of nephron epithelial shuttling and transport induced nephritis, and a mouse model of anti-glomer- in proteinuric diseases. ular basement membrane nephritis (GBM-GN) will be evaluated with biochemical and histochemical methods. In Thy1-GN, nephron segment specific changes

108 Harm Peters – NEPHROLOGY / MOLECULAR FIBROSIS AND HYPOXIA RESEARCH

2. Open European Nephrology Science Center / a Charité renal biopsy metadata base repository for system biology

Project leader Harm Peters, Christian Rosenberger Coworkers Fabian Halleck, Jens Grüger, Lilianna Horoszynska, Sebastian Martini, Birgit Rudolph, Depart. of Pathology Thomas Schrader, Depart. of Pathology Funding - DFG, 10692/1-1 und 1-2, TP GN - Astellas Pharma, München External cooperations - Berlin-Brandenburgische Gesellschaft für Nephrologie, - Deutsche Gesellschaft für Nephrologie

The typical problems of research in biomedical dis- the intelligent Catalogue. Actual projects focus on the ciplines are apparent: a limitation of the amount of epidemiology of primary glomerular diseases in Berlin- cases of more or less rare diseases and complica- Brandenburg and the identification of unusual cases. tions, heterogeneous data types with varying quality Another recent focus has been the HIF activation in of automatically and „by hand“ generated data, and human kidney transplants. In these studies we have decentralized data retention inhibiting the scientific shown that in long-term renal transplants HIF is acti- work. The main Task of Open European Nephrology vated in clinical rejection, but not in subclinical rejec- Science Center (OpEN.SC) is to create a large renal tion or borderline changes [Rosenberger et al, JASN]. biopsy data base of patients with transplanted and We are currently investigating the potential diagnostic native kidneys with implementation of a metadata gain provided by additional HIF stainings in transplant repository for clinical and laboratory data, including biopsies. In a pilot project we have demonstrated that a focus on vascular consequences of impaired renal renal biopsies stained for HIF can be displayed by vir- function. The metadata repository will be stored and tual microscopy. processed by an intelligent data management tool –

HIF-1a immunohistochemistry in a human renal transplant biopsy

109 Harm Peters – NEPHROLOGY / MOLECULAR FIBROSIS AND HYPOXIA RESEARCH

3. Stimulation of soluble guanylate cyclase improves renal recovery following relief of unilateral ureteral obstruction

Project leader Yingrui Wang-Rosenke Coworkers Alice Mika, Tanja Loof, Harm Peters Funding Charité-Forschungsstipendium External cooperations Bayer Schering Pharma GmbH.

Antifibrotic effects of soluble guanylate cyclase (sGC) This was paralleled by significant reductions in systolic activation and cGMP production have been shown blood pressure, tubular diameter, tubular apoptosis, in anti-thy1 renal disease. In this study, the specific tubulointerstitial macrophage infiltration and cell prolif- sGC stimulator Bay 41-8543 was administered to ani- eration, and tubulointerstitial fibrosis. Thus, stimulation mals with UUO after relief of ureteral obstruction in of sGC through Bay 41-8543 increased cGMP produc- order to further observe the renoprotective effects of tion and further prevented the progression of UUO, sGC/cGMP in restoring and preserving progressive via reductions in renal atrophy, tubular apoptosis and renal disease. Untreated UUO rats showed elevation tubulointerstitial fibrosis and macrophage infiltration. of systolic blood pressure, marked tubular atrophy, The findings suggest that sGC stimulator may be an tubulointerstitial apoptosis, macrophage infiltration effective treatment to restore or preserve renal histol- and fibrosis, α-smooth muscle actin (SMA) expres- ogy and renal function in this experimental model of sion and mRNA expression of TGF-β. Administration renal disease. of Bay 41-8543 significantly increased plasma cGMP.

Typical alpha-smooth muscle actin staining in renal tubular tissue of rats subjected unilateral urethral obstruction.

110 Harm Peters – NEPHROLOGY / MOLECULAR FIBROSIS AND HYPOXIA RESEARCH

4. Signal agonistic AT2 receptor stimulation prevents renal alterations in the experimental model of subtotal nephrectomy

Project leader Stephanie Krämer, Harm Peters Coworkers Tanja Loof, Alica Mika, Dmytro Khazhyno, v Ulrike Steckelings, Institute of Pharmacology Thomas Unger, Instititute of Pharmacology External cooperations Vicore Pharma AB, Göteborg, Sweden

A major contributor to progression in chronic renal reduced by C21. Western blot analysis corresponded disease (CKD) is the renin angiotensin system with its with reductions of TGF-ß immunohistological protein main effector peptide angiotensin II, exerting various detection of fibronectin. Histological collagen I pro- pleiotropic actions via binding at Angiotensin type 1 tein expression was lowered as well. Furthermore C21 (AT1) and type 2 (AT2) receptors. AT2 mediated actions reduced renal macrophage and lymphocyte infiltra- were analyzed by applying the novel non-peptide AT2 tion as well as renal cell proliferation. The investigation agonist Compound 21 (C21) in the model of subto- show that C21 significantly reduces the expression of tal nephrectomy (SNX) in male Wistar rats (250-280 renal pro-fibrotic and pro-inflammatory markers at a g BW). After 12 weeks SNX was characterized by molecular and histological level in the model of SNX. increased systolic blood pressure and protein excre- The results underline previous findings pointing at tion. Administration of C21 had no significant effects direct AT2 mediated anti-fibrotic and anti-inflammatory blood pressure, but on proteinuria. On the molecu- properties. Therefore, pharmacological AT2 stimula- lar level, TGF-ß and fibronectin mRNA-expression tion with C21 might represent a novel therapeutic tool was up-regulated in SNX-animals and significantly for the treatment of CKD.

Course of proteinuria in rats subjected to subtotal nephrectomy (SNX) over 12 weeks without of with administration of Compound 21 (C21).

111 Harm Peters – NEPHROLOGY / MOLECULAR FIBROSIS AND HYPOXIA RESEARCH

5. Cardiovascular NO/cGMP signalling in mild renal insufficiency

Project leader Bianca Grosz, Harm Peters Coworkers Stephanie Krämer. Tanja Loof, Yingrui Wang-Rosenke, Dmytro Khadzhynov Funding European Union, Marie Curie program EST-CT-2005-020268 External cooperations Prof. Dr. Kerstin Amann, Inst. of Pathology, Erlangen

This project aims at characterizing cardiovascular and aortic remodelling were found. Compared to uremic renal morphological and molecular changes in a rat male rats, females showed less hypertension, left model of mild renal insufficiency, with a special focus ventricular and aortic hypertrophy but contrastingly on the role played by NO-cGMP signalling pathway higher proteinuria. These discordant results suggest and the modulator effect of gender. The second exper- that progression of cardiovascular and renal disease is imental study was also directed towards investigating differently influenced by sex hormones. Furthermore, the therapeutic potential of BAY 41-8543, a compound this is the first study demonstrating that enhancing which enhances NO-cGMP signal transduction, in NO/GMP signalling by Bay 41-8543 ameliorates aortic the development of cardiovascular and renal disease wall hypertrophy and stiffening significantly and in a in the same animal model. Summarizing the results blood pressure-independent manner, thus proposing obtained, in a rat model of mild renal insufficiency it as a future therapeutic strategy in the treatment of prominent gender effects on the degree of hyperten- vascular remodelling. sion, proteinuria, renal hypertrophy, and cardiac and

Aortic wall elastin staining of 18 weeks after induction of mild renal insufficiency in rats.

112 Harm Peters – NEPHROLOGY / MOLECULAR FIBROSIS AND HYPOXIA RESEARCH

6. Inducible transgenic HIF activation in acute kidney injury

Project leader Christian Rosenberger Coworkers Harm Peters Sebastian Bachmann, Institute of Anatomy Alexander Paliege, Institute of Anatomy Alice Mika, BTA Funding - DFG FOR 1368 External cooperations Prof. Robert Koester, Nephrology, Paris

Rhabdomyolysis is an important cause of acute kid- feature strong and persistent HIF activation in all renal ney injury (AKI) in the human. We have shown that tubules. To test for the potential of a prophylactic or in experimental rhabdomyolysis induced by glycerol therapeutic HIF activation, the transgene is induced at injection into the hind limbs renal tubular hypoxia is different time points before or after AKI. Renal function, deep and protracted, whereas hypoxia adaptation is fine morphology, and HIF target genes are assessed short-lived and limited to a minority of hypoxic cells. at different time points after AKI. We hypothesize that therapeutic activation of HIF may improve renal outcome in experimental rhabdomyolysis. To activate HIF we employ a transgenic mouse model, based on tetracycline-inducible knockout of the von Hippel Lindau (VHL) protein, which is crucial for physiological HIF degradation. Transgenic animals

Immunohistochemistry for HIF and the cell protective HIF target genes glucose transporter-1 (GLUT-1) and heme oxygenase-1 (HO-1) 24 h after rhabdomyolysis-induced acute kidney injury: Asterixes mark proximal convoluted tubules, which are the main sites of hypoxia and injury in this model. Control animals exhibit neither HIF (A), nor GLUT-1 (C) or HO-1 (E) in proximal convoluted tubules. By contrast, VHL knockout animals feature robust activation of all three markers (B, D, F).

113 Harm Peters – NEPHROLOGY / MOLECULAR FIBROSIS AND HYPOXIA RESEARCH

7. Mechanisms of hypoxia tolerance in acute on chronic kidney injury

Project leader Christian Rosenberger Coworkers Harm Peters, Alice Mika External cooperations Prablheen Singh, La Jolla, CA, USA

Statistically, chronic renal insufficiency is a well known renal mass reduction with and without additional renal predisposing factor to acute kidney injury (AKI) in interstitial fibrosis, and that such HIF activation can humans. However, in individual patients with moderate ameliorate super-imposed ischemic AKI. To this end chronic renal insufficiency the impact of acute hypoxic either normal control rats, rats with unilateral nephrec- insults on renal function is hardly predictable. Most tomy, or rats with subtotal nephrectomy are subjected likely, at least in some of these patients, increased to renal warm ischemia and reperfusion. Renal func- hypoxia adaptation may exists at baseline. This is prob- tion, morphology, pimonidazole adducts (which indi- ably due to sub-lethal hypoxia and consequent adap- cate profound hypoxia with tissue pO2 below 10 mm tive responses of remaining hypertrophied tubules. Hg), HIF, and HIF target genes are measured. We are We hypothesize that hypoxia-inducible transcription particularly interested in evidences for hypoxia and factors (HIFs), which are master regulators of hypoxia hypoxia adaptation in S3 proximal tubules, which are adaptation, are activated at baseline in animals with the major site of injury in this model of AKI.

HIF-1a immunohistochemistry in the remnant kidney

114 Harm Peters – NEPHROLOGY / MOLECULAR FIBROSIS AND HYPOXIA RESEARCH

8. The role of HIF in renal cyclosporine toxicity

Project leader Christian Rosenberger Coworkers Harm Peters, Julia Scheidl, Daniel Brand, Alice Mika Sebastian Bachmann, Inst. of Anatomy External cooperations Robert Koesters, Nephrology, Paris

Cyclosporine toxicity is a major course of renal graft ity to study the occurrence of hypoxia (pimonidazole failure, leading to tubular injury, arteriolar hyalinosis adducts), HIF activation, and up-regulation of HIF and interstitial fibrosis. The responsible mechanisms target genes during disease progression. In addition, are incompletely understood, but, most likely, involve we test the impact of an inducible transgenic HIF acti- vasoconstriction and renal hypoxia. Hypoxia-inducible vation in all renal tubular segments on the course of transcription factors (HIFs) are master regulators of cyclosporine-induced renal fibrosis. Micro array tech- hypoxia adaptation. Recent data suggest that HIF nique will reveal the contribution of acknowledged cell may aggravate chronic renal fibrosis. The role of HIF protective vs pro-fibrotic HIF target genes. Candidate in cyclosporine toxicity has not been investigated, so genes will be located within the kidney with help of far. We employ a mouse model of cyclosporine toxic- immunohistochemistry and in situ hybridization.

HIF-1a after cyclosporine treatment

115 Harm Peters – NEPHROLOGY / MOLECULAR FIBROSIS AND HYPOXIA RESEARCH

Publications 2006 – 2010 Rosenberger C, Solovan C, Rosenberger AD, Li J, Treudler R, Frei U, Eckardt KU, and Brown LF (2007): Upregulation Bernhardt WM, Schmitt R, Rosenberger C, Münchenhagen of hypoxia-inducible factors in normal and psoriatic skin. J PM, Gröne HJ, Frei U, Warnecke C, Bachmann S, Wiesener Invest Dermatol 127(10): 2445-52 MS, Willam C, Eckardt KU (2006): Expression of hypoxia- inducible transcription factors in developing human and rat Seiler M, Brabcova I, Viklicky O, Hribova P, Rosenberger kidneys. Kidney Int 69(1):114-22 C, Pratschke J, Lodererova A, Matz M, Schoenemann C, Reinke P, Volk HD, Kotsch K (2007): Heightened expression Wang Y, Krämer S, Loof T, Martini S, Kron S, Shimizu F, of the cytotoxicity receptor NKG2D correlates with acute Kawachi H, Neumayer HH, Peters H (2006): Enhancing cGMP and chronic nephropathy after kidney transplantation. Am J in experimental progressive renal fibrosis: soluble guanylate Transplant 7(2): 423-33 cyclase stimulation versus phosphodiesterase inhibition. Am J Physiol Renal Physiol 290: F167-76 Martini S, Krämer S, Loof T, Wang-Rosenke Y, Daig U, Budde K, Neumayer HH, Peters H (2007): The S1P modulator Goldfarb M, Rosenberger C, Abassi Z, Shina A, Zilbersat F, FTY720 limits matrix expansion in acute anti-thy1 mesangio- Eckardt KU, Rosen S, Heyman SN (2006): Acute-on-chronic proliferative glomerulonephritis. Am J Physiol Renal Physiol renal failure in the rat: Functional compensation and hypoxia 292:F1761-70 tolerance. Am J Nephrol 26:22-33 Schneider M, Thomas K, Liefeldt L, Kindgen-Milles D, Peters Rosenberger C, Shina A, Rosen S, Goldfarb M, Eckardt K, H, Neumayer HH, Morgera S (2007). Efficacy and safety of Heyman SN (2006): Hypoxia inducible factors and tubular cell intermittent hemodialysis using citrate as anticoagulant: a survival in isolated perfused kidneys. Kidney Int 70:60-70 prospective study. Clin Nephrol 68:302-7

Hammer MH, Brestrich G, Andree H, Engelmann E, Rosen- Rosenberger C, Rosen S, Heyman SN (2007): Normotensive berger C, Tillmann H, Zwinger S, Babel N, Nickel P, Volk HD, ischemic acute renal failure. N Engl J Med 357(21): 2204-5 Reinke P (2006): HLA type-independent method to monitor polyoma BK virus-specific CD4 and CD8 T-cell immunity. Am Rosenberger C, Khamaisi M, Abassi Z, Shilo V, Weksler- J Transplant 6(3): 625-31 Zangen S, Goldfarb M, Shina A, Zibertrest F, Eckardt K-U, Rosen S and Heyman SN (2008): Adaptation to hypoxia in the Waiser J, Dell K, Kreutzkamp J, Bohler T, Budde K, Peters H, diabetic rat kidney. Kidney Int 73(1): 34-42 Neumayer HH (2006): FK506, transforming growth factor- beta1 and mesangial matrix synthesis: Parallels and differ- Rosenberger C, Goldfarb M, Shina A, Bachmann S, Frei ences compared with cyclosporine A. Cytokine 21:59-65 U, Eckardt KU, Schrader T, Rosen S, Heyman SN (2008): Evidence for Sustained Renal Hypoxia and Transient Hypoxia Haase M, Morgera S, Bamberg C, Halle H, Martini S, Hocher Adaptation in Experimental Rhabdomyolysis-Induced Acute B, Diekmann F, Dragun D, Peters H, Neumayer HH, Budde K Kidney Injury. Nephrol Dial Transplant 23(4): 1135-43 (2006): A systematic approach to managing pregnant dialysis patients--the importance of an intensified haemodiafiltration May D, Gilon D, Djonov V, Itin A, Lazarus A, Rosenberger protocol. Nephrol Dial Transplant 20: 2537-42 C and Keshet E (2008): A conditional transgenic system for induction and rescue of chronic Myocardial hibernation Rosenberger C, Pratschke J, Rudolph B, Heyman SN, Schin- provides insights into genomic programs of hibernation and dler R, Babel N, Eckardt KU, Frei U, Rosen S, and Reinke P reversible heart remodeling. Proc Nat Acad Sci USA 105(1): (2007): Immunohistochemical detection of hypoxia-inducible 282-7 factor-1alpha in human renal allograft biopsies. J Am Soc Nephrol 18(1): 343-51 Khamaisi M, Raz I, Shilo V, Shina A, Rosenberger C, Dahan R, Abassi Z, Meidan R, Lecht S, Heyman SN (2008): Diabetes and radiocontrast media increase endothelin converting enzyme-1 in the kidney. Kidney Int 74(1):91-100

116 Harm Peters – NEPHROLOGY / MOLECULAR FIBROSIS AND HYPOXIA RESEARCH

Rosenberger C, Rosen S, Shina A, Frei U, Eckardt KU, Flip- Thumfart J,Jung S, Amasheh S, Kraemer S, Peters H, Som- pin LA, Arend M, Klaus SJ, Heyman SN (2008): Activation mer K, Biber J, Murer H, Meij I, Querfeld U, Wagner CA, of hypoxia inducible factors (HIF) ameliorates hypoxic distal Muller DN (2008): Magnesium stimulates renal phosphate tubular injury in the isolated perfused rat kidney. Nephrol reabsorption. Am J Physiol Renal Physiol 295:F1126-33 Dial Transplant 23(11): 3472-8 Rosenberger C, Rosen S, Paliege A, Heyman S (2009): Krämer S, Wang-Rosenke Y, Scholl V, Loof T, Binder E, Pimonidazole adduct immunohistochemistry in the rat kidney: Khadzhynov D, Kawachi H, Shimizu F, Diekmann F, Neumayer detection of tissue hypoxia. Methods Mol Biol 466:161-74 HH, Peters H (2008): Low-dose mTOR inhibition by rapamycin attenuates progression in anti-thy1-induced chronic glomerulo- Paliege A, Rosenberger C, Bondke A, Sciesielski L, Shina sclerosis of the rat. Am J Physiol Renal Physiol 294:F440-9 A, Heyman SN, Flippin LA, Arend M, Klaus SJ, Bachmann S (2009): Hypoxia-inducible factor-2alpha-expressing interstitial Krämer S, Kron S, Wang-Rosenke Y, Loof T, Khadzhynov D, fibroblasts are the only renal cells that express erythropoi- Morgera S, Kawachi H, Shimizu F, Martini S, Neumayer HH, etin under hypoxia-inducible factor stabilization. Kidney Int. Peters H (2008): Rosuvastatin is additive to high-dose candesartan 77:312-8 in slowing progression of experimental mesangioproliferative glomerulosclerosis. Am J Physiol Renal Physiol 294:F801-11 Gadau J*, Peters H* (* equal contribution), Kasner C, Kühn H, Krämer C, Castrop H, Bachmann S, Theilig F (2009): Câmpean V, Karpe B, Haas C, Atalla A, Peters H, Rupprecht Tubular involvement of volume retention in acute experimental H, Liebner S, Acker T, Plate K, Amann K (2008): Angiopoietin glomerulonephritis Kidney Int 75: 699-710 1 and 2 gene and protein expression is differentially regulated in acute anti-Thy1.1 glomerulonephritis. Am J Physiol Renal Krämer S, Binder E, Loof T, Wang-Rosenke Y, Khadzhynov Physiol 294:F1174-1184 D, Budde K, Neumayer HH, Peters H (2009): The lymphocyte migration inhibitor FTY720 attenuates experimental hyperten- Schneider M, Liefeldt L, Slowinski T, Peters H, Neumayer HH, sive nephropathy. Am J Physiol Renal Physiol 297:F218-27 Morgera S (2008). Citrate anticoagulation protocol for slow extended hemodialysis with the Genius dialysis system in Morgera S, Schneider M, Slowinski T, Vargas-Hein O, Zuck- acute renal failure. Int J Artif Organs 31:43-8 ermann-Becker H, Peters H, Kindgen-Milles D, Neumayer HH (2009). A safe citrate anticoagulation protocol with variable Rosenberger C, Khamaisi M, Goldfarb M, Shina A, Shilo V, treatment efficacy and excellent control of the acid-base Zilbertrest F, Rosen S, Heyman SN (2008): Acute kidney injury status. Crit Care Med 37:2018-24 in the diabetic rat: studies in the isolated perfused and intact kidney. Am J Nephrol 28(5): 831-9 Kleeberg L, Morgera S, Jakob C, Hocher B, Schneider M, Peters H, Rötzer S, Müller C, Kaiser M, Fleissner C, Heider U, Babel N, Eibl N, Ulrich C, Bold G, Sefrin A, Hammer MH, Rosen- Neumayer HH, Sezer O (2009): Novel renal replacement strat- berger C, Reinke P (2008): Development of Kaposi’s sarcoma egies for the elimination of serum free light chains in patients under sirolimus-based immunosuppression and successful treat- with kappa light chain nephropathy. Eur J Med Res 18: 47-54 ment with imiquimod. Transpl Infect Dis 10(1): 59-62 Knebel F, Schimke I, Schroeckh S, Peters H, Eddicks S, Welker P,Krämer S, Groneberg D, Neumayer HH, Bachmann Schattke S, Brechtel L, Lock J, Wernecke KD, Dreger H, Gru- S, Amann K, Peters H (2008): Increased mast cell number bitz S, Schmidt J, Baumann G, Borges AC (2009). Myocardial in human hypertensive nephropathy. Am J Physiol Renal function in older male amateur marathon runners: assess- Physiol 295:F1103-9 ment by tissue Doppler echocardiography, speckle tracking, and cardiac biomarkers. J Am Soc Echocardiogr 22:803-9 Riad A,Van Linthout S, Mohra Z, Rütten H, Peters H, Schultheiss HP, Tschöpe C (2008). Role of pharmacological enhancement of eNOS in experimental diabetes mellitus. Diabetologia 51:2325-32

117 Harm Peters – NEPHROLOGY / MOLECULAR FIBROSIS AND HYPOXIA RESEARCH

Liefeldt L, Rylski B, Walcher F, Manhart J, Kron S, Wang- Martini S, Peters H, Böhler T, Budde K (2007): Current per- Rosenke Y, Paul M, Neumayer HH, Amann K, Peters H spectives on FTY720. Expert Opin Investig Drugs 16:505- (2009). Transgenic overexpression of endothelin-2 aggravates 518 moderately diabetic myocardiopathy in rats. Eur J Clin Invest 40:203-10 Martini S, Peters H (2007): Medical treatment in retroperito- neal fibrosis: The difficulty to provide good evidence in rare Zebger-Gong H, Kampmann J, Kong L, Sommer K, Akashi Y, diseases. J Urol 178, 143-144 Janke M, Krämer S, Peters H, Roigas J, Müller DN, Dragun D, Querfeld U (2009): Decreased Transplant Arteriosclerosis Heyman SN, Rosen S, Rosenberger C (2008): Renal paren- in Endothelial Nitric Oxide Synthase-deficient Mice. Trans chymal hypoxia, hypoxia adaptation, and the pathogenesis plantation 89:518-26 of radiocontrast nephropathy. Clin J Am Soc Nephrol 3(1): 288-96 37.Werth N, Beerlage C, Rosenberger C, Yazdi AS, Edel- mann M, Amr A, Bernhardt W, von Eiff C, Becker K, Schäfer Heyman SN, Khamaisi M, Rosen S, Rosenberger C (2008): A, Peschel A, Kempf VA (2010): Activation of hypoxia induc- Renal Parenchymal Hypoxia, Hypoxia Response and the ible factor 1 is a general phenomenon in infections with Progression of Chronic Kidney Disease. Am J Nephrol 28(6): human pathogens. PLoS One Jul 14;5(7):e11576 998-1006

Unger JK and Peters H (2008): Hepatitis B in chronic kidney Reviews & book chapters (2006-20010) disease: moving toward effective prevention. Kidney Int 2008;73:799-801 Gaedeke J, Peters H (2006): Pharmacological management of fibrotic renal disease. Expert Opin Pharmacother 7:377-86 Wang-Rosenke Y, Neumayer HH, Peters H (2008): NO signaling through cGMP in renal tissue fibrosis and beyond: Budde K, Schütz M, Glander P, Peters H, Waiser J, Liefeldt L, key pathway and novel therapeutic target. Curr Med Chem Neumayer HH, Böhler T (2006): FTY720 (fingolimod) in renal 15:1396-406 transplantation. Clin Transplant 20 Suppl 17:17-24 Heyman, SN, Rosen S, Rosenberger C (2009): Critical Goldfarb M, Rosenberger C, Shina A, Rosen S, Heyman SN assessment of experimental models of acute renal failure. In: (2006): A Role for Erythropoietin in the Attenuation of Con- Critical Care Nephrology, 2nd Ed; Ronco C, Bellomo R & trast Medium-Induced Acute Renal Failure in Rats? Renal Kellum J (Eds), Springer/Kluwer Acad Pub Failure 28: 345-350 Slowinski T, Schneider H, Peters H (2009): Akutes Nierenver- Rosenberger C, Rosen S, Heyman SN (2006): Renal Paren- sagen: Fragen und Probleme im intensivmedizinischen Alltag. chymal Oxygenation and Hypoxia Adaptation in Acute Kidney In: Akutes Nierenversagen bei Intensivpatienten, 1st Ed: Injury. Clin Exp Pharmacol Physiol 33: 980-988 Jörres A. Deutscher Ärzteverlag

Hampl H, Hennig L, Rosenberger C, Gogoll L, Riedel E, Peters H, Martini S (2008): Glomeruläre Erkrankungen (Über- Scherhag A (2006): Proven strategies to reduce cardiovascular setzung Lewis JB, Neilson EG) mortality in hemodialysis patients. Blood Purif 24(1): 100-6 In: Harrison Innere Medizin, 17 Ed. Zeitz M, Dietel M, Suttorp N; ABW-Wissenschaftsverlag Peters H, Neumayer HH, Gaedeke J (2006): Hochdruckthera- pie und frühe Nephroprotektion: eine hausärztliche Heraus- Martin DR, Semelka RC, Chapman A, Peters H, Finn PJ, Kalb forderung. Hausarzt- und Notfallmedizin 32:138-42 B, Thomsen H (2009): Nephrogenic systemic fibrosis versus contrast-induced nephropathy: risks and benefits of contrast- Peters H, Unger T (2007): Mast cells and the power of local enhanced MR and CT in renally impaired patients. J Magn RAS activation. Nephrol Dial Transplant 22:40-2 Reson Imaging 30:1350-6

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Heyman SN, Rosenberger C, Rosen S (2010): Experimental Heyman SN, Rosen S, Khamaisi M, Idée JM, Rosenberger ischemia-reperfusion: biases and myths - the proximal vs. C (2010): Reactive oxygen species and the pathogenesis of distal hypoxic tubular injury debate revisited. Kidney Int 77(1): radiocontrast-induced nephropathy. Invest Radiol 45(4): 9-16 188-95

General information Third party funding (2006-2010)

Project leader Project title Sponsor Period Theilig F. Zelluläre Translokation tubulärer Membrant- Deutsche Forschungsgemein- 2006-2008 Peters H. ransporter bei Proteinurie“ Forschergruppe schaft, Bonn 667, Teilprojekt 10 Peters H. „Wachstumsfaktor-Rezeptor-Inhibition und Deutscher Akademischen 2006 renale Progression“ (Dmytro Khadzhynov, Austauschdienst, Bonn A/06/09454) Peters H. Teilprojekt: „Glomerulonephritis-Register“ Deutsche Forschungsgemein- 2006-2008 Budde K. Forscherverbundes „Open schaft, Bonn Nephrology Sciences Center“ (OpEN.SC). (AZ Inst/10692/1-1) Peters H. “Cardiovascular NO/cGMP signaling in Marie Curie, Early Stage Train- 2006-2010 renal insufficiency“ CARDIOVASC ing, Brüssel, EU (EU-Nr. 020268) Peters H. Nephrologische Forschung Sonnenfeld-Stiftung Berlin 2006-2012 Rosenberger C. Teilprojekt: “HIF in renal biopsies” For- Deutsche Forschungsgemein- 2007-2009 scherverbund „Open Nephrology Sciences schaft, Bonn Center“ (OpEN.SC). (AZ Inst/10692/1-2) Peters H. „AT2 Agonismus und Progression von chro- Deutscher Akademischen 2009-2011 nischer Anti-Thy1-induzierter Nierenfibrose“ Austauschdienst, Bonn (Anna Abadyjan, A/08/77466) Theilig F. „Glomerulonephritis, tubuläre Endozytose Deutsche Forschungsgemein- 2009-2011 Peters H. und Proteinurie“ Forschergruppe 667, schaft, Bonn Teilprojekt 8: Peters, H. Teilprojekt: „Glomerulonephritis-Register“, Deutsche Forschungsgemein- 2009-2011 Budde K. Forscherverbund „Open Nephrol- schaft, Bonn ogy Sciences Center“ (OpEN.SC). (AZ Inst/10692/1-2) Peters H. Experimentelle nephrologische Forschung Industrie, verschiedene 2006-2010 Peters H. Klinische nephrologische Forschung Industrie, verschiedene 2006-2010

119 Harm Peters – NEPHROLOGY / MOLECULAR FIBROSIS AND HYPOXIA RESEARCH

Awards

2006 Best Poster Award. ISN Forefronts Conference “Endothelial Biology and Renal Disease”, New York; USA. Harm Peters

2006 Rainer Greger - Promotionspreis, Gesellschaft für Nephrologie Yingrui Wang-Rosenke

2006 Georg Haas-Doktorandenpreis des Verbandes Deutscher Nierenzentren, DDnÄ e.V. Yingrui Wang-Rosenke

2007 Hans-U. Zollinger-Preis der Gesellschaft für Nephrologie Harm Peters

2007 „Lehrbär“ für beste Unterrichtsveranstaltung, Reformstudiengang Charité Harm Peters

2008 Best Poster Presentation. Annual Meeting of the European Council for Cardiovascular Research; Nice, France Bianca Grosz

2010 Best Poster Presentation. Annual Meeting of the European Council for Cardiovascular Research; Nice, France Tanja Loof

120 Axel Pries – PHYSIOLOGY

Head of the group

Prof. Dr. med. Axel R. Pries

Curriculum Vitae: From 1973 to 1980 Axel Pries studied medicine at the medical school University of Cologne, and also worked on his doctoral thesis on ‘Model studies on phase separation at a capillary orifice’. He finished his studies with the approbation as medical doctor in 1980 and his doctoral thesis was ranked ‘summa cum laude’. In the following years he worked as postdoctoral fellow at the Institute for Physiology, University of Cologne and Free University of Berlin. After a positions as lecturer and associate professor, he had an appointment in 1997-1998 at the Institute of Anesthesiology of German Heart Center Berlin. In 1998, hebecame full professor at the Department of Physiology of the Free University Berlin. Since 2001, he is Professor of Physiology and head of the Institute for Physiology (one of three physiological institutes) of the Charité, Berlin. His main tasks involve organization, teaching and research. He is also involved in work for national and international scientific organisations, including the ESC (European Society for Cardiology) and the ESM (European Society for Mirocirculation). In the ESC, he is chairing the council for basic cardiovascular science (CBCS) while in the ESM, he is serving as general secretary. Scientifically, his key areas of interest include microcirculation, and tumor vasculature, blood rheology, vascular adaptation, angiogenesis, ischemia/ reperfusion and the endothelial surface. Recently, he worked on the mechanisms preventing maldistribution and shunting in microvascular networks related to the differences in normal and tumor vascular beds (Pries et. al Plos Comp Biol, 2009 and Nat Rev Cancr 2010).

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Members of the group

Office Technicians Bünsch, Brigitte Hofmann, Eveline Becker, Angela Marruhn, Cornelia Wisniewski, Susanne Himmelsbach, Bärbel Hoffmann, Björn Research group leaders Noske-Reimers, Renate Gunga, Hanns-Christian Prof. Dr. med. Plog, Sylvia Habazettl, Helmut Prof. Dr. med. Höpfner, Michael PD Dr. rer. nat. Students Kübler, Wolfgang Prof. Dr. med. Phillip Zöller Munz, Barbara Prof. Dr. rer. nat. Christian Hoffmann Preissner, Robert PD Dr. rer. nat. Bernd Vorderwülbecke Siegel, Günther Prof. Dr. med Julian Maroski Zakrzewicz, Andreas Dr. med Margret Hohberg Sven Chlench Scientists Christoph Glösenkamp Berger, Felicitas Dr. rer. nat. Felix Landmann Ermilov, Eugeny Dr. rer. nat. Julian Lenk Hoffmann, Julia Bianca Nitzsche Klippel, Nina Kera Westphal Márki, Alex Dr. med. Dr. rer. nat. Florian Thilo Neye, Nils Nickles, Hannah Nitzsche, Bianca Opatz, Oliver Dr. med. Samapati, Rudi Dr. med. vet. Reglin, Bettina Dr. rer. medic. Schlabs, Thomas Stahn, Alexander Supe, Steffi

122 Axel Pries – PHYSIOLOGY

Summary

Integrative Physiology and cular networks was established (Pries et al. Nature Organ Perfusion Reviews Cancer 2010). The group of W. Kübler uses intravital microscopy with advanced imaging tech- The research profile of the section ‘Integrative Phys- niques together with molecular biology to investi- iology and Organ Perfusion’ including the groups gate relevant pathophysiological mechanisms in the Pries, Kübler, Gunga, Kunz, Preissner und Höpfner alveolo-capillary units of the lung. integrates physiological research from the molecular level to human physiology according to the motive of The exponents for investigation of human physiology the American Physiological Society „Integrating Life in the spectrum,are the groups Gunga and Kunz. Sciences from Molecule to Organism“. The devel- H.-C Gunga and his coworkers are longstanding opment of integrative concepts is a prerequisite players in the field of integrative regulatory phe- for the fast and efficient translation of mechanistic nomena during space flight (space medicine) and knowledge back into the complex environment of in extreme environments. The concept „from Mol- human physiology and pathophysiology. For this ecule to Organism“. is completed by the integrative kind of research, there is also a high demand by research of circadian phenomena and sleep disor- extra-university institutions. ders in healthy people and in patients by the group Kunz. Within the section, the group Preissner is located at the molecular end of the spectrum, investigating molecular interactions with bioinformatic approaches. Some of the studies are performed together with the group of M. Hoepfner which is interested in the molecular and functional events in Tumor cells and the influence of growth factors and growth factor inhibitors. In the investigation of tumor microvasculature, the cooperation of the groups Hoepfner and Pries has added the techniques of intravital microscopy and computer simulation. With this approach, the importance of conducted responses for the characteristics of tumor microvas-

123 Axel Pries – PHYSIOLOGY

Zusammenfassung

Integrative Physiologie flächendeckend bearbeitet werden. Daher ist der und Organperfusion inhaltliche Zusammenhang der beteiligten Arbeits- gruppen von besonderer Bedeutung. Am moleku- Das übergreifende Forschungsprofil der Abteilung laren und submolekularen Ende des Integrationsbo- „Integrative Kreislauf-Physiologie und Organperfu- gens angesiedelt ist die Arbeitsgruppe Preissner, die sion’“ mit den Arbeitsgruppen Pries, Kübler, Gunga, mit bioinformatischen Methoden molekulare Interak- Kunz, Preissner und Höpfner ist die Integration phy- tionen untersucht. Basierend auf entsprechenden siologischer und pathophysioloigscher Grundlagen- Datenbanken und Suchalgorithmen erlaubt dieser forschung von der molekularen Ebene bis hin zum Ansatz die z.B. Identifikation geeigneter Kandiaten- Menschen in Anlehnung an das Leitmotiv der Ame- moleküle für gezielte pharmakologische Beeinflus- rikanischen Gesellschaft für Physiologie „Integrating sung relevanter zellulärer Abläufe. Ein Schwerpunkt Life Sciences from Molecule to Organism“. liegt hierbei in der Suche nach kleinen molekularen Inhibitoren von zellulären Wachstumsfaktor-Rezep- Die Entwicklung leistungsfähiger molekularbiolo- toren und von nachgeschalteten Signalwegen, z.B. gischer Methoden und die Erfolge der biomedizi- für die Tumortherapie. Diese Studien werden in enger nischen Forschung in molekularen und zellulären Zusammenarbeit mit der AG Höpfner durchgeführt, Bereichen haben zu einem enormen Erkenntniszu- die primär an zellulären Modellen die Wirksamkeit wachs über molekulare Mechanismen geführt. Dies unterschiedlicher antioangionetischer Tumorzell- macht die Entwicklung integrativer Konzepte zum Inhibitoren untersucht. Zusammenwirken der molekularen Einzelmecha- nismen im gesunden oder kranken Individuum oder Eine wesentliche Erweiterung der methodischen Organ dringend erforderlich. Integrative grundlagen- Möglichkeiten für die AG Höpfner ergibt sich wiede- medizinische Forschung ist eine Voraussetzung für rum aus der Nutzung der intravitalmikroskopischen einen schnellen und erfolgreichen Wissenstransfer Ansätze der AG’s Pries und Kübler. So kann z.B. das von der molekularen/zellulären Ebene zurück in Wachstum und die Vaskularisierung unterschiedli- das komplexe Gesamtsystem und die Lösung der cher Tumorzelllinien mit und ohne Applikation der ursprünglichen Fragestellungen. Die Bedeutung zu testenden Substanzen in situ in der Rücken- integrativer, anwendungsorientierter Forschungs- hautkammer der Maus untersucht werden. In der ansätze wird auch durch die erhebliche Nachfrage Arbeitsgruppe Pries werden zur Analyse derartiger gesellschaftlicher Institutionen nach konkreten Pro- Daten und zur Entwicklung weitergehender Kon- blemlösungen und nach langfristiger wissenschaft- zepte mathematische Simulationsverfahren einge- licher Kooperation deutlich. setzt. Mit Hilfe dieser Ansätze können Hypothesen generiert werden, die dann experimentell untersucht Selbstverständlich kann mit einer begrenzten Zahl werden können. So wurde in den letzten Jahren die von Arbeitsgruppen ein derartiger Ansatz nicht Bedeutung intravaskulärer Kommunikation über

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Gap-Junctions für Funktionsfähigkeit mikrovasku- Der Schwerpunkt der Arbeitsgruppe Gunga am lärer Grefäßnetzwerke untersucht. In einem Artikel Physiologischen Institut der Charité in Dahlem liegt in Nature Reviews Cancer (Pries et al. 2010) wird auf dem Gebiet integrative Humanphysiologie, im die Hypothese ausgestellt, dass eine Fehlfunktion speziellen die Anpassungen des Herz-Kreislauf dieser Kommunikation wesentlich an der Maldistri- systems, der Thermoregulation sowie der Chro- bution der Perfusion in Tumoren beteiligt ist. Eine nobiologie unter klinischen, labor- und feldphysio- Verbesserung dieser Funktion wäre demnach ein logischen extremen Bedingungen. Die Projekte Ansatz um den Effekt pharmakologischer Therapien werden finanziell bzw. logistisch gefördert durch zu erhöhen. das Bundesministerium für Wirtschaft (BMWi)/Deut- sches Zentrum für Luft- und Raumfahrt (DLR), das Die quantitative Analyse der funktionellen Integra- Alfred-Wegener-Institut für Polarforschung in Bre- tion relevanter Mechanismen auf vaskulärer Ebene merhaven, die European Space Agency (ESA), die im Organismus mit Hilfe der quantitativen Intravi- National Space Administration (NASA), das Karls- talmikroskopie ist auch für andere Fragestellungen ruher Institute of Technology (KIT), das österreichi- von zunehmender Bedeutung und stellt eine zen- sche Bundesheer und die deutsche Bundeswehr, trale Kompetenz der Gruppen Pries, Kübler und den Deutschen Schwimmverband, die Deutsche Habazettl dar. Durch eine deutliche Verbesserung Lebensrettungsgesellschaft (DLRG) sowie die Hum- der Meßmöglichkeiten dieses Ansatzes haben sich boldt- und Nathan-Zuntz-Stiftung. Mit integrativer neue Anwendungsfelder in der Untersuchung funk- Forschung auf der Ebene der Humanphysiologie tioneller, zellulärer und molekularer Mechanismen auch an Patienten durch die AG Kunz, die sich mit erschlossen. Hierzu haben die genannten Arbeits- circadianen Rhythmen und ihre Beeinflussung durch gruppen durch Entwicklung eigener Tiermodelle, Lichteposition beschäftigt, vervollständigt sich der Meßverfahren und Bildanalysesoftware beigetragen. Bogen „from Molecule to Organism“.

Der Arbeitsschwerpunkt der Gruppen Pries, Kübler und Habazettl liegt im Bereich mikrovasku- lärer Adaptationsvorgänge, der pulmonalen End- strombahn und Untersuchungen der Mikrozirkula- tion am Menschen.

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Research Projects

1. The shunt problem: control of functional shunting in normal and tumour vasculature

Project leader Axel R Pries Coworkers Micheal Höpfner, Ferdiand Le Noble

Researchers know that the blood vessels in tumors are poor in distriubuting oxygen and drugs, and comparing vascular networks in tumors with normal tissues may suggest new approaches to improving drug delivery in tumors. Axel Pries, Michael Höpfner, and Ferdinand le Noble of the CCR, Mark Dewhirst of Duke University Medical Center and Timothy Secomb of the University of Arizona have offered a new hypothesis. Their argument, published in Nature Reviews Cancer1, is that impaired communication along vessels in tumor microvascular networks can lead to shunting of blood away from some regions of the tumor and the generation of hypoxic regions (blue and purple in the graph)

There are always both long pathways and shortcuts from the arteries to the veins. These shortcuts in tumors tend to grow in diameter, shunting the flow away from the long pathways.

One line of attack would be to give a VEGF antagonist, to improve communication and reduce shunting, which enables better delivery of anti-tumor therapies. It is assumed that approaches targeted at improving gap Anti-angiogenic treatments (treatments that inhibit the growth of new vessels) may help to restore communication and junction communication may be able to take advantage improve flow distribution in tumor vessels. of this concept in a more predictable manner. In detail, the communication along blood vessel walls, which is needed to coordinate the distribution of blood flow was (1) Pries AR, Hopfner M, Le Noble F., Dewhirst MW, Secomb investigated. Gap junctions connect the endothelial cells of the vessels.It is theorized that the gap junctions do not exist or TW. The shunt problem: control of functional shunting in don’t work well in tumors, which leads to loss of communica- normal and tumour vasculature. Nat Rev Cancer 2010 August tion. The vessel structure in the tumor reflects this breakdown 1;10(8):587-93. of communication.

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2. Metabolic control of microvascular structure: Where are the oxygen sensors?

Project leader Bettina Reglin Coworkers Axel R Pries, Tim Secomb

Generation and maintenance of functional vascu- sel walls or in RBC. Resulting network structures were lar networks requires structural adaptation of vessel analyzed with regard to tissue oxygenation and to the diameters in response to the metabolic state of tissue. difference between estimated blood flow velocities The exact mechanism of this response is not known and corresponding experimental data (velocity error). but may involve the release of vasoactive substances Wall signaling led to the highest hemodynamic simi- in response to low oxygen by tissue (“tissue signaling”, larity and low oxygen deficit. Tissue signaling also led e.g. CO2, adenosine), by vessel walls (“wall signaling”, to low oxygen deficit, but resulted in higher velocity e.g. prostaglandins, adenosine) and/or by red blood error and less realistic diameters. RBC signaling led to cells, RBCs (“RBC signaling”, e.g. ATP and NO). widespread hypoxia, unrealistic velocity distributions and shrinkage of small vessels. The results suggest that the metabolic signal for structural adaptation of vessel diameters originates mainly in vessel walls. Such data are used to develop an integrated view of control of vascular structure and function, including short term changes of vessel diameter effected by vascular smooth muscle and tone (right), long term changes of vessel diameter and wall mass by structural adaptation (middle), and changes in vessel number by elimination of unnecessary vessels by pruning or generation of new vessels by the sprouting and splitting modes of angiogenesis left).

In this project, the goal was to test the capability of these three modes of oxygen sensing to adequately control vascular diameters and tissue oxygenation. A theoretical model of structural diameter adaptation based on experimental data on microvascular network structure and hemodynamics was used considering oxygen-dependent metabolic signals in tissue, in ves-

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3. Regulation of microvascular Permeability by Sphingolipids

Project leader Wolfgang Kübler Coworkers Yang Yin Funding DFG- Focus “Sphingolipids“ Cooperations Institut für Pharmakologie und Toxikologie, RWTH Aachen

Platelet-activating factor (PAF) increases pulmonary membrane fraction. Endthelial NOS was quantitatively micrivascular permeability within minutes. This effect assessed by in situ fluoreszenz-microscopy. Caveo- equally depnds on activation of cyclooxygenase and lae, isolated 10 min after PAF stimulation showed an acicd sphingomyelinase (ASM). Correspondingly, increase in Caveolin-1, eNOS und ASM activity. PAF pharmacological inhibition of ASM by imipramin in parralel reduced endothelial NO synthesis. These reduces acute lung injury in a number of preclinical effects could be reproduced by direct lung-perfusion animal models. However, the mechanisms underlying with ASM,while inhibition of the ASM signal pathway this new signal transduction pathway are still unre- by imipramin, D609 or dexamethason blocked PAF- solved. induced increase in Caveolin-1 and eNOS in caveolae, Caveolae are plasma domains which are speciffically as well as the simultaneous decline in NO production, rich in sphingomyelin (the substrate of ASM), and and the generation of a PAF-induced lung oedma.. they contain Caveo- Restitution of NO levels by application of exogene- lin-1 (cav1) which ous NO donors reduce PAF-induced microvascular binds and blocks barrier defect. endothelial NO synthase (eNOS). There- Thus PAF perturbates the microvascular barrier result- fore we analyzed the ing in lug oedema. The mechanism includes activation relationship between of ASM leading to ioncreased levels of Caveolin-1 and ASM, cav1 und eNOS eNOS in caveoli. This results in reduced endothelial in the context of PAF- NO synthesis, which contributes to microvascular bar- induced lung oedema rier defect. in the isolatd perfused Yang Y, Yin J, Baumgartner W, Samapati R, Solymosi EA, mouse and rat-lung. Reppien E, Kuebler WM*, Uhlig S*: Platelet-activating factor Caveolae were iso- reduces endothelial NO production - role of acid sphingomy- lated from pulmonary elinase. Eur Respir J 2010;36:417-27 (*shared senior author- endothelial cells after ship) sowie in Kuebler WM, Yang Y, Samapati R, Uhlig S: Vascular barrier regulation by PAF, ceramide, caveolae, and marking with silica NO - an intricate signaling network with discrepant effects beads preparing the in the pulmonary and systemic vasculature. Cell Physiol Bio- detergent-resistant chem 2010;26:29-40.

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4. Novel antiangiogenic compounds with antitumor activity for innovative treatment approaches in cisplatin resistant urologic tumors

Project leader Michael Höpfner Coworkers Bianca Nitzsche, Christoph Gloesenkamp, Björn Hoffmann Funding Stiftung Urologische Forschung Berlin Collaborators Prof. Dr. Mark Schrader, Department of Urology, University of Ulm Prof. Dr, Matthias Ocker, Institute of Surgical Research, Philipps University Marburg

Testicular germ cell tumor (TGCT) is the most common tion with platinum compounds in both platinum sensitive cause of death from solid tumors in young men and and –resistant TGCT cell lines. For in vivo evaluation of especially for platinum-refractory TGCT patients novel the antiangiogenic effects of the new compounds the so treatment approaches are urgently needed. As angio- called CAM assay (chicken chorioallantoic membrane genesis is essential for the development, growth and assay) was employed. 48 h incubation of the CAM of metastases of tumors we hypothesised that targeting fertilized chicken eggs with HP-2 or HP14 resulted in angiogenic growth factor receptor signalling pathways a pronounced arrest in microvessel formation (Fig. 1). may be a promising approach for novel treatment Moreover TGCT cells inoculated onto the CAM were approaches of platinum resistant testicular germ cell treated with the HP-14. As depicted in Figure 2 a marked tumors (TGCT). Two VEGFR-blocking antiangiogenic inhibition of TGCT growth was observed upon treatment. compounds, HP-2 and HP-14, that were recently iden- The novel compounds effectively suppressed the growth tified by our group, were evaluated for their suitability to of both platinum- sensitive as well as –resistant TGCTs. inhibit the formation of tumor microvasculature as well Together, these data suggest that HP-2 and HP-14 may as the growth of normal and platinum-resistant TGCTs in be interesting new drugs for targeted therapy of urologic vitro and in vivo. Proliferation assays revealed the antine- cancers, particularly for those being resistant to the usu- oplastic effects of HP-2 and HP-14 alone or in combina- ally successful platinum-based interventions.

HP-14 induced inhibition of tumor growth of TGCT cells on the chicken chorioallantoic membrane (CAM). Cells were transplanted onto the CAM and treated HP-2 and HP-14 induced vasodegeneration of with HP-14 for 96h. Left side: Grown tumors were photographed (x6 the developing CAM. Upper panel depicts CAMs magnification, representative images) and tumor volumes were calcu- before treatment. In the lower panel effects of 48 lated. Right side: Growth inhibition by HP-14 is depicted as the percent- h of treatment with PBS, HP-2 (10µM) or HP-14 age of growth compared to those of untreated controls (means of n=3 (10µM) are shown. (a = artery and v = vein). independent experiments)

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5. Connecting the regulation of angiogenesis to shear stress – a new role for ADAMTS1

Project leader Andreas Zakrzewicz Coworkers Margret Hohberg, medical student Christian Hoffmann, medical student Sven Chlench, physician Luis Da Silva-Azevedo, PhD Axel R. Pries External cooperations Robert Lehmann

ADAMTS1 inhibits capillary sprouting, and since blocked by a knockdown of ADAMTS1. Nonperfused capillary sprouts do not experience the shear stress capillary sprouts in rat mesenteries stained negative caused by blood flow, this study undertook to clarify for ADAMTS1, while vessels in the microcirculation that the relationship between shear stress and ADAMTS1. had already experienced blood flow yielded the oppo- It was found that endothelial cells exposed to shear site results. The shear stress-dependent expression stress displayed a strong upregulation of ADAMTS1, of ADAMTS1 in vitro was therefore also demonstrated dependent upon both the magnitude and duration of in vivo and thereby confirmed as a mechanism con- their exposure. Investigation of the underlying path- necting blood flow with the regulation of angiogenesis. ways demonstrated involvement of phospholipase C, phosphoinositid 3-kinase and nitric oxide. Forkhead box protein O1 was identified as a likely inhibitor of the system, as its knockdown was followed by a slight increase in ADAMTS1 expression. In silico prediction displayed a transcriptional binding site for forkhead box protein O1 in the promotor region of the ADAMTS1 gene, as well as sites for nuclear factor 1, SP1 and AP 1. The anti-angiogenic effects of ADAMTS1 were attributed to its cleavage of thrombospondin 1 into a 70-kDa fragment, and a significant enhancement of this fragment was indeed demonstrated by immunoblotting shear stress-treated cells. Accordingly, scratch wound closure displayed a slowdown in con- Sprouting capillaries (visualized by staining with ditioned medium from shear stress-treated endothe- GS-IB4) in rat mesenteries stain positive for Angi- lial cells, an effect that could be completely blocked opoietin-2 but negative for ADAMTS1. by a knockdown of thrombospondin 1 and partially

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6. Possible atheroprotective effects of hyaluronic acid from the endothelial surface layer and its modulation by shear stress

Project leader Andreas Zakrzewicz, Günter Siegel Coworkers Julian Maroski, medical student Bernd Vorderwülbecke, medical student Eugeny Ermilov, PhD Axel R. Pries External cooperations M. Malmsten, Uppsala University Biomedical Center, Uppsala

The endothelium is covered with a relatively thick (0.5 to oscillating (“atheroprone”) flow did not induce any dif- 1 µm) surface layer (ESL) which contains hyaluronic acid. ferences towards static control cultures. Nanoplaque In arteries, there is a correlation between disturbed blood formation was measured by in situ ellipsometry. The flow (reduced wall shear stress), reduction of the ESL and atherogenic lipoprotein fraction VLDL/IDL/LDL forms a predilection for arteriosclerosis. This research project is ternary complexes (nanoplaques) on proteoheparan designed to investigate if endothelial hyaluronan synthases sulfate-coated surfaces in a calcium dependant man- (HAS) are regulated by shear stress and if hyaluronic acid ner. Hyaluronic acid proved to be similarly effective in has a role to prevent nanoplaque formation. inhibiting this ternary complex formation as HDL. So far, human umbilical vein endothelial cells, were These data support the hypothesis that the synthesis exposed to distinct flow conditions in a cone-and- of hyaluronic acid by endothelial cells contributes to plate system, and analysed for hyaluronan synthase 2 shear stress-dependent athero-protective mecha- expression by real-time RT-PCR and immunoblotting, nisms especially during the initiation of arteriosclerosis as well as for hyaluronan by ELISA, both in cell cul- by blocking ternary complex formation and do thus ture supernatant and in a cell surface derived fraction. encourage further investigation. Thereby hyaluronan synthase 2 and hyaluronan were found to be shear stress-dependently increased via the PI3K-Akt-pathway. Especially atheroprotective flow was found to induce both – enzyme and Monomolecular coating of a methylated silica surface with proteoheparan sulfate molecules and their interaction both with Ca2+ and Na+ ions and lipoprotein particles in blood substitute solution (not to scale). hyaluronan – effectively, while low and Siegel et al.: Biosens Bioelectron.18 (2003) 635-647; Siegel et al.: Biosens Bioelectron.24 (2009) 1512-1517

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7. Thermoregulation and cardiovascular adaptation in extreme environments

Project leader Hanns-Christian Gunga, PhD, MD, Dipl. Geol. Coworkers Oliver Opatz, Alexander Stahn, Mathias Steinach, MD, Andreas Werner, MD, LtCol MC

Space (Micro-g) Data recorder (Flashmaster), temperature satellites The current research projects of the group are structured and two double sensors. To compare the size of the in studies under simulated (immersion, isolation, bed technical systems, a 1 Euro coin is depicted. rest, head-down-tilt) and real (parabolic flights, Inter- national Space Station ISS) micro-g conditions. The Cold climate and hypothermia immersion-, isolation-, bed rest- and head-down-tilt The studies in this field of research are divided into two studies under principal- and co-investigator guidance of main center points: On the one hand field physiological the group take place in close cooperation with the Ger- studies in the Georg Neumayer and Concordia Stations man Federal Armed Forces, the IMBP, ESA and NASA in in the Antarctic (cold climate), on the other hand clinical Manching (Flight-Medical Research Institute of the Ger- investigations on thermoregulation under moderate and man Air Force), in Moscow (MARS 500), Berlin (Berlin deep hypothermia during heart transplantations in the Bed Rest Study) and in Galveston (Mood Bed Study, German Heart Institute Berlin, the latter again in coop- Texas). With the aid of the double sensor for non-invasive eration with the Pries group (Opatz et al. Resuscitation, determination of the body core temperature at the head 2010). In the Antarctic, changes in body composition (Gunga et al., J Thermal Biology 33: 297-307, 2008), developed in close cooperation with the company Drägerwerke in Lübeck in the course of seven years of intensive research work (Figure 1), the effectiveness of protective suits as well as the effect of long-time isolation, confinement and immobilization on the temperature regulation and chronobiology of the human organism are investigated. Similar problems are addressed on the International Space Station ISS by current studies on astronauts as well as in parabolic flights with short- time micro-g expositions. The latter studies are complemented by infrared photographs and Laser Doppler method for the recording of changes in microcirculation. The projects mentioned are carried out in close cooperation with the groups of Pries/Habazettl and Kunz. Joint continuative studies are planned for 2011 in the human centrifuge of the DLR in Cologne-Porz.

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(muscle mass, fat mass, body water), various hormones of the German Federal Armed Forces at Königsbrück, and circadian rhythm of the core temperatures under in order to elucidate the effects of hypobaric hypoxia long-time isolation and confinement are analyzed at sea on disagreeable thermic sensations at the head. These level (Georg Neumayer Station) and in comparison also investigations are done in cooperation with the Flight at an elevation of 3,800 m (Concordia Station). Medical Service of the German Federal Armed Forces. Hyperbaric studies are carried out in cooperation with Barophysiology the German Life Saving Association (DLRG) to enable a This research area as well is structured in two parts: hypo- continuous measurement of the body core temperature and hyperbaric physiology. Hypobaric studies are carried of divers. The development of this sensor is based upon out under laboratory conditions in the height chamber the further development of the double sensor.

Publications (2006-2010)

Akram A, Han B, Masoom H, Peng C, Lam E, Litvack L, Bai Hohberg M, Knochel J, Hoffmann CJ, Chlench S, Wunderlich X, Shan Y, Hai T, Batt J, Slutsky AS, Zhang H, Kuebler WM, W, Alter A, Maroski J, Vorderwulbecke BJ, Silva-Azevedo Haitsma JJ, Liu M, dos Santos CC (2010): Activating tran- LD, Knudsen R, Lehmann R, Fiedorowicz K, Bongrazio M, scription factor 3 confers protection against ventilator-induced Nitsche B, Hoepfner M, Styp-Rekowska B, Pries AR, Zakrze- lung injury. Am J Respir Crit Care Med 182(4):489-500 wicz A (2010): Expression of ADAMTS1 in endothelial cells is induced by shear stress and suppressed in sprouting capillar- Boning D, Maassen N, Pries A (2010): The Hematocrit Para- ies. J Cell Physiol dox - How Does Blood Doping Really Work? Int J Sports Med Epub ahead of print Kerem A, Yin J, Kaestle SM, Hoffmann J, Schoene AM, Singh B, Kuppe H, Borst MM, Kuebler WM (2010): Lung endothe- Buschmann I, Pries A, Styp-Rekowska B, Hillmeister P, lial dysfunction in congestive heart failure: role of impaired Loufrani L, Henrion D, Shi Y, Duelsner A, Hoefer I, Gatzke Ca2+ signaling and cytoskeletal reorganization. Circ Res N, Wang H, Lehmann K, Ulm L, Ritter Z, Hauff P, Hlush- 106(6):1103-16 chuk R, Djonov V, van Veen T, Le Noble F. (2010): Pulsatile shear and Gja5 modulate arterial identity and remodeling Kuebler WM, Yang Y, Samapati R, Uhlig S (2010): Vascular events during flow-driven arteriogenesis. Development barrier regulation by PAF, ceramide, caveolae, and NO - an 137(13):2187-96 intricate signaling network with discrepant effects in the pulmonary and systemic vasculature. Cell Physiol Biochem Fung YL, Kim M, Tabuchi A, Aslam R, Speck ER, Chow L, 26(1):29-40 Kuebler WM, Freedman J, Semple JW (2010): Recipient T lymphocytes modulate the severity of antibody-mediated Meissner S, Tabuchi A, Mertens M, Kuebler WM, Koch E transfusion-related acute lung injury (TRALI). Blood Epub (2010): Virtual four-dimensional imaging of lung parenchyma ahead of print by optical coherence tomography in mice. J Biomed Opt 15(3):036016 Habazettl H, Athanasopoulos D, Kuebler WM, Wagner H, Roussos C, Wagner PD, Ungruhe J, Zakynthinos S, Vogiatzis Nickles HT, Kuebler WM (2010): Take my breath away: I (2010): Near-infrared spectroscopy and indocyanine green perivascular fluid cuffs impair lung mechanics. Crit Care derived blood flow index for noninvasive measurement of mus- Med 38(6):1494-6 cle perfusion during exercise. J Appl Physiol 108(4):962-7

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Nitzsche B, Gloesenkamp C, Schrader M, Ocker M, Preissner Zhou C, Chen H, King JA, Sellak H, Kuebler WM, Yin J, R, Lein M, Zakrzewicz A, Hoffmann B, Hopfner M (2010): Townsley MI, Shin HS, Wu S (2010): Alpha1G T-type calcium Novel compounds with antiangiogenic and antiproliferative channel selectively regulates P-selectin surface expression in potency for growth control of testicular germ cell tumours. Br pulmonary capillary endothelium. Am J Physiol Lung Cell J Cancer 103(1):18-28 Mol Physiol 299(1):L86-L97

Preissner S, Kroll K, Dunkel M, Senger C, Goldsobel G, Alter A, Schmiedeck D, Fussnegger MR, Pries AR, Frees- Kuzman D, Guenther S, Winnenburg R, Schroeder M, meyer WB, Zakrzewicz A (2009): Angiopoietin-1, but not Preissner R (2010): SuperCYP: a comprehensive database platelet-derived growth factor-AB, is a cooperative stimulator on Cytochrome P450 enzymes including a tool for analysis of vascular endothelial growth factor A-accelerated endothe- of CYP-drug interactions. Nucleic Acids Res 38(Database lial cell scratch closure. Ann Vasc Surg 23(2):239-45 issue):D237-D243 Bauer RA, Gunther S, Jansen D, Heeger C, Thaben PF, Pre- Pries AR, Hopfner M, Le Noble F., Dewhirst MW, Secomb TW issner R (2009): SuperSite: dictionary of metabolite and drug (2010): The shunt problem: control of functional shunting in nor- binding sites in proteins. Nucleic Acids Res 37(Database mal and tumour vasculature. Nat Rev Cancer 10(8):587-93 issue):D195-D200

Sander PM, Christian A, Clauss M, Fechner R, Gee CT, Bergmann F, Breinig M, Hopfner M, Rieker RJ, Fischer L, Griebeler EM, Gunga HC, Hummel J, Mallison H, Perry SF, Kohler C, Esposito I, Kleeff J, Herpel E, Ehemann V, Friess Preuschoft H, Rauhut OW, Remes K, Tutken T, Wings O, Wit- H, Schirmacher P, Kern MA (2009): Expression pattern and zel U (2010): Biology of the sauropod dinosaurs: the evolution functional relevance of epidermal growth factor receptor and of gigantism. Biol Rev Camb Philos Soc cyclooxygenase-2: novel chemotherapeutic targets in pancreatic endocrine tumors? Am J Gastroenterol 104(1):171-81 Sodian R, Schaefermeier P, begg-Zips S, Kuebler WM, Shak- ibaei M, Daebritz S, Ziegelmueller J, Schmitz C, Reichart B Bickenbach J, Dembinski R, Czaplik M, Meissner S, Tabuchi (2010): Use of human umbilical cord blood-derived progenitor A, Mertens M, Knels L, Schroeder W, Pelosi P, Koch E, Kue- cells for tissue-engineered heart valves. Ann Thorac Surg bler WM, Rossaint R, Kuhlen R (2009): Comparison of two in 89(3):819-28 vivo microscopy techniques to visualize alveolar mechanics. J Clin Monit Comput 23(5):323-32 Valois CR, Braz JM, Nunes ES, Vinolo MA, Lima EC, Curi R, Kuebler WM, Azevedo RB (2010): The effect of DMSA-func- Bueltmann M, Kong X, Mertens M, Yin N, Yin J, Liu Z, Koster tionalized magnetic nanoparticles on transendothelial migra- A, Kuppe H, Kuebler WM (2009): Inhaled milrinone attenuates tion of monocytes in the murine lung via a beta2 integrin- experimental acute lung injury. Intensive Care Med 35(1):171-8 dependent pathway. Biomaterials 31(2):366-74 Da Silva-Azevedo L, Jahne S, Hoffmann C, Stalder D, Heller Von EJ, Gunther S, Preissner R (2010): Structural features M, Pries AR, Zakrzewicz A, Baum O (2009): Up-regulation and evolution of protein-protein interactions. Genome Inform of the peroxiredoxin-6 related metabolism of reactive oxygen 22:1-10 species in skeletal muscle of mice lacking neuronal nitric oxide synthase. J Physiol 587(Pt 3):655-68 Yang Y, Yin J, Baumgartner W, Samapati R, Solymosi EA, Reppien E, Kuebler WM, Uhlig S (2010): Platelet-activating Dunkel M, Schmidt U, Struck S, Berger L, Gruening B, factor reduces endothelial nitric oxide production: role of acid Hossbach J, Jaeger IS, Effmert U, Piechulla B, Eriksson R, sphingomyelinase. Eur Respir J 36(2):417-27 Knudsen J, Preissner R (2009): SuperScent--a database of flavors and scents. Nucleic Acids Res 37(Database Yin J, Kuebler WM (2010): Mechanotransduction by TRP issue):D291-D294 channels: general concepts and specific role in the vasculature. Cell Biochem Biophys 56(1):1-18

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Eisenreich A, Bogdanov VY, Zakrzewicz A, Pries A, Anto- Lipnicki DM, Gunga HC (2009): Physical inactivity and cogni- niak S, Poller W, Schultheiss HP, Rauch U (2009): Cdc2- tive functioning: results from bed rest studies. Eur J Appl like kinases and DNA topoisomerase I regulate alternative Physiol 105(1):27-35 splicing of tissue factor in human endothelial cells. Circ Res 104(5):589-99 Mecha DN, Styp-Rekowska B, Hinz B, Da Silva-Azevedo L, Pries AR, Zakrzewicz A (2009): Differential expression of Franz R, Hummel J, Kienzle E, Kolle P, Gunga HC, Clauss VEGFA, TIE2, and ANG2 but not ADAMTS1 in rat mesenteric M (2009): Allometry of visceral organs in living amniotes microvascular arteries and veins. Physiol Res 58(2):193-202 and its implications for sauropod dinosaurs. Proc Biol Sci 276(1662):1731-6 Mertens M, Tabuchi A, Meissner S, Krueger A, Schirrmann K, Kertzscher U, Pries AR, Slutsky AS, Koch E, Kuebler WM Fullbeck M, Gebhardt N, Hossbach J, Daniel PT, Preissner (2009): Alveolar dynamics in acute lung injury: heterogeneous R (2009): Computer-assisted identification of small-molecule distension rather than cyclic opening and collapse. Crit Care Bcl-2 modulators. Comput Biol Chem 33(6):451-6 Med 37(9):2604-11

Fullbeck M, Dunkel M, Hossbach J, Daniel PT, Preissner R Paris S, Wolgin M, Kielbassa AM, Pries A, Zakrzewicz A (2009): Cellular fingerprints: a novel approach using large- (2009): Gene expression of human beta-defensins in healthy scale cancer cell line data for the identification of potential and inflamed human dental pulps. J Endod 35(4):520-3 anticancer agents. Chem Biol Drug Des 74(5):439-48 Pries AR, Cornelissen AJ, Sloot AA, Hinkeldey M, Dreher MR, Gunga HC, Werner A, Stahn A, Steinach M, Schlabs T, Hopfner M, Dewhirst MW, Secomb TW (2009): Structural Koralewski E, Kunz D, Belavy DL, Felsenberg D, Sattler F, adaptation and heterogeneity of normal and tumor microvas- Koch J (2009): The Double Sensor-A non-invasive device to cular networks. PLoS Comput Biol 5(5):e1000394 continuously monitor core temperature in humans on earth and in space. Respir Physiol Neurobiol 169 Suppl 1:S63- Pries AR, Secomb TW (2009): Origins of heterogeneity in tis- S68 sue perfusion and metabolism. Cardiovasc Res 81(2):328- 35 Gunther S, Von EJ, May P, Preissner R (2009): JAIL: a structure-based interface library for macromolecules. Nucleic Reglin B, Secomb TW, Pries AR (2009): Structural adaptation Acids Res 37(Database issue):D338-D341 of microvessel diameters in response to metabolic stimuli: where are the oxygen sensors? Am J Physiol Heart Circ Hildebrand PW, Goede A, Bauer RA, Gruening B, Ismer J, Physiol 297(6):H2206-H2219 Michalsky E, Preissner R (2009): SuperLooper--a prediction server for the modeling of loops in globular and membrane Rother K, Hildebrand PW, Goede A, Gruening B, Preissner proteins. Nucleic Acids Res 37(Web Server issue):W571- R (2009): Voronoia: analyzing packing in protein structures. W574 Nucleic Acids Res 37(Database issue):D393-D395

Hossbach J, Michalsky E, Henklein P, Jaeger M, Daniel PT, Schmidt U, Struck S, Gruening B, Hossbach J, Jaeger Preissner R (2009): Inhibiting the inhibitors: retro-inverso IS, Parol R, Lindequist U, Teuscher E, Preissner R (2009): Smac peptides. Peptides 30(12):2374-9 SuperToxic: a comprehensive database of toxic compounds. Nucleic Acids Res 37(Database issue):D295-D299 Lipnicki DM, Gunga HC, Belavy DL, Felsenberg D (2009): Bed rest and cognition: effects on executive functioning and reac- Verdant CL, De BD, Bruhn A, Clausi CM, Su F, Wang Z, Rod- tion time. Aviat Space Environ Med 80(12):1018-24 riguez H, Pries AR, Vincent JL (2009): Evaluation of sublingual and gut mucosal microcirculation in sepsis: a quantitative Lipnicki DM, Gunga HC, Belavy DL, Felsenberg D (2009): analysis. Crit Care Med 37(11):2875-81 Decision making after 50 days of simulated weightlessness. Brain Res 1280:84-9

135 Axel Pries – PHYSIOLOGY

Vogiatzis I, Athanasopoulos D, Habazettl H, Kuebler WM, Hildebrand PW, Gunther S, Goede A, Forrest L, Frommel C, Wagner H, Roussos C, Wagner PD, Zakynthinos S (2009): Preissner R (2008): Hydrogen-bonding and packing features Intercostal muscle blood flow limitation in athletes during of membrane proteins: functional implications. Biophys J maximal exercise. J Physiol 587(Pt 14):3665-77 94(6):1945-53

Yin J, Kuebler WM (2009): New targets in pulmonary Hopfner M, Schuppan D, Scherubl H (2008): Treatment of hypertension--another ACE up the sleeve. Am J Respir Crit gastrointestinal neuroendocrine tumors with inhibitors of Care Med 180(5):481-2 growth factor receptors and their signaling pathways: recent advances and future perspectives. World J Gastroenterol Yin N, Kaestle S, Yin J, Hentschel T, Pries AR, Kuppe H, 14(16):2461-73 Kuebler WM (2009): Inhaled nitric oxide versus aerosolized iloprost for the treatment of pulmonary hypertension with left Hopfner M, Schuppan D, Scherubl H (2008): Targeted medi- heart disease. Crit Care Med 37(3):980-6 cal therapy of biliary tract cancer: recent advances and future perspectives. World J Gastroenterol 14(46):7021-32 Bauer RA, Rother K, Bujnicki JM, Preissner R (2008): Suffix techniques as a rapid method for RNA substructure search. Hopfner M, Schuppan D, Scherubl H (2008): Growth factor Genome Inform 20:183-98 receptors and related signalling pathways as targets for novel treatment strategies of hepatocellular cancer. World J Gas Bauer RA, Bourne PE, Formella A, Frommel C, Gille C, Goede troenterol 14(1):1-14 A, Guerler A, Hoppe A, Knapp EW, Poschel T, Wittig B, Ziegler V, Preissner R (2008): Superimpose: a 3D structural Huebener N, Fest S, Strandsby A, Michalsky E, Preissner R, superposition server. Nucleic Acids Res 36(Web Server Zeng Y, Gaedicke G, Lode HN (2008): A rationally designed issue):W47-W54 tyrosine hydroxylase DNA vaccine induces specific antineu- roblastoma immunity. Mol Cancer Ther 7(7):2241-51 Boldt LH, Fraszl W, Rocker L, Schefold JC, Steinach M, Noack T, Gunga HC (2008): Changes in the haemostatic sys- Kuebler WM (2008): Hitting new barriers in ventilator-induced tem after thermoneutral and hyperthermic water immersion. lung injury. Intensive Care Med 34(4):592-4 Eur J Appl Physiol 102(5):547-54 Kuebler WM (2008): How NIR is the future in blood flow moni- Boning D, Maassen N, Pries A (2008): No proof for augmented toring? J Appl Physiol 104(4):905-6 arterial oxygen content as only factor influencing exercise capacity after Epo doping. J Appl Physiol 105(6):1988 Le NF, Klein C, Tintu A, Pries A, Buschmann I (2008): Neural guidance molecules, tip cells, and mechanical factors in vas- Dunkel M, Gunther S, Ahmed J, Wittig B, Preissner R (2008): cular development. Cardiovasc Res 78(2):232-41 SuperPred: drug classification and target prediction. Nucleic Acids Res 36(Web Server issue):W55-W59 Pries AR, Habazettl H, Ambrosio G, Hansen PR, Kaski JC, Schachinger V, Tillmanns H, Vassalli G, Tritto I, Weis M, de Frassl W, Kowoll R, Katz N, Speth M, Stangl A, Brechtel L, WC, Bugiardini R (2008): A review of methods for assessment Joscht B, Boldt LH, Meier-Buttermilch R, Schlemmer M, of coronary microvascular disease in both clinical and experi- Roecker L, Gunga HC (2008): Cardiac markers (BNP, NT-pro- mental settings. Cardiovasc Res 80(2):165-74 BNP, Troponin I, Troponin T, in female amateur runners before and up until three days after a marathon. Clin Lab 54(3-4):81-7 Pries AR, Secomb TW (2008): Modeling structural adaptation of microcirculation. Microcirculation 15(8):753-64 Gunther S, Kuhn M, Dunkel M, Campillos M, Senger C, Petsalaki E, Ahmed J, Urdiales EG, Gewiess A, Jensen LJ, Schneider R, Pries AR, Mulvany MJ, Bakker EN (2008): MBEC special Skoblo R, Russell RB, Bourne PE, Bork P, Preissner R (2008): issue on microcirculation “engineering principles of vascular SuperTarget and Matador: resources for exploring drug-target networks”. Med Biol Eng Comput relationships. Nucleic Acids Res 36(Database issue):D919-D922

136 Axel Pries – PHYSIOLOGY

Redlin M, Koster A, Huebler M, Boettcher W, Nagdyman N, Baradari V, Hopfner M, Huether A, Schuppan D, Scherubl H Hetzer R, Kuppe H, Kuebler WM (2008): Regional differences (2007): Histone deacetylase inhibitor MS-275 alone or comin tissue oxygenation during cardiopulmonary bypass for bined with bortezomib or sorafenib exhibits strong antiprolif- correction of congenital heart disease in neonates and small erative action in human cholangiocarcinoma cells. World J infants: relevance of near-infrared spectroscopy. J Thorac Gastroenterol 13(33):4458-66 Cardiovasc Surg 136(4):962-7 Chlench S, Mecha DN, Hohberg M, Hoffmann C, Pohlkamp Schmidt U, Ahmed J, Michalsky E, Hoepfner M, Preissner R T, Beyer G, Bongrazio M, Da Silva-Azevedo L, Baum O, (2008): Comparative VEGF receptor tyrosine kinase modeling Pries AR, Zakrzewicz A (2007): Regulation of Foxo-1 and for the development of highly specific inhibitors of tumor the angiopoietin-2/Tie2 system by shear stress. FEBS Lett angiogenesis. Genome Inform 20:243-51 581(4):673-80

Schobersberger W, Toff WD, Eklof B, Fraedrich G, Gunga Gunga HC, Kirsch KA, Roecker L, Kohlberg E, Tiedemann HC, Haas S, Landgraf H, Lapostolle F, Partsch H, Perschler F, J, Steinach M, Schobersberger W (2007): Erythropoietin Schnapka J, Schobersberger B, Scurr JH, Watzke H (2008): regulations in humans under different environmental and Traveller’s thrombosis: international consensus statement. experimental conditions. Respir Physiol Neurobiol 158(2- VASA 37(4):311-7 3):287-97

Schwarzer R, Kaiser M, Acikgoez O, Heider U, Mathas S, Pre- Gunga HC, Suthau T, Bellmann A, Friedrich A, Schwanebeck issner R, Sezer O, Doerken B, Jundt F (2008): Notch inhibition T, Stoinski S, Trippel T, Kirsch K, Hellwich O (2007): Body blocks multiple myeloma cell-induced osteoclast activation. mass estimations for Plateosaurus engelhardti using laser Leukemia 22(12):2273-7 scanning and 3D reconstruction methods. Naturwissen schaften 94(8):623-30 Secomb TW, Beard DA, Frisbee JC, Smith NP, Pries AR (2008): The role of theoretical modeling in microcirculation Gunther S, May P, Hoppe A, Frommel C, Preissner R (2007): research. Microcirculation 15(8):693-8 Docking without docking: ISEARCH--prediction of interactions using known interfaces. Proteins 69(4):839-44 Struck S, Schmidt U, Gruening B, Jaeger IS, Hossbach J, Preissner R (2008): Toxicity versus potency: elucidation of Gunther S, Hempel D, Dunkel M, Rother K, Preissner R toxicity properties discriminating between toxins, drugs, and (2007): SuperHapten: a comprehensive database for small natural compounds. Genome Inform 20:231-42 immunogenic compounds. Nucleic Acids Res 35(Database issue):D906-D910 Tabuchi A, Kuebler WM (2008): Endothelium-platelet interactions in inflammatory lung disease. Vascul Pharmacol Hentschel T, Yin N, Riad A, Habbazettl H, Weimann J, Koster A, Tschope C, Kuppe H, Kuebler WM (2007): Inhalation of the Tabuchi A, Mertens M, Kuppe H, Pries AR, Kuebler WM phosphodiesterase-3 inhibitor milrinone attenuates pulmo- (2008): Intravital microscopy of the murine pulmonary micro- nary hypertension in a rat model of congestive heart failure. circulation. J Appl Physiol 104(2):338-46 Anesthesiology 106(1):124-31

Yin J, Hoffmann J, Kaestle SM, Neye N, Wang L, Baeurle J, Huether A, Hopfner M, Baradari V, Schuppan D, Scherubl Liedtke W, Wu S, Kuppe H, Pries AR, Kuebler WM (2008): H (2007): Sorafenib alone or as combination therapy for Negative-feedback loop attenuates hydrostatic lung edema growth control of cholangiocarcinoma. Biochem Pharmacol via a cGMP-dependent regulation of transient receptor poten- 73(9):1308-17 tial vanilloid 4. Circ Res 102(8):966-74 Kaestle SM, Reich CA, Yin N, Habazettl H, Weimann J, Kue- Ahmed J, Gunther S, Moller F, Preissner R (2007): A structural bler WM (2007): Nitric oxide-dependent inhibition of alveolar genomics approach to the regulation of apoptosis: chimp vs. fluid clearance in hydrostatic lung edema. Am J Physiol human. Genome Inform 18:22-34. Lung Cell Mol Physiol 293(4):L859-69

137 Axel Pries – PHYSIOLOGY

Kuebler WM, Parthasarathi K, Lindert J, Bhattacharya Bongrazio M, Da Silva-Azevedo L, Bergmann EC, Baum J (2007): Real-time lung microscopy. J Appl Physiol O, Hinz B, Pries AR, Zakrzewicz A (2006): Shear stress 102(3):1255-64 modulates the expression of thrombospondin-1 and CD36 in endothelial cells in vitro and during shear stress-induced Meinke M, Schroder M, Schutz R, Netz U, Helfmann J, Dor- angiogenesis in vivo. Int J Immunopathol Pharmacol schel K, Pries A, Muller G (2007): Frequency weighted laser 19(1):35-48 Doppler perfusion measurements in skin. Laser Physics Letters 4(1):66-71 Brueckl C, Kaestle S, Kerem A, Habazettl H, Krombach F, Kuppe H, Kuebler WM (2006): Hyperoxia-induced reactive Mittermayr M, Fries D, Gruber H, Peer S, Klingler A, Fisch- oxygen species formation in pulmonary capillary endothelial bach U, Gunga HC, Koralewski E, Faulhaber M, Simmer cells in situ. Am J Respir Cell Mol Biol 34(4):453-63 M, Schobersberger W (2007): Leg edema formation and venous blood flow velocity during a simulated long-haul flight. Dunkel M, Fullbeck M, Neumann S, Preissner R (2006): Thromb Res 120(4):497-504 SuperNatural: a searchable database of available natural compounds. Nucleic Acids Res 34(Database issue):D678- Mulder ER, Kuebler WM, Gerrits KH, Rittweger J, Felsenberg D683 D, Stegeman DF, De HA (2007): Knee extensor fatigability after bedrest for 8 weeks with and without countermeasure. Fest S, Huebener N, Weixler S, Bleeke M, Zeng Y, Strandsby Muscle Nerve 36(6):798-806 A, Volkmer-Engert R, Landgraf C, Gaedicke G, Riemer AB, Michalsky E, Jaeger IS, Preissner R, Forster-Wald E, Jensen- Schobersberger W, Mittermayr M, Fries D, Innerhofer P, Jarolim E, Lode HN (2006): Characterization of GD2 peptide Klingler A, Faulhaber M, Gunga HC, Streif W (2007): Changes mimotope DNA vaccines effective against spontaneous neu- in blood coagulation of arm and leg veins during a simulated roblastoma metastases. Cancer Res 66(21):10567-75 long-haul flight. Thromb Res 119(3):293-300 Fullbeck M, Michalsky E, Jaeger IS, Henklein P, Kuhn H, Secomb TW, Styp-Rekowska B, Pries AR (2007): Two-dimen- Ruck-Braun K, Preissner R (2006): Design and biological sional simulation of red blood cell deformation and lateral evaluation of photo-switchable inhibitors. Genome Inform migration in microvessels. Ann Biomed Eng 35(5):755-65 17(1):141-51

Spohr F, Busch CJ, Reich C, Motsch J, Gebhard MM, Kuebler Goede A, Michalsky E, Schmidt U, Preissner R (2006): WM, Bloch KD, Weimann J (2007): 4-Aminopyridine restores SuperMimic--fitting peptide mimetics into protein structures. impaired hypoxic pulmonary vasoconstriction in endotoxemic BMC Bioinformatics 7:11 mice. Anesthesiology 107(4):597-604 Greie S, Humpeler E, Gunga HC, Koralewski E, Klingler A, Struijker-Boudier HA, Rosei AE, Bruneval P, Camici PG, Christ Mittermayr M, Fries D, Lechleitner M, Hoertnagl H, Hoffmann F, Henrion D, Levy BI, Pries A, Vanoverschelde JL (2007): G, Strauss-Blasche G, Schobersberger W (2006): Improve- Evaluation of the microcirculation in hypertension and cardio- ment of metabolic syndrome markers through altitude specific vascular disease. Eur Heart J 28(23):2834-40 hiking vacations. J Endocrinol Invest 29(6):497-504

Styp-Rekowska B, Disassa NM, Reglin B, Ulm L, Kuppe Gunther S, Senger C, Michalsky E, Goede A, Preissner R H, Secomb TW, Pries AR (2007): An imaging spectroscopy (2006): Representation of target-bound drugs by computed approach for measurement of oxygen saturation and hematocrit conformers: implications for conformational libraries. BMC during intravital microscopy. Microcirculation 14(3):207-21 Bioinformatics 7:293

Baradari V, Huether A, Hopfner M, Schuppan D, Scherubl H Habazettl H, Kukucka M, Weng YG, Kuebler WM, Hetzer R, (2006): Antiproliferative and proapoptotic effects of histone Kuppe H, Pries AR (2006): Arteriolar blood flow pulsatility in deacetylase inhibitors on gastrointestinal neuroendocrine a patient before and after implantation of an axial flow pump. tumor cells. Endocr Relat Cancer 13(4):1237-50 Ann Thorac Surg 81(3):1109-11

138 Axel Pries – PHYSIOLOGY

Herre S, Schadendorf T, Ivanov I, Herrberger C, Steinle W, Roecker L, Kowoll R, Fraszl W, Battal K, Brechtel L, Brach- Ruck-Braun K, Preissner R, Kuhn H (2006): Photoactivation mann S, Meier-Buttermilch R, Gunga HC, Stangl A, Kiesewet- of an inhibitor of the 12/15-lipoxygenase pathway. Chembio ter H (2006): Observation of serum erythropoietin concentra- chem 7(7):1089-95 tions in female athletes for up to eight days after a marathon run. Clin Lab 52(9-10):511-3 Hildebrand PW, Lorenzen S, Goede A, Preissner R (2006): Analysis and prediction of helix-helix interactions in mem- Secomb TW, Hsu R, Pries AR (2006): Tribology of capillary brane channels and transporters. Proteins 64(1):253-62 blood flow. Proceedings of the Institution of Mechani cal Engineers Part J-Journal of Engineering Tribology Hopfner M, Baradari V, Huether A, Schofl C, Scherubl H 220(J8):767-74 (2006): The insulin-like growth factor receptor 1 is a promising target for novel treatment approaches in neuroendocrine Sodian R, Lueders C, Kraemer L, Kuebler W, Shakibaei M, gastrointestinal tumours. Endocr Relat Cancer 13(1):135-49 Reichart B, Daebritz S, Hetzer R (2006): Tissue engineering of autologous human heart valves using cryopreserved vascular Hopfner M, Huether A, Sutter AP, Baradari V, Schuppan umbilical cord cells. Ann Thorac Surg 81(6):2207-16 D, Scherubl H (2006): Blockade of IGF-1 receptor tyrosine kinase has antineoplastic effects in hepatocellular carcinoma Steinbrink J, Fischer T, Kuppe H, Hetzer R, Uludag K, Obrig cells. Biochem Pharmacol 71(10):1435-48 H, Kuebler WM (2006): Relevance of depth resolution for cerebral blood flow monitoring by near-infrared spectroscopic Hopfner M, Sutter AP, Huether A, Baradari V, Scherubl H bolus tracking during cardiopulmonary bypass. J Thorac (2006): Tyrosine kinase of insulin-like growth factor recep- Cardiovasc Surg 132(5):1172-8 tor as target for novel treatment and prevention strategies of colorectal cancer. World J Gastroenterol 12(35):5635-43 Valenti G, Fraszl W, Addabbo F, Tamma G, Procino G, Satta E, Cirillo M, De Santo NG, Drummer C, Bellini L, Kowoll R, Hudlicka O, Brown MD, May S, Zakrzewicz A, Pries AR Schlemmer M, Vogler S, Kirsch KA, Svelto M, Gunga HC (2006): Changes in capillary shear stress in skeletal muscles (2006): Water immersion is associated with an increase in exposed to long-term activity: role of nitric oxide. Microcir aquaporin-2 excretion in healthy volunteers. Biochim Bio culation 13(3):249-59 phys Acta 1758(8):1111-6

Huether A, Hopfner M, Sutter AP, Baradari V, Schuppan D, Williams JL, Weichert A, Zakrzewicz A, Da Silva-Azevedo L, Scherubl H (2006): Signaling pathways involved in the inhibi- Pries AR, Baum O, Egginton S (2006): Differential gene and tion of epidermal growth factor receptor by erlotinib in hepato- protein expression in abluminal sprouting and intraluminal cellular cancer. World J Gastroenterol 12(32):5160-7 splitting forms of angiogenesis. Clin Sci (Lond) 110(5):587-95

Kuebler WM (2006): Selectins revisited: the emerging role Book chapters of platelets in inflammatory lung disease. J Clin Invest 116(12):3106-8 Pries AR, Secomb TW. Blood flow in microvascular networks. In: Tuma RF, Durán WN, Ley K, editors. Handbook of Physi Opitz B, Vinzing M, van L, V, Schmeck B, Heine G, Gunther S, ology: Microcirculation. 2 ed. San Diego: Academic Press, Preissner R, Slevogt H, N’Guessan PD, Eitel J, Goldmann T, Elsevier; 2008. p. 3-36 Flieger A, Suttorp N, Hippenstiel S (2006): Legionella pneumophila induces IFNbeta in lung epithelial cells via IPS-1 and IRF3, which Secomb TW, Pries AR. Basic Principles of Hemodynamics. also control bacterial replication. J Biol Chem 281(47):36173-9 In: Baskurt OK, hardeman MW, Rampling MW, Meiselman HJ, editors. Handbook of Hemorheology and Hemody Redlin M, Boettcher W, Huebler M, Berger F, Hetzer R, Koster A, namics. Amsterdam: IOS Press; 2007 Kuebler WM (2006): Detection of lower torso ischemia by near-infrared spectroscopy during cardiopulmonary bypass in a 6.8-kg infant Pries AR, Kuebler WM (2006): Normal endothelium. Handb with complex aortic anatomy. Ann Thorac Surg 82(1):323-5 Exp Pharmacol (176 Pt 1):1-40

139 Axel Pries – PHYSIOLOGY

General information Third party funding (2006-2010)

Project leader Project title Sponsor Period Kübler W. SFB Transregio 19 DFG 2008-2012 Kübler W. Protektive Beatmung TP1 DFG 2005-2007 Kübler W. Protektive Beatmung TP2 DFG 2007-2009 Kübler W. Strömungsmechanik DFG 2009-2011 Kübler W. Sphingolipide FP 1 DFG 2007-2010 Kübler W. Sphingolipide FP 2 DFG 2010-2013 Kübler W. Angiotensin(1-7) DFG 2009-2011 Kübler W. Totraumventilation DFG 2009-2011 Kübler W. Vscular regulation DFG (GRK865) 2003-2008 Kübler W. Pulmotension EU 2006-2009 Kübler W. Vasodilatory effect of RO50241 Hoffmann–La Roche Inc 2009-2011 Kübler W. Therapeutic potential of oral LF1166910 Solvay Pharmaceuticals 2009-2011 Kübler W. Sildenafil citrate Pfizer Inc 2004-2011 Kübler W. Connexin 40 CIHR 2009-2012 Kübler W. Pulmonary hypertension HSFC 2010-2012 Blottner D. Molekulare Zellstrukturen BMBF 2005-2008 Blottner D. Bed Rest Study EU 2007-2010 Siegel G. Fluvastatin Industrie 2001-2005 Kunz D. PLACAR BMBF 2006-2010 Kunz D. Energieoptimiertes Bauen: Beleuchtung BMWi 2007-2010 Kunz D. Nikotin und Schlaf DFG 2006-2011 Kunz D. Fit for School BMBF 2007-2010 Gunga H.C. Langzeitaufenthalte unter Schwerelosigkeit BMBF 2002-2007 Gunga H.C. MILIEU Exzellenzcluster FU 2009-2011 Gunga H.C. Spacelife Graduiertenkolleg DLR 2008-2010 Gunga H.C. Brain function and physical exercise Karlsruhe Institute of Technology (KIT) 2010 Gunga H.C. Fluid Shift - Thermolab BMBF 2007-2010 Gunga H.C. Thermolab BMBF 2010-2013 Munz B. Bedeutung von sKNac DHSF 2007-2010 Munz B. Rhabdomyosarkomzellen Kutzner Stiftung 2009-2011 Munz B. Rolle von Rip-Protein_2 DFG 2006-2009 Munz B. ZFP 36 Proteine DFG 2010-2014

140 Uwe Querfeld – PEDIATRIC NEPHROLOGY

Head of the group

Prof. Dr. med. Uwe Querfeld

Curriculum Vitae: 1979‑1984 Training in Pediatrics at the Children’s Hospital Heidelberg University. Specialization in Pediatric Nephrology 1984‑1986 Fellow, Division for Pediatric Nephrology, UCLA Medical School, Los Angeles, Bench training in lipid research at Los Angeles Veterans Administration Medical Center. 1987‑1988 Advanced Research Fellowship grant by the American Heart Association 1987-1988 Fellow, Division for Pediatric Nephrology, Cedars‑Sinai Medical Center, Los Angeles 1991-1999 Attending physician in Pediatrics, University Children’s Hospital, Cologne, Germany Since 10/99 Director, Department of Pediatric Nephrology, Charité Berlin, Germany. 2003 European Soecity for Pediatric Nephrology (ESPN) Congress President, World Congress of Nephrology Since 2004 Head of a research group at the Center for Cardiovascular Research, Berlin

Main scientific interest: Cardiovascular disease in children and adolescents with chronic kidney disease, prevention of atherosclerosis, non-invasive imaging methods of arterial structure and function, vascular calcification, vitamin D metabolism, lipid metabolism

Members of the group

Scientists Müller, Dominik PD Dr. med. Stuiver, Marchel Dipl. – Molbiol. Thumfart, Julia Dr. med.

Technicians Sommer, Kerstin MTA

Students Diercke, Michaela Medical Student Jung, Susanne PhD student Kopplin, Kathrin PhD student Hecht, Eva PhD student Will, Constanze PhD student

141 Uwe Querfeld – PEDIATRIC NEPHROLOGY

Summary

Our group conducts translational research in car- metalloproteinases (MMP-2 and MMP-9) on high- diovascular aspects of clinical pediatric nephrology dose calcitriol induced vascular calcifications in and vascular and renal physiology. uremic animals.

Vitamin D- induced vascular calcification in The Cardiovascular Comorbidity in Children chronic kidney disease with Chronic Kidney Disease Study (4 C Study) Whether treatment with vitamin D receptor activators In this prospective observational study of children (VDRA) contributes to cardiovascular disease (CVD) in and adolescents with CKD, a comprehensive study patients with chronic kidney disease (CKD) is a matter of arterial biopsy specimens, usually taken at the of debate. Observational studies in adult CKD patients time of AV fistula surgery or pre-emptive renal show that VDRA provide a survival benefit associated transplantation, is performed for histopathological with a decrease in CVD-related mortality. These data assessment in a biopsy sub-study. Data will be ana- are in contrast to studies in children and adolescents lyzed for associations with the clinical progression and in young adults with childhood-onset CKD, where of renal and cardiovascular disease as monitored treatment with 1-alpha or 1,25(OH)2D3 is associated prospectively by non-invasive imaging methods. with the occurrence of arterial calcifications. As a rule, children and adolescents require comparatively high Claudin tight junction proteins in renal ion doses of VDRA to prevent the development of rickets homeostasis in the growing skeleton. Tight junction proteins of the Claudin family are tet- We are therefore interested in studying the mecha- raspanning transmembrane proteins which serve as nisms leading to vitamin D-induced calcifications by a semipermeable barrier in epithelia. They regulate using an animal model and cell culture experiments. the paracellular diffusion of solutes through epithelial and endothelial cell layers. The different family We are currently studying the relative contribution of members render the epithelia leaky or tight and calcitriol treatment and elevated PTH levels to vas- contribute to the specifity of ions and molecules cular calcifications. In animal experiments, we have that are absorbed. In the kidney, Claudins show a investigated the effect of cinacalcet, a modulator distinct expression pattern along the tubule and are of the calcium-sensing receptor in the parathyroid involved in paracellular ion resorption and contribu- gland, and of parathyroidectomy (PTX), on high- tion to urine formation, thus they contribute to vol- dose calcitriol induced vascular calcifications in ume regulation and blood pressure control. We are uremic rats. In addition, we are studying the effect evaluating the physiological and pathophysiological of high-dose calcitriol, Cinacalcet and PTX on ure- role Claudin-3, -10 and -16 which are all expressed mic bone disease In cooperation with Laboratory in the thick ascending limb (TAL) of Henle´s loop a of Pathophysiology, University of Antwerp, Belgium. crucial region of volume regulation and blood pres- We have also initiated studies of the role of matrix sure control.

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Zusammenfassung

Unsere Arbeitsgruppe befasst sich mit kardiovasku- Außerdem gehen wir der Frage nach, ob Metallpro- lären Aspekten der klinischen pädiatrischen Nephro- teinasen (MMP-2, MMP-9) eine maßgebliche Rolle logie und der Gefäß- und Nierenphysiologie. in der Pathogenese von Vitamin D-induzierten Ver- kalkungen spielen. Vitamin D-induzierte vaskuläre Verkalkungen bei chronischer Niereninsuffizienz Studie zur kardiovaskulären Komorbidität bei Ob Vitamin D-Präparate zur hohen kardiovaskulä- Kindern mit chronischen Nieren-erkrankungen ren Morbidität und Mortalität bei chronischer Nie- (The Cardiovascular Comorbidity in Children reninsuffizienz beitragen, wird gegenwärtig kont- with Chronic Kidney Disease, 4 C Study) rovers diskutiert. Einerseits fanden epidemiologi- Die 4C-Studie ist eine prospektive Beobachtungs- schen Studien bei erwachsenen Patienten einen studie an Kindern und Jugendlichen mit chroni- günstigen Effekt in Hinblick auf kardiovaskuläre scher Niereninsuffizienz, in der die kardiovaskuläre Komplikationen und Überlebensstatistiken; ander- Komorbidität mit nicht-invasiven vaskulären Unter- seits haben Studien bei Kindern gezeigt, dass das suchungsmethoden charakterisiert wird. In einer Auftreten von koronaren Verkalkungen (unabhängig Sub-Studie wird bei Patienten, die das Terminalsta- von anderen Risikofaktoren) mit der Dosierung von dium der Niereninsuffizienz erreichen, zum Zeitpunkt Vitamin D-Präparaten verbunden war. Im Prinzip der Anlage einer AV-Fistel für die Hämodialyse oder müssen Kinder und Jugendliche dauerhaft und mit einer präemptiven Nierentransplantation eine Arte- höheren Dosen als Erwachsene behandelt werden, rienbiopsie durchgeführt. Im CCR werden Biopsien um das Auftreten von rachitischen Erkrankungen im mit färberischen Methoden, der Histochemie, der wachsenden Skelett zu verhindern. Wir sind des- Elektronenmikroskopie, der Mineralzusammenset- wegen daran interessiert, die zugrunde liegenden zung von Verkalkungen sowie durch Vergleich der Mechanismen der Vitamin D-induzierten Gefäßver- Genexpression untersucht. Die Daten werden hin- kalkung besser zu charakterisieren. sichtlich einer Assoziation mit klinischen Variabeln, d.h. der Progression der Niereninsuffizienz und der Gegenwärtig untersuchen wir die Frage, in welchem kardiovaskulären Komorbilität ausgewertet. Ausmaß erhöhte PTH-Spiegel bei der Entstehung von Vitamin D-induzierten Verkalkungen beteiligt Claudine: Ionerenabsorption, Volumenregula sind. Zu diesem Zweck werden in Experimenten an tion und Blutdruckkontrolle niereninsuffizienten Ratten, die unter einer hochdo- Claudine sind membranständige Tight Junction Pro- sierten Calcitriol Gabe ausgeprägte Verkalkungen teine mit 4 transmembranen Domänen, die wichtig entwickeln, parathyreoidektomierte und (mit dem für epitheliale und endotheliale Zell-Zell Kontakte Calcimimetikum) Cinacalcet- behandelte Tiere mit sind. Sie begünstigen oder verhindern parazelluläre urämischen Kontrolltieren verglichen. Transportvorgänge indem sie das Epithel abdichten

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oder durchgängig machen. Dabei weisen die einzel- gesamten Ionenhomöostase bei. Unsere Arbeits- nen Mitglieder der Claudin-Familie eine Substrat-, gruppe beschäftigt sich mit der Funktion der renal Ladungs- und Größenspezifität für Substrate auf. exprimierten Claudine -3, -10 und-16, die im aufstei- In der Niere sind die Claudine an der Rückresorp- genden Ast der Henleschen Schleife exprimiert sind tion glomerulär filtrierter Substanzen beteiligt, ihre – einem Bereich des Tubulussystems, der beson- Expression variiert mit dem reginalen Ionenresorp- ders eng mit der Blutdruckregulation korreliert ist. tionsprofil und der Entwicklung. Durch diese Fähig- Um Funktion und Fehlfunktion besser zu verstehen, keiten tragen Claudine maßgeblich zur Volumenre- entwickeln wir für diese Claudine murine Knock-out gulation und damit zur Blutdruckkontrolle und der Modelle, bei denen das jeweilige Gen inaktiviert ist.

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Research projects

1. Vitamin D-induced vascular calcification in uremic rats -Effect of cinacalcet (CINA) and parathyroidectomy (PTX)

Project leader Uwe Querfeld Coworkers Dominik Müller, Susanne Jung, Kerstin Sommer Cooperations AG Peters, CCR: Harm Peters, Stephanie Krämer

Pediatric patients with CKD need treatment with pared to controls (CON). Growth and weight gain active vitamin-D preparations to prevent the develop- was not suppressed in the U+vitD+CINA group. We ment of rickets and to suppress hyperparathyroidism. conclude that in uremic rats, CINA attenuates vita- The therapeutic window, however, is small, and clini- min D- induced calcification.Neither CINA nor PTX cal and experimental data show that both calcitriol can completely prevent vitamin-D induced calcifica- and PTH are involved in the pathogenesis of vascular tion. Calcification occurs by direct vascular effects of calcifications. We have studied the question to what calcitriol, independent of serum calcium and serum extent calcitriol-induced vascular calcifications can be phosphorus concentrations. prevented by parathyroidectomy (PTX) and cinacalcet (CINA) in a rat model of uremia (5/6 nephrectomy). Five groups of animals were studied: sham- operated (CON), uremic (U), uremic + calcitriol (U+vitD), uremic+calcitriol+PTX (U+vitD+PTX), uremic+calcitriol+CINA (U+vitD+CINA). We found that vascular calcifications (%area of aortic wall) were not seen in CON and U animals, present (>20%) in U+vitD animals, and reduced to a similar degree (<10%) in both, the U+vitD+PTX and the U+vitD+CINA group. PTH was significantly lower in U+D rats compared to U rats, and further suppressed to a similar degree in animals treated with PTX or CINA. The serum calcium-phosphate product was elevated in all groups receiving calcitriol, without a significant difference between groups. The creatinine clearance We could show that calcitriol treatment induces endo- was significantly decreased only in U+vitD animals chondral differentiation in vascular smooth muscle compared to uremic controls (U). Systolic blood pres- cells (Figure below). sure was elevated in all experimental groups com-

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mental design, it may be possible to identify the therapeutic window for active vitamin d preparations, which is essential for patients with chronic kidney disease, especially children and adolescents. Furthermore, current reasearch is aimed at characterizing the role of matrix metalloproteinases (MMP-2 and MMP-9) on high-dose calcitriol induced vascular calcifications in The experimental model used in these studies (high- uremic animals. dose vitamin D-induced vascular calcifications in uremic animals) is potentially suitable for identifying In addition, we are studying the effect of high-dose molecular mechanisms for vascular calcification, calcitriol, Cinacalcet and PTX on uremic bone disease which may provide therapeutic targets for intervention in cooperation with Laboratory of Pathophysiology, studies. Thus, by further modifications of the experi- University of Antwerp, Belgium.

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2. The Cardiovascular Comorbidity in Children with Chronic Kidney Disease Study (4 C Study)

Project leader Uwe Querfeld Franz Schaefer (Heidelberg) Coworkers Dominik Müller, Kerstin Sommer, The 4 C Study group

The pediatric population appears uniquely suited to In the CCR, a comprehensive study of arterial study the effects of CKD on the cardiovascular system biopsy specimens, usually taken at the time of AV due to the virtual absence of vascular morbidity related fistula surgery or pre-emptive renal transplantation, to ageing, diabetes and smoking. is performed for histopathological assessment in a biopsy sub-study. Biopsy samples will be ana- The 4 C Study is a 3+ year prospective observational lyzed by conventional staining, histochemistry and study in up to 625 children and adolescents with CKD. by electron microscopy, physicochemical charac- Enrollment has started in 2010 and thus far includes terisation of mineral precipitates, and comparative > 500 children aged 6 to 17 years with a glomerular gene expression profiling. Data will be analyzed for filtration rate of 10 to 45 ml/min/1.73 m² in 51 pediatric associations with the clinical progression of renal nephrology units in 14 European countries. Patients and cardiovascular disease obtained from the 4 C are studied by annual non-invasive assessment of Study database. arterial and cardiac morphology and function, Monitoring of putative anthropometric and biochemical predictor variables, and by whole-genome study of common genetic variants for association with progression of cardiovascular comorbidity and renal failure progression. “Functional“ endpoints are the increase of local (Carotid, M-mode) and systemic arterial stiffness (pulse wave velocity) and myocardial dysfunction. „Morphology“ endpoints are the increase of carotid IMT and left ventricular mass index .

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3. Claudin-3 tight junction protein in renal ion homeostasis

Project leader Dominik Müller Coworkers Kathrin Kopplin,Marchel Stuiver, Kerstin Sommer, Uwe Querfeld Collaborations Thomas Willnow (MDC Berlin), Markus Bleich (University Kiel), Dorothee Günzel, Michael Fromm (Charite CBF) Funding DFG (FOR 721) Eunefron (EU, FP 7)

Claudin-3 is ubiquitously expressed in various epithe- ron, where the resorption processes of ions like Na+, lia. The functional and physiological role of claudin-3 Mg2+, Ca2+ occur. We are interested in the impact is still unclear, however, its distinct expression profile of claudin-3 for ion and water homeostasis and thus and widespread distribution in the body makes it likely blood pressure regulation. A claudin KO strain has to be important for the integrity of epithelia. Here we been generated successfully and is currently being investigate the function of claudin-3 with a special investigated. Additionally a double KO (cldn16 / 3 KO) focus on its role in the kidney. It is known that clau- has been obtained. din-3 is expressed in the tighter segments of the neph-

Immunohistochemistry of Claudin-3 in mouse kidney demonstrating its expression along the nephron

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4. Claudin-10 tight junction protein in renal ion homeostasis

Project leader Dominik Müller Coworkers Constanze Will, Marchel Stuiver, Kerstin Sommer, Uwe Querfeld Collaborations Thomas Willnow (MDC Berlin), Dorothee Günzel, Michael Fromm (Charite CBF) Funding: DFG (FOR 721) Eunefron (EU, FP 7)

The Cldn10 gene consists of two alternative first exons, tissue specific deficiency as well as gene deletion at which give rise to the expression of two Claudin-10 certain timepoints of pre- or postnatal development. isoforms. Of these isoforms, Claudin-10a shows exclu- Our first data from Cldn10 total KO animals suggests sive expression in the kidney and has previously been that Claudin-10 deficiency leads to vital impairment reported to influence anion permeability in epithelia. within the first days after birth. Subsequent expres- Claudin 10b, however, is ubiquitously expressed in sion studies, investigation of organ morphology and various epithelia and has been functionally character- body fluid composition analysis will provide us with ized as pore former. The expression of both isoforms information about the physiological defects in this along the nephron makes Claudin-10 an attractive mouse model. Furthermore, conditional gene dele- candidate for distinct renal ion resorption processes tions will offer the possibility to establish vital mouse and thereby the regulation of sodium in body fluids, models which will allow investigate special salt and water homeostasis and blood pressure. water stress conditions to further characterize the role The goal of our study is to evaluate the contribution of Claudin-10 in vivo. Due to the widespread tissue of Claudin-10 to epithelial transport processes and distribution of Claudin-10, this mouse model approach the effect of Claudin-10 deficiency on the organisms´ is an excellent basis for interdisciplinary analysis with physiology. To obtain a suitable in vivo model, we have experts on the fields of cardiology, pulmonology and successfully generated a murine conditional knockout nephrology. strain which offers the possibility to analyze total or

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5. Claudin-16 tight junction protein in renal ion homeostasis

Project leader Dominik Müller Coworkers Constanze Will, Marchel Stuiver, Kerstin Sommer, Uwe Querfeld Collaborations Thomas Willnow (MDC Berlin), Markus Bleich (Universitiy of Kiel) Michael Fromm (Charite CBF) Funding: Eunefron (EU, FP 7) DFG (FOR 721)

Claudin-16 is exclusively expressed in the kidney in the perform pharmacological and salt stress models on thick ascending limb of Henle´s loop, where it serves as Cldn16 KO mice, where we hope to have insights into a cation channel during the resorption of mono- and compensative/regulative mechanisms in metabolism bivalent ions. Mutations in the human CLDN16 gene which might be applicable for patients with FHHNC. have been reported to come out with the salt loss syn- We already identified differences of acid-base han- drome FHHNC (Familial hypomagnesemia with hyper- dling in these animals. Another goal of our studies is to calciuria and nephrocalcinosis), which cause a severe study Cldn16 deficiency at different timepoints of pre- loss of Ca and Mg and an imbalance of hormone levels and postnatal development, to further understand the regulating the homeostasis of bivalent ions. To fur- role of Claudin-16 in organogenesis and ontogenesis. ther analyse ion handling in vivo and to obtain a suit- For this purpose a Tamoxifen inducible cldn16 cre line able mouse model for FHHNC, we have targeted the has been successfully established. Cldn16 gene to set up a conditional murine Claudin-16 deficiency strain, which allows to study total loss of Claudin-16 as well as temporo-spatial deficiency. Metabolic analysis of Claudin-16 null mice reveals hypomagnesemia and hypercalciuria in these animals, as well as elevated levels of parathyroid hormone and Calcitriol to overcome salt loss. These data confirm the human phenotype and point out the impact of Claudin-mediated paracellular transport mechanisms on ion homeostasis and the composition of body fluids. Subsequent investigations will focus on morphologic and physiologic analysis of the kidneys, as well as counteracting processes, e.g. renal transcellular Cldn-16 KO mice exhibit severe renal Ca2+- and Mg2+ waste ion transport mechanisms. Furthermore, we plan to and serve as a robust model for the human disorder FHHNC.

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Publications 2006 - 2010 Kempf C, Winkelmann B, Roigas J, Querfeld U, Müller D (2010): Severe complications after endoscopic injection of Milatz S, Krug SM, Rosenthal R, Günzel D, Müller D, Schulzke polydimethylsiloxane for the treatment of vesicoureteral reflux JD, Amasheh SS, Fromm M (2010): Claudin-3 acts as a seal- in early childhood. Scand J Urol Nephrol Jun 14. [Epub ing component of the tight junction for ions of either charge ahead of print] and uncharged solutes.Biochem Biophys Acta – Biomem branes: July 22 [Epub ahead of print] Zipfel PF, Mache C, Müller D, Licht C, Wigger M, Skerka C; for the European DEAP-HUS Study Group (2010): DEAP-HUS: Querfeld U, Anarat A, Bayazit AK, Bakkaloglu AS, Bilginer Deficiency of CFHR plasma proteins and autoantibody-pos- Y, Caliskan S, Civilibal M, Doyon A, Duzova A, Kracht D, itive form of hemolytic uremic syndrome. Pediatr Nephrol Litwin M, Melk A, Mir S, Sözeri B, Shroff R, Zeller R, Wühl E, Feb 16. [Epub ahead of print] Schaefer F; the 4C Study Group (2010): The Cardiovascular Comorbidity in Children with Chronic Kidney Disease (4C) Hoppe A, von Puttkamer C, Linke U, Kahler C, Booß M, Brau- Study: Objectives, Design, and Methodology. Clin J Am Soc nauer-Kolberg R, Hofmann K, Joachimsky P, Hirte I, Schley S, Nephrol Jun 24. [Epub ahead of print] Utsch B, Thumfart J, Briese S, Gellermann J, Zimmering M, Querfeld U, Müller D. (2010): A Hospital-Based Intermit Querfeld U, Mak RH. (2010): Vitamin D deficiency and toxicity tent Nocturnal Hemodialysis Program for Children and in chronic kidney disease: in search of the therapeutic win- Adolescents. J Pediatr Aug 5. [Epub ahead of print] dow. Pediatr Nephrol Jun 22. [Epub ahead of print] Zebger-Gong H, Kampmann J, Kong L, Roigas J, Som- Höcker B, Weber LT, Feneberg R, Drube J, John U, Fehren- mer K, Krämer S, Peters H, Müller D, Dragun D, Querfeld U. bach H, Pohl M, Zimmering M, Fründ S, Klaus G, Wühl E, Decreased Transplant Arteriosclerosis in Endothelial Nitric Tönshoff B. (2010): Improved growth and cardiovascular risk Oxide Synthase-Deficient Mice. Transplantation 2009 Dec after late steroid withdrawal: 2-year results of a prospective, 16. [Epub ahead of print] randomised trial in paediatric renal transplantation. Nephrol Dial Transplant 25(2):617-24. Epub 2009 Sep 30 Günzel D, Stuiver M, Kausalya PJ, Haisch L, Hunziker W, Krug S, Meij IC, Fromm M, Müller D (2009): Identification and Will C, Breiderhoff T, Thumfart J, Stuiver M, Kopplin K, Som- characterization of six novel Claudin-10 splice variants J Cell mer K, Günzel D, Querfeld U, Meij IC, Shan Q, Bleich M, Will- Sci 15: 1507-15017 now TE, Müller D (2010): Targeted deletion of murine Cldn16 identifies extra- and intrarenal compensatory mechanisms of ESCAPE Trial Group, Wühl E, Trivelli A, Picca S, Litwin M, Ca2+ and Mg2+ wasting. Am J Physiol Renal Physiol Feb Peco-Antic A, Zurowska A, Testa S, Jankauskiene A, Emre 10. [Epub ahead of print] S, Caldas-Afonso A, Anarat A, Niaudet P, Mir S, Bakkaloglu A, Enke B, Montini G, Wingen AM, Sallay P, Jeck N, Berg Schaefer F, van de Walle J, Zurowska A, Gimpel C, van Hoeck U, Caliskan S, Wygoda S, Hohbach-Hohenfellner K, Dusek K, Drozdz D, Montini G, Bagdasorova IV, Sorof J, Sugg J, J, Urasinski T, Arbeiter K, Neuhaus T, Gellermann J, Drozdz Teng R, Hainer JW (2010): Candesartan in Children with D, Fischbach M, Möller K, Wigger M, Peruzzi L, Mehls O, Hypertension Investigators. Efficacy, safety and pharmacoki- Schaefer F. (2009): Strict blood-pressure control and progres- netics of candesartan cilexetil in hypertensive children from 1 sion of renal failure in children. N Engl J Med 361:1639-50 to less than 6 years of age. J Hypertens 28(5):1083-90. Gimpel C, Wühl E, Arbeiter K, Drozdz D, Trivelli A, Charbit M, Franke D, Völker S, Haase S, Pavicic L, Querfeld U, Ehrich JH, Gellermann J, Dusek J, Jankauskiene A, Emre S, Schaefer F; Zivicnjak M (2010) Prematurity, small for gestational age and ESCAPE Trial Group (2009): Superior consistency of ambula- perinatal parameters in children with congenital, hereditary tory blood pressure monitoring in children: implications for and acquired chronic kidney disease. May 31. [Epub ahead clinical trials. J Hypertens 27:1568-74 of print]

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Raile K, Klopocki E, Holder M, Wessel T, Galler A, Deiss D, König S, Ringe H, Dorner BG, Diers A, Uhlenberg B, Müller D, Müller D, Riebel T, Horn D, Maringa M, Weber J, Ullmann R, Varnholt V, Gaedicke G (2007): Atypical tetanus in a completely Grüters A (2009): Expanded clinical spectrum in HNF1B- immunized 14-year old boy. Pediatrics 120: e1355-1358 MODY. J Clin Endocrinol Metab 94: 2658-2664 Hinkes B, Wiggins RC, Gbadegesin R, Vlangos CN, Seelow Offner G, Toenshoff B, Höcker B, Krauss M, Bulla M, Cochat D, Nurnberg G, Garg P, Verma R, Chaib H, Hoskins BE, P, Fehrenbach H, Fischer W, Foulard M, Hoppe B, Hoyer PF, Ashraf S, Becker C, Hennies HC, Goyal M, Wharram B, Sch- Jungraithmayr TC, Klaus G, Latta K, Leichter H, Mihatsch MJ, achter AD, Drummond I, Kerjaschki D, Waldherr R, Dietrich A, Misselwitz J, Montoya C, Müller-Wiefel DE, Neuhaus TJ, Pape Ozaltin F, Bakkaloglu A, Cleper R, Basel-Vanagaite L, Pohl M, L, Querfeld U, Plank C, Schwarke D, Wygoda S, Zimmerhackl Griebel M, Tsygin AN, Soylu, Muller D, Katan M, Liu J, Attana- LB (2008): Efficacy and safety of basiliximab in pediatric renal sio M, O’Toole JF, Hasselbacher K, Mucha M, Otto EA, Airik transplant patients receiving cyclosporine, mycophenolate R, Kispert A, Kelley GG, Smrcka AV, Gudermann T, Holzman mofetil, and steroids. Transplantation 86:1241-8 LB, Nurnberg P, Hildebrandt F (2006): Positional cloning of PLCE1 mutations as the first cause of a nephrotic syndrome Thumfart J, Jung S, Amasheh S, Krämer S, Peters H, Som- variant which may be reversible. Nat Genet 38: 1397-1404 mer K, Biber J, Murer H, Meij I, Querfeld U, Wagner CA, Müller D (2008): Magnesium stimulates renal phosphate Müller D, Kausalya PJ, Meij IC, Hunziker W (2006): Familial reabsorption. Am J Physiol Renal 295: F126-133 hypomagnesemia with hypercalciuria and nephrocalcinosis: blocking endocytosis restores surface expression of a novel Raile K, Klopocki E, Wessel T, Deiss D, Horn D, Müller D, Claudin-16 mutant that lacks the entire C-terminal cytosolic Ullmann R, Grüters A (2008): HNF1B abnormality (MODY5) in tail. Hum Mol Genet 15: 1049-1058 children and adolescents: high prevalence in autoantibody- negative type 1 diabetes with kidney defects. Diabetes Care Kausalya JP, Amasheh S, Günzel D, Wurps H, Müller D, 31: e83 Fromm M, Hunziker W (2006): Disease-associated mutations affect intracellular traffic and paracellular Mg2+ transport Briese S, Claus M, Querfeld U (2008): Arterial stiffness in function of Claudin-16 J Clin Invest 116: 878-891 children after renal transplantation. Pediatr Nephrol Jun 27. [Epub ahead of print] Müller D, Kausalya PJ, Bockenhauer D, Thumfart J, Meij IC, Dillon M, van’t Hoff W, Hunziker W (2006): Unusual clinical Müller D, Zimmering M, Chan CT, McFarlane PA, Pierratos presentation and possible rescue of a novel Claudin-16 muta- A, Querfeld U (2008):.Intensified hemodialysis regimens: tion. J Clin Endocrinol Metab 91: 3076-3079 neglected treatment options for children and adolescents. Pediatr Nephrol Mar 12. [Epub ahead of print] Müller D, Klopocki E, Taupitz M, Mundlos S, Schulze I, Ropers HH, Querfeld U, Ullmann R (2006): A complex phenotype with Nissel R, Faraj S, Sommer K, Henning L, van der Giet M, cystic renal disease. Kidney Int 70: 1656-1660 Querfeld U (2008): Oxidative stress markers in young hemodialysis patients – a pilot study. Clin Nephrol 70, 2:135-143 Fliegauf M, Horvath J, von Schnackenburg C, Olbrich H, Müller D, Thumfart J, Schermer B, Pazour GJ, Neumann Robben J, Sze M, Eggert P, Knoers NV, Deen PM, Müller D HPH, Zentgraf H, Benzing T, Omran H (2006): Nephrocystin (2007): Relief of nocturnal enuresis by desmopressin is kidney specifically localizes to the transition zone of renal and res- and Vasopressin type-2 receptor independent. J Am Soc piratory cilia and photoreceptor connecting cilia. J Am Soc Nephrol 18:1534-1539 Nephrol 17: 2424-2433

Verberckmoes S, Persy V, Behets GJ, Hufkens A, Müller D, Pieper AK, Haffner D, Hoppe B, Dittrich K, Offner G, Bonzel Haffner D, Querfeld U, Bohic S, De Broe ME, D’Haese KE, John U, Frund S, Klaus G, Stubinger A, Duker G, PC (2007): Uremia-related Vascular Calcification: More than Querfeld U (2006): A randomized crossover trial comparing Apatite Deposition. Kidney Int 71: 298-303 sevelamer with calcium acetate in children with CKD. Am J Kidney Dis 47:625-35

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Briese S, Wiesner S, Will JC, Lembcke A, Opgen-Rhein B, Nis- Günzel D, Haisch L, Pfaffenbach S, Krug S, Milatz S, sel R, Wernecke KD, Andreae J, Haffner D, Querfeld U (2006): Amasheh S, Hunziker W, Müller D (2008): Claudin function in Arterial and cardiac disease in young adults with childhood- the thick ascending limb of Henle’s loop. Ann NY Acad Sci onset end-stage renal disease-impact of calcium and vitamin D 1165: 152-162 therapy. Nephrol Dial Transplant 21:1906-14 (2006) Thumfart J, Müller D (2008): Hämolytisch-urämisches Syn- Reviews & book chapters (2006-20010) drom im Kindesalter. Nephrologe 3: 297-307

Querfeld U: Dialyse im Kindesalter Müller D, Zimmering M, Chan CT, McFarlane PA, Pierratos In: F. Keller, M. Ketteler, R. Schindler, U. Heemann, G. Wolf A, Querfeld U (2008): Intensified hemodialysis regimens: (Hrsgb): Manuale Nephrologicum, 2nd edition, Dustri-Ver- Neglected treatment options for children and adolescents. lag Dr. Karl Feistle, München-Orlando, 2009, p. 427-443 Pediatr Nephrol 23: 1729-1736

Querfeld U: Urämische Vaskulopathie im Kindesalter Müller D (2007): Das hämolytisch-urämische Syndrom im In: Pourhassan S, Sandmann W (Hrsgb): Gefäßerkrankun Kindes- und Jugendalter. Päd 13: 403-406 gen im Kindes- und Jugendalter, p. 250-256, Springer- Verlag Berlin Heidelberg New York 2009 Giessing D, Müller D, Winkelmann B, Roigas J, Loening SA (2007): Kidney Transplantation in Children and Adolescents. Zipfel PF, Mache C, Müller D, Licht C, Wigger M, Skerka C Transplant Proceedings 39: 2197-2201 How Deficiency of CFHR1 and CFHR3 Plasma Proteins and Autoantibodies to Factor H Contribute Hemolytic Uremic Müller D (2007): Rapid progressive Glomerulonephritis: Syndrome: THE DEAP HUS Subtype- Form Pediatr Nephrol Schnittstelle zwischen pädiatrischer Nephrologie und in press pädiatrischer Rheumatologie. Aktuelle Rheumatologie 32: 149-153 Devuyst O, Meij I, Jeunemaitre X, Ronco P, Antignac C, Chris- tensen EI, Knoers NV, Levtchenko EN, Deen PM, Müller D, Winkelmann B, Thumfart J, Müller D, Giessing M, Wille A, Wagner CA, Rampoldi L, Van’t Hoff WG (2009): EUNEFRON, Deger S, Schnorr D, Querfeld U, Loening S, Roigas J.(2006): the European Network for the Study of Orphan Nephropa- Nierentransplantation im Kindes- und Jugendalter. Urologe thies Nephrol Dial Transplant 24: 2011-2015 A. 45: 18-24

Müller D, Müller DN (2009): Battle against the renin-agiotensin Müller D (2007): Nierenersatztherapie bei terminaler Niere- system: Help from an unexpected party. Nephrol Dial ninsuffizienz. In: S. Wirth (Hrsg.): Leitlinien Kinder- und Transplant 24: 1110-1112 Jugendmedizin P11: 36-39, Urban und Fischer, München

Will C, Fromm M, Müller D (2008): Claudin Tight Junction Querfeld U (2006): Adipocyte signaling: at the crossroads Proteins – Novel Aspects in paracellular transport. Peritoneal of metabolism, inflammation, and vascular function. Pediatr Dial Int 28: 577-584 Transplant Mar; 10(2) 136-9

Cardiovascular Disease in Pediatric Chronic Kidney Disease Querfeld U, Mitsnefes M (2008): In: Geary D, Schaefer FS (eds): Clinical Pediatric Nephrology, Mosby-Elsevier, Phila delphia p. 793-810

153 Uwe Querfeld – PEDIATRIC NEPHROLOGY

General information Third party funding (2006-2010)

Project leader Project title Sponsor Period Querfeld U Taurolidine-citrate vs heparin as catheter Tauropharm 2006-2010 lock solutions in paediatric patients Querfeld U The Cardiovascular Comorbidity in Kuratorium für Heimdialyse (KfH) 2009-2014 Children with Chronic Kidney Disease Stiftung Präventivmedizin („4C“) Study Müller D Spatio-temporal aspects in renal para- EU: The European Network of Orphan 2008-2012 cellular transport Nephropathies (EUNEFRON, FP 7) Müller D Evaluation of in-center nocturnal hemo- Else Kröner-Fresenius Stitung 2006-2009 dialysis Müller D Genetically modified mouse modelst o EU: European Renal Genome Project 2005-2009 study paracellular ion transport (EUREGENE, FP 6) Müller D Rolle in der Ionenhomöostase und Blut- DFG: Molekulare Struktur und Funk- 2006-2010 druckregulation tion der Tight Junction (FOR 721)

Awards (2006-2009)

2010 Else Kröner Fresenius-Preis Zentrumsbasierte intermittierende nächtliche Hämodialyse für Kinder und Jugendliche Gesellschaft für Pädiatrische Nephrologie Hamburg Anne Hoppe

2009 Arbeitsgemeinschaft Pflegekräfte in der Dialyse, 3. Preis Etablierung eine nächtlichen Hämodialyseprograms für Kinder und Jugendliche. Fulda Christina von Puttkamer

2009 Shortterm Fellowship European Network of Orphan Nephropathies (EUNEFRON) Brüssel Kathrin Kopplin

2009 Else Kröner Fresenius-Preis Gesellschaft für Pädiatrische Nephrologie Functional characterization of six alternative CLDN10 splice variants found in renal epithelia; 40. Jahrestagung der Gesellschaft für Pädiatrische Nephrologie Amsterdam Lea Haisch

154 Uwe Querfeld – PEDIATRIC NEPHROLOGY

2009 Reisestipendium der Gesellschaft für Pädiatrische Nephrologie An in-center based intermittent nocturnal hemodialysis program for children and adolescents, 40. Jahrestagung der Gesellschaft für Pädiatrische Nephrologie, Amsterdam Anne Hoppe

2009 Reisestipendium der Gesellschaft für Pädiatrische Nephrologie Generation and characterization of a CLDN-16 KO model 40. Jahrestagung der Gesellschaft für Pädiatrische Nephrologie Amsterdam Constanze Will

2008 Short term Fellowship European Renal Genome Project (EUREGENE) Paris Constanze Will

2007 Forschungsstipendium der Deutschen Nierenstiftung Ca2+- und Mg2+ Regulation am transgenen Tiermodell Jahrestagung der deutschen Gesellschaft für Nephrologie, München Julia Thumfart

2007 Else Kröner Fresenius-Preis Arbeitsgemeinschaft für pädiatrische Nephrologie Magnesium stimuliert die renale Phosphatexkretion 38. Jahrestagung der Arbeitsgemeinschaft für Pädiatrische Nephrologie Stuttgart Julia Thumfart

2006 Poster Preis Mouse models to study renal paracellular solute transport 2nd Summerschool of the European Renal Genome Project Oxford Constanze Will

155

VERA REGITZ-ZAGROSEK – GENDER in CARDIOVASCULAR MEDICINE

Head of the group

Prof. Dr. med. Vera Regitz-Zagrosek

Curriculum Vitae: She received her medical degree in 1979 from Saarland Uni- versity and completed her fellowship in clinical cardiology at Dept. of Biochemistry, University of Madison, Wisconsin. She also completed her residency at the German Heart Institute Munich, where she served as Chief Medical Resident. 1985 she became senior fellow at the German Heart Institute Berlin with responsibility for a large cardiovascular outpatient department. In 1995 she became professor in Internal Medicine and in 2002 she became the first and only German professor of Cardiovascular Disease in Women at the Charite Berlin in cooperation with the German Heart Institute Berlin. In 2003, she founded the Berlin Institute for Gender in Medicine (GiM) and is President of the International Society for Gender in Medicine (IGM). The aim of the GiM is the systematic analysis of sex and gender differences in medicine and its introduction into medical education. The research interests of Vera Regitz-Zagrosek include gender differences in cardiovascular disease, estrogen receptors and molecular and genetic mechanisms in heart failure. She leads and participates in major projects funded by European Union, Federal Ministry of Education and Research (BMBF), German Research Foundation (DFG) and in industry sponsored projects. She is the founder, project leader and speaker of the Graduate Course GK 754 “Sex-specific mechanisms of myocardial hypertrophy” (2001-2010) and project leader and speaker of the Research Group FOR 1054 on the same subject. She is project leader of EUGene Heart Task Gender and the pilot project “Gender Medicine” in cooperation with German Heart Institute Berlin. Since 2009 she is task force leader of the ESC’s Committee to develop the new version of the Guidelines for the Management of Cardiovascular Diseases during Preg- nancy (New Version 2011). She is reviewer for the German Research Foundation (DFG), the European Union and several journals. Her scientific publication record consists of over 150 scientific publications, more than 200 abstracts, and some book chapters. Her work has been published in numerous peer-reviewed journals including Nature Reviews.

157 VERA REGITZ-ZAGROSEK – GENDER in CARDIOVASCULAR MEDICINE

Members of the group

Office Students Schmidt, Stefanie Brunhuber, Claudia Medical student Brzank, Maria Medical student Scientists Eschen, Claudia PhD student Becher, Eva Dr. rer. nat. Fischer, Susanne Medical student Brožová, Eva Dr. rer. nat. Fritschka, Stephan PhD student Dworatzek, Elke Dr. rer. medic. Hampl, Hannah Medical student Fliegner, Daniela Dr. rer. nat. Leber, Joachim PhD student Kararigas, Georgios Dr. rer. nat. Penkalla, Adam Medical student Kendel, Friederike Psychologist Petrov, Georgi Medical student Lehmkuhl, Elke Dr. med. Pham, Thi Hang PhD student Mahmoodzadeh, Shokoufeh Dr. rer. nat. Queiros, Ana PhD student Sanchez Ruderisch, Hugo Dr. rer. nat. Schanz, Miriam Medical student Schubert, Carola Dr. rer. nat. Urbschat, Annika Medical student Westphal, Christina PhD student Technicians Zhang, Xiang Guest scientist, Angelov, Anja University of Bejing Brincker, Sven Kühne, Arne Fielitz, Britta Niehage, Sylke Riese, Vanessa Thomas, Jenny

158 VERA REGITZ-ZAGROSEK – GENDER in CARDIOVASCULAR MEDICINE

Summary

The group of Prof. Regitz-Zagrosek investigates the its influence on heart function. Furthermore, matrix gender specific aspects of cardiovascular disease, synthesis and its control by estrogen is analyzed. focussing on regulation of myocardial hypertrophy, Another goal is the identification of transcriptional heart failure and vascular mechanisms. Our projects factors and their potential binding sites within the stretch from basic research to clinical research and promoter region of matrixmetalloproteinases (MMP- clinical trials. 2), as well as the characterization of other matrix- - Research Group Myocardial Hypertrophy related genes. (DFG, FOR 1054) - EuGeneHeart European Excellence Project The main aim of the research group “Myocardial Heart Research Hypertrophy” (FOR 1054) is the analysis of gender The team of Prof. Regitz-Zagrosek participates in differences in the adaptation of the heart to mechan- the EU-funded EUGeneHeart project. The project ical stress. In this research group, we perform a aims at the elucidation of the mechanisms leading series of mechanical analysis using cell cultures and from myocardial hypertrophy to heart failure. Task animal models. This includes models of physiologi- number 4 “gender” is coordinated by Prof. Regitz- cal and pathological myocardial stress, e.g. strength Zagrosek and includes collaboration with research training and hypertension. The role of estrogen and groups at the INSERM (Institut national de la santé et androgen receptors is selectively analyzed. Moreo- de la recherche médicale) and the Karolinska Insti- ver, the effect of sex hormones on the lipid turnover tute. Aim is the investigation of gender differences in is investigated. Several collaborations with clinical myocardial hypertrophy induced by stress, training, projects exist; in this area we mainly focus on gender infarction and pressure. Experiments are performed differences in myocardial reaction to overload due to using cell cultures and animal models and focus on aortic valve disease. the role of sex hormones. - Graduate Group 754 - Gender differences in human aortic stenosis Graduate group GK 754 „Gender-specific mecha- In 2006 a single project has been designed to link nisms of myocardial hypertrophy“ (DFG) originates basic and clinical research in analysing gender dif- from the collaboration of 12 principal investigators ferences in human aortic stenosis. The project was within Charité, 8 at the Center for Cardiovascular funded by the DFG, German Research Foundation Research (CCR), and the German Heart Institute in (DFG-Re 662/6-1). Gender-specific differences Berlin. It comprises four interconnected modules: 1) after aortic valve transplantation are investigated in Animal models of hypertrophy, 2) functional genom- patients and in experimental animal models. ics and proteomics, 3) gender-specific molecular - CARDIOVASC mechanisms and 4) gender aspects in the clinics of Several projects at the CCR/GiM are conducted by cardiovascular diseases. In this project, the research investigators funded through the Marie Curie Early group of Prof. Regitz-Zagrosek is analyzing the sex- Stage Research Training Program CARDIOVASC. specific regulation of the energy metabolism and Cardiovascular diseases display significant sex and

159 VERA REGITZ-ZAGROSEK – GENDER in CARDIOVASCULAR MEDICINE

gender differences. Sexual hormones, especially collaboration with the German Heart Institute in Berlin. estrogens, are believed to play a major role in these We also collaborate with the German Competence differences. To define the effects of estrogen on tran- Network “Heart Failure” and were able to introduce scription regulation in the human heart, systematic our sex/gender-specific point of view, especially comparison between microarray gene expression through project TP13. We also work at the definition in estrogen-stimulated and non-stimulated cardiac of the young discipline of gender medicine and its cur- tissue was performed. This project should identify rent state-of-the-art through the Pilot project “Gender and elucidate molecular mechanisms influencing the Medicine” funded by The BMBF. Within this project incidence and progression of cardiac disease. we test scientific publications for gender relevance - Clinical Research and systematically classify the results. In the area of clinical research we conduct studies with primarily cardiologic focus and maintain intense

Zusammenfassung

Die Gruppe Prof. Regitz-Zagrosek untersucht Lipidstoffwechsel. In Kooperation mit verschiedenen Geschlechterunterschiede in der Entstehung von klinischen Projekten untersuchen wir insbesondere Herz-Kreislauferkrankungen mit besonderer Beach- die Reaktion des männlichen und weiblichen Myo- tung von Herzhypertrophie, Herzinsuffizienz und kards auf Druckbelastungen bei Erkrankungen der vaskulären Mechanismen. Unsere Projekte reichen Aortenklappe. von der Grundlagenforschung bis hin zur klinischen - Graduiertenkolleg 754 Forschung sowie klinischen Studien. Im GK 754 „Geschlechtsspezifische Mechanismen - Forschergruppe Myokardhypertrophie (FOR bei Myokard-Hypertrophie“ (DFG) kooperieren 1054) Arbeitsgruppen der Charité, davon 8 des Center Das vordringliche Ziel der Forschergruppe „Myo- for Cardiovascular Research (CCR), und des Deut- kardhypertrophie“ (FOR 1054) ist es, die Anpassung schen Herzzentrums Berlin. Es besteht aus vier des Herzens an mechanische Belastung in beiden eng miteinander verbundenen Modulen: 1) Tiermo- Geschlechtern zu untersuchen. Dazu werden eine delle bei Hypertrophie, 2) funktionelle Genomik und Reihe mechanistischer Untersuchungen an Zellkul- Proteomik, 3) geschlechtsspezifische molekulare tur- und Tiermodellen durchgeführt. Dies beinhal- Mechanismen und 4) klinische Geschlechtsaspekte tet Modelle für physiologische und pathologische bei Herz-Kreislauferkrankungen. In diesem Projekt Belastungen des Myokards, zum Beispiel Ausdau- analysiert die Arbeitsgruppe von Prof. Regitz-Zag- ertraining und Bluthochdruck. Die Rolle von Östro- rosek die geschlechtsspezifische Regulierung des gen- und Androgenrezeptoren sowie Sexualhormo- Energiemetabolismus und seinen Einfluss auf die nen wird gezielt analysiert. Darüber hinaus untersu- Herzfunktion. Darüber hinaus werden die Matrix- chen wir die Effekte von Sexualhormonen auf den synthese und ihre Kontrolle durch Östrogen analy-

160 VERA REGITZ-ZAGROSEK – GENDER in CARDIOVASCULAR MEDICINE siert. Ein weiteres Ziel ist es, Transkriptionsfaktoren - CARDIOVASC zu identifizieren und ihre mutmaßlichen Bindungs- Im Rahmen der Marie Curie Early Stage Research stellen innerhalb der Promoterregion von Matrix- Training Programme CARDIOVASC sind mehrere Metallproteinasen (MMP-2) und anderen matrixbe- Projekte im CCR/GiM angesiedelt. Herzkreislaufer- zogenen Genen zu charakterisieren. krankungen weisen deutliche Geschlechterunter- - EUGeneHeart - Europäisches Exzellenzpro- schiede auf. Es wird angenommen, dass Sexual- jekt Herzforschung hormone, speziell Östrogen hierbei eine wesentliche Die Arbeitsgruppe Regitz-Zagrosek nimmt an dem Rolle spielen. Um die Funktion von Östrogen für die durch die EU geförderten Projekt EUGeneHeart teil, Regulation der Transkription im humanen Herzen zu das sich mit dem Übergang der Herzhypertrophie zur untersuchen, wird in diesem Projekt ein systemati- Herzinsuffizienz befasst. Die Koordination des Tasks scher Vergleich der Genexpression von mit Östro- Nr. 4 “Geschlecht“ hat Prof. Regitz-Zagrosek inne. gen stimuliertem mit unstimuliertem Herzgewebe Es beinhaltet die Kooperation mit Forschergruppen ex vivo mittels DNA Microarrays vorgenommen. des INSERM (Institut national de la santé et de la Dieses Projekt soll molekulare Mechanismen, die recherche médicale) und des Karolinska Instituts bei zu geschlechtsspezifischen Unterschieden in Ent- der Untersuchung von Geschlechterunterschieden stehung und Verlauf von Herzerkrankungen führen, bei Myokardhypertrophie, die durch Stress, Trai- identifizieren und aufklären. ning, Infarkt oder Druckbelastung ausgelöst wird. - Klinische Forschung Dies geschieht in Zellkultur- und Tiermodellen und Im Rahmen der klinischen Forschung führen wir Studien fokussiert auf die Wirkung von Sexualhormonen. mit primär kardiologischem Fokus durch und pflegen - Geschlechterunterschiede bei menschlicher dabei sehr enge Kollaborationen mit dem Deutschen Aortenstenose Herzzentrum Berlin. Wir kooperieren weiterhin mit dem Im Jahre 2006 wurde in der Schnittstelle zwischen Kompetenznetz „Herzinsuffizienz“ und bringen dabei Grundlagen- und klinischer Forschung ein DFG- unseren geschlechtsspezifischen Gesichtspunkt ein, Einzelprojekt (DFG-Re 662/6-1) konzipiert und ein- besonders durch das Teilprojekt 13. geworben, in dem es um Geschlechterunterschiede Weiterhin befassen wir uns in einem vom BMBF bei menschlicher Aortenstenose geht. Hier werden geförderten Projekt, mit der Definition des jungen sowohl geschlechtsspezifische Verläufe nach Aor- Fachbereiches Gendermedizin und dessen aktuellen tenklappenersatz am Patienten untersucht als auch Standes durch das Pilotprojekt „Gender Medicine“, experimentelle Untersuchungen am Tiermodell bei dem Fachliteratur auf ihre Geschlechtsspezifik hin durchgeführt. überprüft und systematisch analysiert wird.

161 VERA REGITZ-ZAGROSEK – GENDER in CARDIOVASCULAR MEDICINE

Research projects

1. 17β-oestradiol (E2) effects and mechanisms of sex-specific gene regulation in the heart

Project leader Georgios Kararigas Coworkers Eva Becher, Daniela Fliegner, Shokoufeh Mahmoodzadeh, Karina Nawrath, Vanessa Riese Funding EC: MEST-CT-2005-020268 CARDIOVASC (2006-2009) EC: EUGeneHeart EC-018833 (WP18; currently) Boehringer Ingelheim Fonds (2007) External cooperations Dr. Ba Tiep Nguyen & Prof. Hubertus Jarry, Goettingen University Dr. Virginie Bito & Prof. Karin Sipido, Leuven University Prof. Roland Hetzer, German Heart Institute Berlin

Cardiovascular disease (CVD) manifests differently in so far there has been no report of E2-dependent gene men and women. Sex steroid hormones are gener- regulation in a sex-specific manner in the heart, while ally believed to play a major role in the occurrence such effects have been described in other organs. of sex-related differences observed in the develop- The main goal here is to investigate the effect(s) of ment of CVD. In particular, a large body of evidence E2 in the heart. To achieve this, we are comparing from several observational and experimental studies the transcriptomes of human cardiac tissues and of indicates that E2 might be involved in reducing risk hearts from mice treated with E2. We combine these for cardiovascular disease. However, harmful effects analyses with data collected from in vivo studies and of E2 have also been reported leading to the current assays on cellular effects generated in vitro. controversy regarding the actions of E2. In addition,

Sex-specific PGR (progesterone receptor) regulation by E2 in human cardiac tissues. (HBC: control) From Kararigas et al. Biology of Sex Differences 2010 Accepted

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2. Role of sex and sex hormones on extracellular matrix remodelling in pressure overload induced myocardial hypertrophy

Project leader Elke Dworatzek Coworkers George Petrov, Carola Schubert, Shokoufeh Mahmoodzadeh, Elke Lehmkuhl, Irina Wenzel, Anja Angelov Funding DFG: GK II/III 754 TP4 DFG: Re662/6-1

Patients with pressure overload induced left ventricular collagen I and III expression in isolated cardiac fibrob- (LV) hypertrophy show sex-differences in cardiac func- last. Furthermore, we showed for the first time the tion and extracellular matrix (ECM) remodeling, with inhibitory effect of E2 on MMP-2 expression in cardiac more pronounced fibrosis in male hearts. Goal of the fibroblast via the activation of estrogen receptors (ER) study was to identify clinical parameters that determine and MAP kinase-ERK1/2 signalling (Figure 1). Based LV hypertrophy, and to correlate underlying mecha- on these data, we speculate that E2 regulates ECM nisms of ECM remodeling with clinical endpoints under turnover and may be, at least partially, responsible for gender specific aspects. In order to investigate the role sex-differences in cardiac remodelling. Further work of 17β-Estradiol (E2) in ECM remodeling, we performed will analyze the sex-differences in ECM remodeling in in-vitro studies in isolated adult rat cardiac fibroblasts. LV hypertrophy more detailed, and identify underlying We found that women with aortic (AS) adapt differently molecular mechanisms by which E2 regulates ECM to pressure overload compared to men. Male patients components in a sex-specific manner. This could con- had a significant higher gene expression of ECM- tribute to a better understanding of sex-differences in associated genes, e.g. collagen I and III compared to cardiac remodeling and help to design sex-specific females. We could show a sex-specific regulation of pharmacological interventions.

Proposed model of the E2 regulation on human MMP-2 promoter activity by ER alpha and MAP kinase-ERK1/2 signaling pathway (Mahmoodzadeh S. & Dworatzek E. et al., Cardiovas- cular Research 2010).

163 VERA REGITZ-ZAGROSEK – GENDER in CARDIOVASCULAR MEDICINE

3. Estrogen receptor modulation of myocardial energy metabolism contributes to sex differences in myocardial hypertrophy

Project leader Vera Regitz-Zagrosek, Shokoufeh Mahmoodzadeh Coworkers Elke Dworatzek, Eva Brozova, Georgios Kararigas, Karina Nawrath, Carola Schubert, Sylke Niehage Funding DFG: FOR 1054 / TP1

Females develop a more physiological myocardial chondrial function and metabolic genes are probably hypertrophy (MH) than males. Male hearts have a involved in these processes. Therefore in this study, higher incidence of cardiac dysfunction and heart we investigate: failure in response to stress. Exercise leads to more 1) sex differences in cardiac function in physiological physiological MH in female than in male mice. Female (exercise) MH model. 2) Effect of E2 and pharmacologi- sex or estrogens may lead to an improved cardiac cal/mechanical load on cellular growth and metabolic response to stress. Based on our previous findings, we genes. 3) Effect of E2-activated ERa and ERb on gene hypothesize that estrogen (E2) and its receptors ERa expression and biological activity of PGC1a and MEF2 and ERb improve the cardiac response to mechanical transcription factors that control mitochondrial activity. load by regulating mitochondrial function and energy The project will identify sex-specific MH mechanisms metabolism. AKT signalling and key regulators of mito- with therapeutic potential relevant to both genders.

A: Experimental setup for voluntary cage running, B: Female mice show higher exercise performance and C: a significant increase in left ventricular mass / Tibila length.

164 VERA REGITZ-ZAGROSEK – GENDER in CARDIOVASCULAR MEDICINE

4. Analysis of the role of estrogen receptor a in the heart in a transgenic mouse model

Project leader Shokoufeh Mahmoodzadeh Coworkers Joachim Leber, Britta Fielitz, Arne Kühne, Funding DFG: GK III 754 TP4 EC: EUGeneHeart EC-018833 (WP17) External cooperations Dr. F. Jaisser, INSERM, France

Estrogen exerts its beneficial effects on the myocar- notype of a transgenic mouse model with an inducible, dium during stress via estrogen receptors (ER) a and myocardial ERa-overexpression (ERa-OE). Further- b. During and after myocardial infarct (MI), administra- more, we perform functional and biochemical analysis tion of ERa-selective agonist has protective effects in of the transgenic mice in unstressed condition and the heart. ERa-KO animals have a poorer outcome after induction of MI, in a sex-specific context. We and abnormal mitochondrial morphology after MI. expect major deviations from WT at base line condi- We hypothesize that ERa contributes to protection tions and after induction of MI, possibly leading to the of ischemic myocardium and this differs in male and discovery of major cellular pathways influenced by ERa female. we therefore generate and evaluate the phe- and thereby to new therapeutic targets.

Transactivator mouse

MHC tTA

tetO ERα Responder mouse

tetO ERα Conditional mouse model (generated by Frederic Jaisser, INSERM, Paris)

A: Mouse model with inducible heart specific C expression of the ERα gene using the Tet-Off B system (collaboration with Dr. F. Jaisser, myocardial infarction INSERM, Paris). B: Westernblot analysis Western-Blot ERa of heart tissue from double transgenic M WT ERα-overexpresser

(ERα-OE) and wild type mice using antibody ER α, against ERα. C: Representative micrographs 66 kD of a left ventricular cross section of Masson trichrome staining at 14 days post-MI.

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5. Sex differences and the influence of estrogen receptor b

Project leader Daniela Fliegner, Carola Schubert Coworkers Adam Penkalla Funding DFG: GK 754 II/III TP2 EC: EuGeneHeart EC-018833 (WP19) Charité: Post-doctoral fellowship External Collaborations Prof. Dr. Ulrich Kintscher (CCR), Prof. Jan-Ake Gustafsson, Karolinska Institute Stockholm

Myocardial hyper- tribute to the maintenance of energy homeostasis and trophy (MH) rep- attenuate the development of fibrosis and apoptosis, resents the car- thus slowing the progression to heart failure. diac response to different stimuli, Current ongoing studies are focused now on the such as pressure effects of sex and sexual hormones on the cardiac Development of myocardial overload, hyper- mitochondrial function in physiological and pathologi- hypertrophy. tension, diet or cal conditions. aging and can result in heart failure (HF). The development of MH is associated with alterations in cardiac geometry (size and shape), which is characteristically referred to as ventricular remodeling and appears to be different between the sexes. Sex differences in the cardiovascular system have largely been attributed to the effects of sex steroid hormones such as estrogen, which are mainly mediated by their nuclear receptors: ERα, ERβ. The goal of this study was to investigate the molecular mechanisms underlying these sex differences. We used a mouse model of pressure overload induced myocardial hypertrophy in both sexes with or without deletion of ERb. In this project our findings indicate that female sex offers protection against ventricular chamber dilation in Genes involved in oxidative phosphorylation were regulated in a sex-specific the TAC model. Both female sex and ERb con- manner. (Fliegner et al. Am J Physiol Regul Integr Comp Physiol (June 2010).

166 VERA REGITZ-ZAGROSEK – GENDER in CARDIOVASCULAR MEDICINE

6. Influence of different estrogen receptor modulators on pressure overload-induced myocardial hypertrophy

Project leader Carola Schubert Coworkers Christina Westphal Funding DFG: GK 754 II/III TP2 EC: EuGeneHeart EC-018833 (WP19) Bayer Schering AG External Collaborations Dr. Katja Prelle (Bayer Schering AG)

Estrogens influence many physiological processes In the current project we wanted to identify the estro- in mammals, including cardiovascular health. It is gen receptor, which is responsible for the protection of also implicated in the development or progression the heart against pressure-overload. Ovariectomiced of cardiovascular diseases. In many of the diseases, (OVX) female mice were pressure loaded by trans- 17b-estradiol (E2) mediates its effects through its estro- verse aortic constriction (TAC) and treated with E2 gen receptors a and b (ERa and ERb), which serves and specific estrogen receptor agonists (ERaA and as the basis for many therapeutic interventions. Selec- ERbA) or raloxifen to prevent cardiac hypertrophy and tive estrogen receptor modulators (SERMs) such as heart failure. tamoxifen and raloxifen are examples of compounds that exhibit tissue-specific estrogenic activity. They may be useful for the reduction of cardiovascular risk.

Time flow of the animal experiment (OVX/ovariectomy; PI/pellet implantation with treatment substances; TAC/transverse aortic constriction; Echo/echocardiographic measurements; OW/organ withdrawal)

167 VERA REGITZ-ZAGROSEK – GENDER in CARDIOVASCULAR MEDICINE

7. Sex specific differences in microRNA expression and their role in heart diseases

Project leader Hugo Sanchez-Ruderisch Coworkers Claudia Eschen, Ana Queiros, Daniela Fliegner Funding DFG: GK 754 II/III

MicroRNAs (miRs) are small non-coding RNAs that partially rescued by estrogen administration. Based control gene expression by duplex formation with on these data and on our previous results showing 3´ untranslated regions of target mRNAs. Dysregu- gender differences after TAC we hypothesise that an lation of miRs has been reported in various cardiac estradiol-dependent gender different expression of diseases. Most of the miRNAs expressed in mouse miRs is in part responsible for the observed gender cardiac tissue are regulated during cardiomyocytes different development of cardiac hypertrophy after hypertrophy. However, nothing is known about the pressure overload. The aims of this project are the regulation of expression of miRs considering the gen- identification of those miRNAs expressed in a gender- der aspect. Gender differences in the development specific manner in the mice heart and elucidate their and progression of cardiovascular diseases such as role in heart hypertrophy comparing male and female myocardial infarction, hypertension, heart failure and Endogenousmice after TAC miR-1 treatment. inhibits Preliminary the expression results indicated of miR1-targeted Renilla in sudden death have been described. In mouse model sex different expression of variousHL-1 miRs. cells The influence of cardiomyopathies, a more severe cardiovascular of altered miRs expression on putative target genes phenotype is observed in male animals and can be involved in fibrosis is under current investigation.

2,5

2,0

1,5

(Renilla/Luc) 1,0

RLU 0,5

0,0 psiCHECK psiCheckmiR1 psiCheckmiR1 + mimic1

168 VERA REGITZ-ZAGROSEK – GENDER in CARDIOVASCULAR MEDICINE

8. Melusin protects against cardiac rupture and maladaptive remodeling after myocardial infarction sex dependently

Project leader Vera Regitz-Zagrosek, Carola Schubert Coworkers Susette Bernert, Jenny Thomas Funding EC: EUGeneHeart EC-018833 (WP3.1) External cooperations MD Bernhard Unsöld (Göttingen), Prof. Karin Sipido (Leuven), Prof. Guido Tarone (Turin)

The muscle specific ß1 integrin interacting protein cardiac remodeling and early death after MI and sex Melusin has been found to be upregulated during differences therein. For that reason an animal model cardiac hypertrophy and protects against the transi- for MI was performed in male and female WT mice tion into heart failure after pressure overload. Attenu- and in mice overexpressing the melusin gene (TG). ation of cardiac hypertrophy was accompanied by Animals were killed 3 or 14 days after induction of MI. lower tissue deposition and apoptosis of cardiomyo- The experiments showed that melusin protects male cytes. A careful analysis of the influence of melusin on animals against early cardiac rupture by decreasing remodeling processes after myocardial infarction (MI) MMP9 activity. In later maladaptive processes follow- and sex differences therein is needed. We wanted to ing MI the TG affects mainly females and protects them understand the protective effects of melusin against against heart failure, while in males it does not.

Survival after MI in wildtype (WT) and melusin transgene (TG) animals

169 VERA REGITZ-ZAGROSEK – GENDER in CARDIOVASCULAR MEDICINE

Publications 2006 - 2010 Kendel F, Dunkel A, Lehmkuhl E, Hetzer R, Regitz-Zagrosek V (2008): Does time spent on household activities or housework Mahmoodzadeh S, Eder S, Nordmeyer J, Ehler E, Huber O, stress complicate recovery following coronary artery bypass Martus P, Weiske J, Pregla R, Hetzer R, Regitz-Zagrosek V surgery? Women Health 48(3) 325-38 (2006): Estrogen receptor alpha up-regulation and redistribution in human heart failure. Faseb J 20(7) 926-34 Witt H, Schubert C, Jaekel J, Fliegner D, Penkalla A, Tiemann K, Stypmann J, Roepcke S, Brokat S, Mahmoodzadeh S, Brozova Philipp S, Jürgensen JS, Fielitz J, Bernhardt WM, Weidemann A, E, Davidson MM, Ruiz Noppinger P, Grohé C, Regitz-Zagrosek V Schiche A, Pilz B, Dietz R, Regitz-Zagrosek V, Eckardt KU, Willen- (2008): Sex-specific pathways in early cardiac response to pres- brock R (2006): Stabilization of hypoxia inducible factor rather than sure overload in mice. J Mol Med 86(9) 1013-24 modulation of collagen metabolism improves cardiac function after acute myocardial infarction in rats. Eur J Heart Fail 8(4) 347-54 Boengler K, Buschmann I, Deindl E, Gottwik M, Hoffmeister HM, Ito W, Klein H, Mauser M, Nienaber C, Regitz-Zagrosek V, Regitz-Zagrosek V, Hocher B, Bettmann M, Brede M, Had- Sack S (2009): On the occasion of Wolfgang Schaper’s 75th amek K, Gerstner C, Lehmkuhl HB, Hetzer R, Hein L (2006): birthday. Basic Res Cardiol 104(1) 2-4 {alpha}2C-Adrenoceptor polymorphism is associated with improved event-free survival in patients with dilated cardiomy- Dunkel A, Kendel F, Lehmkuhl E, Babitsch B, Oertelt-Prigione opathy. Eur Heart J 27(4) 454-459 S, Hetzer R, Regitz-Zagrosek V (2009) Predictors of preoperative depressive risk in patients undergoing coronary artery Schupp M, Kintscher U, Fielitz J, Thomas J, Pregla R, Hetzer R, bypass graft surgery. Clin Res Cardiol 98 643-650 Unger T, Regitz-Zagrosek V (2006): Cardiac PPARalpha expression in patients with dilated cardiomyopathy. Eur J Heart Fail 8(3) 290-4 Friedrichs F, Zugck C, Rauch GJ, Ivandic B, Weichenhan D, Müller-Bardorff M, Meder B, El Mokhtari NE, Regitz-Zagrosek Brokat S, Thomas J, Herda LR, Knosalla C, Pregla R, Bran- V, Hetzer R, Schäfer A, Schreiber S, Chen J, Neuhaus I, Ji R, caccio M, Accornero F, Tarone G, Hetzer R, Regitz-Zagrosek Siemers NO, Frey N, Rottbauer W, Katus HA, Stoll M (2009): V (2007): Altered melusin expression in the hearts of aortic HBEGF, SRA1, and IK: Three cosegregating genes as determi- stenosis patients. Eur J Heart Fail 9(6-7) 568-73 nants of cardiomyopathy. Genome Res 19(3) 395-403

Diedrich M, Tadic J, Mao L, Wacker MA, Nebrich G, Hetzer R, Hassel D, Dahme T, Erdmann J, Meder B, Huge A, Stoll M, Regitz-Zagrosek V, Klose J (2007): Heart protein expression Just S, Hess A, Ehlermann P, Weichenhan D, Grimmler M, related to age and sex in mice and humans. Int J Mol Med Liptau H, Hetzer R, Regitz-Zagrosek V, Fischer C, Nürnberg P, 20(6) 865-74 Schunkert H, Katus HA, Rottbauer W (2009): Nexilin mutations destabilize cardiac Z-disks and lead to dilated cardiomyopathy. Fielitz J, Philipp S, Herda LR, Schuch E, Pilz B, Schubert C, Nat Med 15(11) 1281-8 Günzler V, Willenbrock R, Regitz-Zagrosek V. (2007): Inhibition of prolyl 4-hydroxylase prevents left ventricular remodelling in Mahmoodzadeh S, Fritschka S, Dworatzek E, Pham TH, Becher rats with thoracic aortic banding. Eur J Heart Fail 9(4):336-42 E, Kuehne A, Davidson MM, Regitz-Zagrosek V (2009): Nuclear factor-Kappa B regulates estrogen receptor-alpha transcription Fliegner D, Westermann D, Riad A, Schubert C, Becher E, Fielitz in the human heart. J Biol Chem 284(37) 24705-24714 J, Tschöpe C, Regitz-Zagrosek V (2008): Up-regulation of PPAR- gamma in myocardial infarction. Eur J Heart Fail 10(1) 30-8 Fliegner D, Schubert C, Penkalla A, Witt H, Kararigas G, Dworatzek E, Staub E, Martus P, Ruiz Noppinger P, Kintscher Isensee J, Witt H, Pregla R, Hetzer R, Regitz-Zagrosek V, Nop- U, Gustafsson JA, Regitz-Zagrosek V (2010): Female sex and pinger PR (2008): Sexually dimorphic gene expression in the estrogen receptor-beta attenuate cardiac remodeling and heart of mice and men. J Mol Med 86(1) 61-74 apoptosis in pressure overload. Am J Physiol-Reg I 298(6) R1597-606 Karatas A, Hegner B, de Windt LJ, Luft FC, Schubert C, Gross V, Akashi YJ, Gürgen D, Kintscher U, da Costa Goncalves AC, Kendel F, Wirtz M, Dunkel A, Lehmkuhl E, Hetzer R, Regitz- Regitz-Zagrosek V, Dragun D (2008): Deoxycorticosterone Zagrosek V (2010): Screening for depression: Rasch analysis acetate-salt mice exhibit blood pressure-independent sexual of the dimensional structure of the PHQ‑9 and the HADS-D. J dimorphism. Hypertension 51(4) 1177-83 Affect Disorders 122 241-246

170 VERA REGITZ-ZAGROSEK – GENDER in CARDIOVASCULAR MEDICINE

Mahmoodzadeh S, Dworatzek E, Fritschka S, Pham TH, Gohlke-Bärwolf C, Pildner von Steinburg S, Kaemmerer H, Regitz-Zagrosek V (2010): 17{beta}-estradiol inhibits matrix Regitz-Zagrosek V (2008): Anticoagulation and thrombophilia in metalloproteinase-2 transcription via MAP kinase in fibroblasts. pregnancy. Internist 49(7) 779-87 Cardiovasc Res 85(4) 719-728 Regitz-Zagrosek V, Gohlke-Bärwolf C, Geibel-Zehender A, Regitz-Zagrosek V, Petrov G, Lehmkuhl E, Smits JM, Babitsch Haass M, Kaemmerer H, Kruck I, Nienaber C (2008): Heart B, Brunhuber C, Jurmann B, Stein J, Schubert C, Merz NB, diseases in pregnancy Clin Res Cardiol 97(9) 630-65 Lehmkuhl HB, Hetzer R (2010): Heart transplantation in women with dilated cardiomyopathy. Transplantation 89(2) 236-44 Regitz-Zagrosek V, Lehmkuhl E, Lehmkuhl HB (2008): Herzin- suffizienz: geschlechts-spezifische Aspekte? Internist 49(4) Ruiz-Holst C, Bölck B, Ghanem A, Tiemann K, Brokat S, 422-8 Regitz-Zagrosek V, Bloch W, Schwinger RHG, Brixius K (2010): eNOS phosphorylation and translocation are altered in male Regitz-Zagrosek V, Schubert C, Krüger S (2008): Gender differ- but not female mice by increased activation of the Galphaq ences in psychopharmacology Internist 49(12) 1516-23 protein. Can J Physiol Pharm 88(2) 121-9 Regitz-Zagrosek V, Schubert C, Krüger S (2008): Sex differ- Toischer K, Rokita AG, Unsöld B, Zhu W, Kararigas G, Sossalla ences in cardiovascular drug targeting Internist 49(11) 1383- S, Reuter SP, Becker A, Teucher N, Seidler T, Grebe C, Preuß 6, 1388-90 L, Gupta SN, Schmidt K, Lehnart SE, Krüger M, Linke WA, Backs J, Regitz-Zagrosek V, Schäfer K, Field LJ, Maier LS, Mehrhof F, Löffler M, Gelbrich G, Özcelik C, Posch M, Hense Hasenfuss G (2010): Differential Cardiac Remodeling in Preload HW, Keil U, Scheffold T, Schunkert H, Angermann C, Ertl Versus Afterload. Circulation 122 993-1003 G, Jahns R, Pieske B, Wachter R, Edelmann F, Wollert KC, Maisch B, Pankuweit S, Erbel R, Neumann T, Herzog W, Katus Petrov G, Regitz-Zagrosek V, Lehmkuhl E, Krabatsch T, Dunkel H, Müller-Tasch T, Zugck C, Düngen HD, Regitz-Zagrosek V, A, Dandel M, Dworatzek E, Mahmoodzadeh S, Schubert C, Lehmkuhl E, Störk S, Siebert U, Wasem J, Neumann A, Göhler Becher E, Hampl H, Hetzer R (2010): Regression of Myocar- A, Anker S, Köhler F, Möckel M, Osterziel KJ, Dietz R, Rauch- dial Hypertrophy After Aortic Valve Replacement. Circulation haus M, on B.o.t.C.N.H.F (2009): A network against failing 122(suppl 1) 23-28 hearts-Introducing the German “Competence Network Heart Failure” Int J Cardiol

Reviews & book chapters (2006-20010) Oertelt-Prigione S, Regitz-Zagrosek V (2009): Women’s cardiovascular health: prevention is key. Arch Intern Med 169(19) Regitz-Zagrosek V (2006): Therapeutic implications of the 1740-1 gender-specific aspects of cardiovascular disease. Nat Rev Drug Discov 5(5) 425-38 Regitz-Zagrosek V, Oertelt-Prigione S, Seeland U, Hetzer R (2010): Sex and gender differences in myocardial hypertrophy Regitz-Zagrosek V, Lehmkuhl E, Weickert MO (2006): Gender and heart failure. Circ J 74(7) 1265-73 differences in the metabolic syndrome and their role for cardiovascular disease. Clin Res Cardiol 95(3) 136-47 Sliwa K, Hilfiker-Kleiner D, Petrie MC, Mebazaa A, Pieske B, Buchmann E, Regitz-Zagrosek V, Schaufelberger M, Tavazzi Regitz-Zagrosek V, Brokat S, Tschöpe C (2007) : Role of L, van Veldhuisen DJ, Watkins H, Shah AJ, Seferovic PM, gender in heart failure with normal left ventricular ejection frac- Elkayam U, Pankuweit S, Papp Z, Mouquet F, McMurray JJV tion. Prog Cardiovasc Dis 49(4) 241-51 (2010): Current state of knowledge on aetiology, diagnosis, management, and therapy of peripartum cardiomyopathy: a Regitz-Zagrosek V, Wintermantel TM, Schubert C (2007): position statement from the Heart Failure Association of the Estrogens and SERMs in coronary heart disease. Curr Opin European Society of Cardiology Working Group on peripartum Pharmacol 7(2) 130-9 cardiomyopathy. Eur J Heart Fail 12(8) 767-78

171 VERA REGITZ-ZAGROSEK – GENDER in CARDIOVASCULAR MEDICINE

General information Third party funding (2006-2010)

Project leader Project title Sponsor Period Regitz-Zagrosek V. Kompetenznetzwerk Herzinsuffizienz (KNHI TP4) BMBF 2003-2008 Regitz-Zagrosek V. Research Training Programm GK 754/2: Gesch- German Research Foundation 2004-2006 Mahmoodzadeh S. lechtsspezifische Mechanismen bei Myokard- hypertrophie Regitz-Zagrosek V. Genstudie Institut für Herz-Kreislaufforschung an 2005-2008 der Universität Witten/Herdecke Regitz-Zagrosek V. IP EUGeneHeart, Task Gender European Commission 2006-2010 Mahmoodzadeh S. EC: 018833 Schubert C. Regitz-Zagrosek V. Research Training Programm GK 754/3: Gesch- German Research Foundation 2006-2010 Schubert C. lechtsspezifische Mechanismen bei Myokard- Mahmoodzadeh S. hypertrophie Regitz-Zagrosek V. Klinische Studie von herzoperierten Frauen Margarete-Ammon-Stiftung 2007-2010 Regitz-Zagrosek V. Kompetenznetzwerk Herzinsuffizienz (KNHI BMBF 2006-2008 TP13) Regitz-Zagrosek V. Satellitensymposium 2007 Regitz-Zagrosek V. GiM Symposium Bundesministerium für Familie, 2008 Senioren & Frauen Regitz-Zagrosek V. Einfluss des Geschlechts bei myokardialer German Research Foundation DFG Re 2007-2010 Mahmoodzadeh S. Druckbelastung 662/6-1 Regitz-Zagrosek V. Research Group: Sex-specific mechanisms in German Research Foundation 1054 2008-2010 Mahmoodzadeh S. myocardial hypertrophy TP1 + Z2 Regitz-Zagrosek V. Crataegus Industry: Dr. Willmar Schwabe 2009-2010 Schubert C. GmbH & Co. KG, Karlsruhe Regitz-Zagrosek V. Research Training Programm GK 754/4: Gesch- German Research Foundation 2010-2011 lechtsspezifische Mechanismen bei Myokard- hypertrophie Regitz-Zagrosek V. Pilotprojekt „Gender Medicine“ (PPGM) BMBF 2008-2011 Regitz-Zagrosek V. EUGIM FP7 2009-2011 Becher E.

Awards 2008 Margherita-von-Brentano-Preis der FU Berlin Joachim Dudenhausen, Marianne Kriszio, Mechthild Koreuber, Vera Regitz-Zagrosek, Martina Dören, Martin Paul

172 MICHAEL SCHUPP – ENDOCRINOLOGY

Head of the group

Michael Schupp, PhD. Emmy-Noether group leader Dept. Endocrinology, Diabetes and Nutrition

Curriculum Vitae: Michael Schupp studied pharmacy in Regensburg, Cardiff, Lon- don, Madrid and Munich. He earned his PhD in 2005 under the supervision of Prof. U. Kintscher and Prof. T. Unger at the Institute of Pharmacology, Charité University Medicine Berlin. Parts of his doctoral thesis were carried out in Prof. B. Staels group at the Pasteur Institute in Lille. He then spent 5 years as a postdoctoral fellow at the University of Pennsylvania, Philadelphia in the laboratory of Prof. M. Lazar in the Division of Endocrinology, Diabetes and Metabolism. He returned August 2010 to Germany to install an Emmy-Noether group at the Charité, Division of Endocrinology, Diabetes and Nutritional Medicine, located in the CCR.

Members of the group

Witte, Nicole Pharmacist, PhD student Münzner, Matthias Technichal Assistant

173 MICHAEL SCHUPP – ENDOCRINOLOGY

Summary is induced during adipocyte differentiation. Strategies that modify the activity of this enzyme could be used After my return from a 5-year postdoctoral fellowship at for the treatment of metabolic diseases. the University of Pennsylvania (Prof. Mitchell A. Lazar laboratory) in August 2010, I am establishing my own We also study the mechanisms by which certain tran- research lab here at the CCR. As part of the Dept. scription factors determine the fate of mesenchymal of Endocrinology (Prof. Andreas Pfeiffer and Prof. stem cells. These cells can give rise to a variety of line- Jochen Spranger), our lab is aimed at understanding ages such as adipocytes, osteoblasts, chondrocytes or the function of the enzyme Retinol Saturase (RetSat) in myogenic precursors. We are particularly interested in the development of obesity and type 2 diabetes. The the interaction of these transcriptional regulators with enzyme catalyzes the reduction of retinol to 13,14-dihy- cofactors, which mediate their activity in the modification droretinol and possibly functions in other yet unknown of histones. We are trying to understand how these tran- reactions. RetSat is expressed at high levels in meta- scriptional networks commit cells to a defined lineage by bolically active tissues like liver and fat. Liver expression the induction of the determining factors PPARg, Runx2, of RetSat is strongly regulated by fasting and feeding, MyoD, Sox proteins, and others. Reprogramming of suggesting that it is involved in hepatic glucose and these pathways may allow us to control disease related fatty acid metabolism. Furthermore, RetSat expression maladaptation in mesenchymal differentiation programs.

Zusammenfassung therapeutische Anwendung bei metabolischen Krank- Nach der Rückkehr von einem 5-jährigen Aufenthalt an heiten haben könnte. der University of Pennsylvania (Prof. Mitchell A. Lazar laboratory) im Sommer 2010, etabliere ich meine Emmy- Weiterhin untersuchen wir die Mechanismen, durch Noether Nachwuchsgruppe hier am CCR. Zugehörig die bestimme Transkriptionsfaktoren die Differenzie- zur Abteilung für Endokrinologie, Diabetes und Ernäh- rung mesenchymaler Stammzellen beeinflussen. Diese rungsmedizin (Prof. Andreas Pfeiffer und Prof. Jochen pluripotenten Zellen können zu verschiedensten Zell- Spranger), untersucht unser Labor die Rolle des Enzy- typen, wie Chondrozyten, Adipozyten, Osteoblasten mes Retinol Saturase (RetSat) in der Entwicklung von oder Myoblasten, differenzieren. Wir sind besonders Typ-2 Diabetes und der Adipositas. RetSat reduziert in an den Wechselwirkungen dieser Transkriptionsfakto- einer NADH-abhängigen Reaktion Retinol zu 13,14-Dihy- ren mit Kofaktoren interessiert, die deren Aktivität in die droretinol. Außerdem scheint das Enzym eine weitere, Modifizierung von Histonen vermitteln. Wir versuchen bisher unbekannte enzymatische Reaktion zu katalysie- zu verstehen, wie diese transkriptionellen Netzwerke zur ren. RetSat wird in metabolisch aktiven Organen, wie Festlegung auf ein spezielles Differenzierungsprogramm z.B. der Leber oder im Fettgewebe, stark exprimiert. Die durch die Expression von PPARg, Runx2, MyoD oder Sox hepatische Expression der RetSat wird zusätzlich durch Proteinen führen. Eine Manipulation dieser Signalwege Nahrungsaufnahme reguliert. Wir erforschen, ob eine könnte uns ermöglichen, krankheitsabhängige Störun- Modulation der enzymatischen Aktivität der RetSat eine gen in der Zelldifferenzierung zu kontrollieren.

174 MICHAEL SCHUPP – ENDOCRINOLOGY

Research project

Role of Retinol Saturase in Liver metabolism

Project leader Michael Schupp Coworkers Nicole Witte, Matthias Münzner Collaborators Prof. Ulrich Kintscher, MD (CCR), Prof. Jochen Spranger, MD (Endocrinology) Funding - DFG SCHU 2546/1-1 External cooperations - Prof. Lorraine J. Gudas, PhD. and Prof. Stephen L. Gross. PhD., Department of Pharmacology, Weill Cornell Medical College New York, United States (Retinoid chemistry, MassSpec) - Dr. Andreas Birkenfeld MD, DIfE Potsdam, Germany (euglycemic-hyperinsulinemic clamp studies)

We initially identified the oxidoreductase RetSat as regulated by PPARa, the key transcription factor in an upregulated gene during adipocyte differentiation. the hepatic fasting response via binding to the same Depletion of RetSat by siRNA in preadipocytes inhib- intronic binding site. Accordingly, it was shown that the ited adipocyte conversion. 13,14-dihydroretinol, the fasting induced increase in hepatic RetSat expression putative product of RetSat’s enzymatic activity, did was blunted in PPARa (-/-) mice. not rescue this defect in adipocyte differentiation, sug- The molecular function of RetSat in liver is unknown. gesting that RetSat catalyzes an additional enzymatic Preliminary studies suggest that RetSat is involved in reaction. Furthermore, we found that adipose RetSat the induction of gluconeogenic gene expression and expression is under the control of the Peroxisome intermediary metabolism during fasting. We will use Proliferator-activated Receptor g (PPARg), the molec- molecular-, cell-, and animal experiments to under- ular target of insulin-sensitizing glitazones, through an stand the physiology of hepatic RetSat in the context intronic binding site in the RetSat gene. of metabolic diseases. In addition to adipose tissue, RetSat is strongly expressed in liver. Hepatic RetSat expression is

175 MICHAEL SCHUPP – ENDOCRINOLOGY

Publications 2006 - 2010 Clemenz M, Frost N, Schupp M, Caron S, Foryst-Ludwig A, Bohm C, Hartge M, Gust R, Staels B, Unger T, Kintscher U Steger DJ, Grant GR, Schupp M, Tomaru T, Lefterova MI, (2008): Liver-specific peroxisome proliferator-activated recep- Schug J, Manduchi E, Stoeckert CJ, Jr., Lazar MA (2010): tor alpha target gene regulation by the angiotensin type 1 Propagation of adipogenic signals through an epigenomic receptor blocker telmisartan. Diabetes 57:1405-1413 transition state. Genes Dev 24:1035-1044 Steger DJ, Lefterova MI, Ying L, Stonestrom AJ, Schupp M, Schupp M, Lefterova MI, Janke J, Leitner K, Cristancho AG, Zhuo D, Vakoc AL, Kim JE, Chen J, Lazar MA, Blobel GA, Mullican SE, Qatanani M, Szwergold N, Steger DJ, Curtin Vakoc CR (2008): DOT1L/KMT4 recruitment and H3K79 JC, Kim RJ, Suh M, Albert MR, Engeli S, Gudas LJ, Lazar methylation are ubiquitously coupled with gene transcription MA (2009a): Retinol saturase promotes adipogenesis and in mammalian cells. Mol Cell Biol 28:2825-2839 is downregulated in obesity. Proc Natl Acad Sci U S A 106:1105-1110 Schupp M, Curtin JC, Kim RJ, Billin AN, Lazar MA (2007): A widely used retinoic acid receptor antagonist induces peroxi- Schupp M, Cristancho AG, Lefterova MI, Hanniman EA, some proliferator-activated receptor-gamma activity. Mol Briggs ER, Steger DJ, Qatanani M, Curtin JC, Schug J, Pharmacol 71:1251-1257 Ochsner SA, McKenna NJ, Lazar MA (2009b): Re-expression of GATA2 Cooperates with Peroxisome Proliferator-activated Kershaw EE, Schupp M, Guan HP, Gardner NP, Lazar MA, Receptor-{gamma} Depletion to Revert the Adipocyte Pheno- Flier JS (2007): PPARgamma regulates adipose triglyceride type. J Biol Chem 284:9458-9464 lipase in adipocytes in vitro and in vivo. Am J Physiol Endo crinol Metab 293:E1736-1745 Lefterova MI, Mullican SE, Tomaru T, Qatanani M, Schupp M, Lazar MA (2009): Endoplasmic reticulum stress regulates Schambach F, Schupp M, Lazar MA, Reiner SL (2007): adipocyte resistin expression. Diabetes 58:1879-1886 Activation of retinoic acid receptor-alpha favours regulatory T cell induction at the expense of IL-17-secreting T helper cell Tomaru T, Steger DJ, Lefterova MI, Schupp M, Lazar MA differentiation. Eur J Immunol 37:2396-2399 (2009): Adipocyte-specific Expression of Murine Resistin Is Mediated by Synergism between Peroxisome Proliferator- Schupp M, Lee LD, Frost N, Umbreen S, Schmidt B, Unger activated Receptor {gamma} and CCAAT/Enhancer-binding T, Kintscher U (2006a): Regulation of peroxisome proliferator- Proteins. J Biol Chem 284:6116-6125 activated receptor gamma activity by losartan metabolites. Hypertension 47:586-589 Moise AR, Dominguez M, Alvarez S, Alvarez R, Schupp M, Cristancho AG, Kiser PD, de Lera AR, Lazar MA, Palczewski Schupp M, Kintscher U, Fielitz J, Thomas J, Pregla R, Hetzer K (2008): Stereospecificity of retinol saturase: absolute con- R, Unger T, Regitz-Zagrosek V (2006b): Cardiac PPARalpha figuration, synthesis, and biological evaluation of dihydroretin- expression in patients with dilated cardiomyopathy. Eur J oids. J Am Chem Soc 130:1154-1155 Heart Fail 8:290-294

Lefterova MI, Zhang Y, Steger DJ, Schupp M, Schug J, Cris- Janke J, Schupp M, Engeli S, Gorzelniak K, Boschmann M, tancho A, Feng D, Zhuo D, Stoeckert CJ, Jr., Liu XS, Lazar Sauma L, Nystrom FH, Jordan J, Luft FC, Sharma AM (2006): MA (2008): PPAR{gamma} and C/EBP factors orchestrate Angiotensin type 1 receptor antagonists induce human in- adipocyte biology via adjacent binding on a genome-wide vitro adipogenesis through peroxisome proliferator-activated scale. Genes Dev 22:2941-2952 receptor-gamma activation. J Hypertens 24:1809-1816

Johnstone CN, Mongroo PS, Rich AS, Schupp M, Bowser Research Highlights (2006-2010) MJ, Delemos AS, Tobias JW, Liu Y, Hannigan GE, Rustgi AK (2008): Parvin-beta inhibits breast cancer tumorigenicity and Schupp M, Lazar MA (2010): Fingered for a fat fate. Cell promotes CDK9-mediated peroxisome proliferator-activated Metab 11:244-245. receptor gamma 1 phosphorylation. Mol Cell Biol 28:687-704,

176 MICHAEL SCHUPP – ENDOCRINOLOGY

General information Third party funding (2006-2010)

Project leader Project title Sponsor Period Michael Schupp Regulierung des hepatischen Glukose- DFG (German Research Society) 2010-2013 und Fettstoffwechsels durch die Retinol Emmy-Noether program Saturase

Awards

2004 New Investigator Award, Council on High Blood Pressure Research, Chicago

2004 German Research Award Obesity and Hypertension (group award), Hannover

2005 Forssmann Award for young scientists, University of Bochum

2008 Scholarship Keystone Conference Diabetes

2009 Scholarship Keystone Conference Diabetes

2010 Emmy-Noether group leader DFG

177

Franz Theuring – Molecular Pharmacology

Head of the group

Prof. Dr. rer. nat. Franz Theuring

Curriculum Vitae: Professor Franz Theuring, PhD, studied electronics and biology at the Universities of Braunschweig and Göttingen. He gained his PhD in Mechanisms of meiotic nondisjunction in mammalian oocytes in 1986 in the Institute of Human Genetics, University of Gottingen, and was postdoctoral research fellow at the Max Planck Institute for Biophysical Chemistry under Professor Peter Gruss, world leader in mouse genetics. Professor Theuring was responsible for the establishment and introduction of transgenic technologies both in the Gruss laboratory, and then from 1988 in the Institute of Cellular and Molecular Biology of Schering AG, Berlin, where he had responsibility for implementation of transgenic technologies in areas of pharmacological research for drug target validation and model development at Schering. He developed transgenic mouse and rat models in eurodegeneration, experimental tumour biology and cardiovascular function. Professor Theuring received his Habilitation from the Free University of Berlin in Molecular Biology and Bio- chemistry in 1996, and after 8 years at Schering AG, was appointed in 1996 to a Professorship in Molecular Pharmacology at the Charite Hospital, which is the Medical Faculty of the Humboldt University Berlin. He is an acknowledged expert in employing transgenic technologies to study gene regulation, gene function and establishment of animal models for human disease. He is consultant to the Berlin State in Biotechnology, to the European Union in Molecular Medicine and to Pharmaceutical Industry,and was elected as Member of the Berlin Scientific Society.

179 Franz Theuring – Molecular Pharmacology

Members of the group

Scientists Animal Care Taker Dietze, Silke Dr.vet.med. Thoma, Anna Fischer, Andreas Dr. med. Marschall, Peter Dr. rer. nat., until May 2010 Trainee (Auszubildende) Zabke, Claudia Dr. rer.nat., until May 2009 Lange, Sebastian 11.2007 – 05.2008, Schwab, Karima Dipl. Ing. from Bayer Schering Pharma, Berlin Osterberg, Stephanie 05.2008 – 10.2008, Technicians from Bayer Schering Pharma, Berlin Magbagbeolu, Mandy Seelhorst, Bettina Tanneberger, Cornelia Kemnitz, Rudi 04.2010 – 09.2010

PhD, Diploma and Master Students Gluth, Markus PhD student Gül, Nadir PhD student Neumann, Boris PhD student Schwab, Karima PhD student Vignon-Zellweger, Nicolas PhD student, until March 2010 Seider, Patrick Diploma student Melis, Valeria PhD student, guest, 03.2008 – 04.2008 and 04.2009 – 05.2009, University of Aberdeen Ilnaz, Jallayier Bachelor Student, 01.2010 – 07.2010 Terzi Menderes, Yusuf Student Apprentice, Molecular Medicine, 11.2007 – 01.2008

180 Franz Theuring – Molecular Pharmacology

Summary

The group is using methods and state-of-the-art successfully tested. TauRx has now initiated prepa- techniques in biomedical research to identify new rations for Phase 3 studies in mild and moderate potential drug targets, to validate drug targets and AD. A very similar approach is used to identify new already known key molecules for their involvement drugs acting on Parkinson’as Disease (PD). Trans- in pathophysiological processes, and, in the frame genic mice modelling PD have been generated and of a R&D program, to characterise and test new a detailed analysis of these model systems identified pharmacological active compounds for their abilities the relevant transgenic lines which will be used to to act as therapeutic drugs in clinical dementia, i.e. test newly synthesized substances for their activity Alzheimer’s Disease. These studies represented the to fight PD. final phase in preclinical drug testing and paved the In parallel, we use different inbred strains of mice to way for the future drugs to enter into clinical trials. analyse for gender and age related effects in cardio- Therefore, transgenic techniques in mice are being vascular function. By employing modern 2-dimen- employed to study gene regulation and function sional gel electrophoresis, mass spectrometry, and and to generate animal models relevant for human phosphoproteomics we hope to identify key proteins diseases. Such animal models have given essential being responsible for and mediating these changes. insights into the regulation and function of genes in the These candidates could then provide an entry point context of the whole organism. They further provide into defining a more specific pharmacotherapy for a basis for transcriptomics and/or proteomics as well these alterations. as for the identification and characterization of genes In addition to their importance as model systems for and proteins involved in human disease processes. human disorders, transgenic animals furthermore In the last CCR report we had reported on a major represent valuable tools for the characterization of breakthrough in the treatment of Alzheimer’s dis- general cell biological processes, providing the pos- ease (AD) in the line of our successful phase II clini- sibility to analyze the role of distinct molecules in an cal trial. The product – rember®, a novel form of in vivo context. Insights gained in these model sys- methylthioninium chloride (MTC) – is the first drug tems will improve our understanding of fundamental to act on the tau tangles discovered by Alois Alzhe- molecular mechanisms governing e.g. the regulation imer. The teams of the University of Aberdeen and of tight junction permeability, thereby facilitating the the Charite, working with Tau Rx Therapeutics – a identification of new pharmacological approaches Singapore-based company spun out of these two for the modulation of these processes. Transgenic Universities – developed a novel treatment based animals expressing various components of the RhoA on an entirely new approach which targets the tan- signalling pathway as well as in vitro systems mim- gles, aggregates of abnormal fibres of Tau protein icking the intestinal barrier furthermore provide an forming inside nerve cells in the brain. By employing insight into the effects of established and innovative our transgenic mice a group of second-generation pharmacological compounds on tight junction per- rember® derivatives had now been discovered and meability and intestinal barrier function.

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Zusammenfassung Weiterhin werden Endothelin 1 transgene und eNOS knockout Mäuse als Modellsysteme für kardiovas- Mittels modernster Methoden und Techniken in der bio- kuläre Erkrankungen eingesetzt. Diese Tiermodelle medizinischen Forschung versuchen wir neue potenzielle stellen die Ausgangsbasis zur detaillierten Analyse drug targets zu identifizieren und zu validieren. Zusätzlich zugrunde liegender molekularer Pathomechanismen beziehen wir bereits bekannte, zentral agierende Mole- dar. Mittels Transkriptom- und Proteom-Analysen und küle in unsere Analysen mit ein, um Aussagen über ihre nach entsprechender Validierung werden anschlie- Beteiligung an pathophysiologischen Prozessen machen ßend Moleküle identifiziert, die eine zentrale Rolle im zu können. Letztendlich, im Rahmen eines Wirkstofffin- Krankheitsgeschehen spielen. Im Rahmen eines Wirk- dungs- und Entwicklungsprogramms zur Entwicklung stofffindungs- und Entwicklungsprogramms könnten eines therapeutisch wirksamen Alzheimer Medikamentes, diese Moleküle als potenzielle drug targets fungieren. charakterisieren und testen wir pharmakologisch aktive Unter Verwendung verschiedener Maus-Inzucht- Substanzen im Rahmen von präklinischen Studien in stämme erfolgt darüber hinaus die Analyse von unseren entsprechenden tierexperimentellen Modellen. geschlechts- und altersspezifischen Veränderungen Zur Realisierung dieser Vorhaben verwenden wir sowohl im kardiovaskulären System. Hierzu werden modernste in vivo (transgene und knockout Mäuse als Modellsys- Proteomtechniken wie 2-Dimensionale Gelelektropho- teme für menschliche Erkrankungen) als auch in vitro rese, massenspektrometrische Analysen und Phos- (Zellinien epithelialen Ursprungs) Modellsysteme. In phoproteomics eingesetzt, um Schlüsselproteine, die Zusammenarbeit mit TauRx Therapeutics - einer Aus- an der Vermittlung dieser Veränderungen beteiligt sind, gründung der Universität Aberdeen und der Charite – zu identifizieren und damit potenzielle Ansatzpunkte für konnten wir im letzten CCR-Report von einem bahn- eine spezifischere Pharmakotherapie aufzuzeigen. brechenden Erfolg unserer klinischen Studie der Phase Neben ihrer Bedeutung als Modellsysteme für spezi- II in der Alzheimerforschung berichten. Die von TauRx fische menschliche Erkrankungen stellen transgene Therapeutics entwickelte neue Form des Wirkstoffes Tiere ebenfalls wertvolle Werkzeuge zur Charakteri- Methylthioniniumchlorid (MTC)- Rember® - scheint die sierung zellbiologischer Prozesse dar und erlauben es erste Substanz zu sein, die in der Lage ist, ein Fortschrei- so, die Funktion spezifischer Moleküle in einem in vivo ten der Demenz aufzuhalten. Die Testung weitere Subs- Kontext zu studieren. Ziel ist es hier, durch ein verbes- tanzen in unseren transgenen Mausmodellen führte nun sertes Verständnis der molekularen Mechanismen, zur Entwicklung von verbesserten second generation die zum Beispiel die tight junction Permeabilität regu- rember® Derivaten, deren Testung in einer Phase III Stu- lieren, neue Ansatzpunkte zur pharmakologischen die vorbereitet wird. Ein ähnlicher Forschungsansatz Modulation dieser Prozesse zu identifizieren. Hierzu bezieht sich auf die Entwicklung neuartiger Therapeu- existieren in unserer Arbeitsgruppe sowohl transgene tika zur Behandlung der Parkinsonschen Erkrankung. Tiermodelle zur Analyse verschiedener Komponenten Auch hier wurden zunächst relevante Tiermodelle eta- des RhoA-Signaltransduktionsweges als auch in vitro bliert und anschließend umfassend charakterisiert, um Modellsysteme der gastrointestinalen Barriere, die in diesen dann unsere Substanzen auf ihre Wirksamkeit ebenfalls die Analyse der Effekte etablierter und inno- in vivo testen zu können. vativer Pharmaka auf die Barrierefunktion gestatten.

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Research projects

1. Adaptation of proteomic techniques for the identification and characterization of protein species from murine heart

Project leader Franz Theuring Coworkers Karima Schwab, Boris Neumann, Ilnaz Jallayier Funding Hypatia-Funding, Projektverbund Chancengleichheit für Frauen Charite, PhD-grant Cooperation Dr. C. Scheler, Proteome Factory, Berlin; Dr. P.R. Jungblut, MPI for Infection Biology, Berlin

Cardiovascular diseases (CVD) are the number one tein emerging from one single protein-coding gene cause of morbidity and mortality with an age and promoted the new term of protein species. While sexual divergence. Premenopausal women are at a genomic and transcriptomic data lack information on lower risk for cardiovascular disease as compared to protein species in a given tissue, advances in pro- age-matched men, but this risk increases dramati- teome analysis and mass spectrometry enabled the cally after menopause, indicating that estrogens may identification and characterization of post-translational play a protective role. However hormone replace- modifications (PTMs) in peptides derived from a pro- ment therapy in humans yielded conflicting results tein species, which lead to an increasing number of and phytoestrogens such as genistein, widely used protein species in databases. Furthermore, advances in traditional asian medicine, could represent alterna- in mass spectrometry allowed the localization of cleav- tive compounds as they are known to exert estrogenic age sites for protein processing, maturation, truncation activity and to have beneficial effect on a wide range and degradation. Protein species resulting from such of cardiovascular parameters. cleavage events play an important role in inflammation, In the cardiovascular context a disturbed energy cell degeneration, apoptosis and oncogenesis. In the metabolism with impaired fatty acid oxidation, ATP cardiovascular context, protein species derived from synthesis and changing levels of contractile proteins modifications such as acetylation, phosphorylation have been observed during diseased conditions. and cleavage are involved in various processes and Whereas numerous studies focussed on gene expres- disease development. sion analyses at the messenger RNA level, other holis- In this project we analyze the effects of a dietary supple- tic and undirected techniques, such as proteomics, ment with the phytoestrogen genistein on the cardiac have been applied to the analysis of CVD. Multiple proteome pattern of young, adult and castrated male identifications of a single protein from various spots and female mice. Our analysis demonstrates consider- on 2-DE gels revealed that the suggestion of a sin- able changes of the heart proteome with dietary gen- gle gene or transcript, encoding for a single protein, istein administration for both male and female animals. is obsolete. The diversity in different forms of a pro- A changing abundance, of among others metabolic,

183 Franz Theuring – Molecular Pharmacology

energetic and contractile proteins, was observed. Ini- tially, we focussed on the identification of PTMs of four selected proteins, using a multiple digestion protocol to enhance sequence coverage (see figure). PTMs were identified by standard NanoLC electrospray ionization ion trap mass spectrometry (nanoLC-ESI-MS/ MS) and linear ion trap fourier transform ion cyclotron resonance mass spectrometry (LTQ-FT-ICR-MS/MS) and revealed several modified and truncated species. Protein species resulting from all protein modifications, post-translational chemical modifications and protein truncation are different products of one single gene. These modifications influence subcellular location, degradation, subunit assembly, tertiary structure or enzyme activity and thus protein function. Therefore, prime importance should be rather given to systematically identify and specify proteins at their species level than to quantify total protein amount. The newly detected protein species were regulated in the myocardium of mice related to age, sex and oral genistein treatment. Therefore those species could be relevant in cardiac disease and should be taken into consideration for the molecular understanding of pathological processes.

Workflow of 2-DE and MS strategy for identification of proteins and protein species. 2-DE two-dimensional gel electrophoresis, ESI–MS/MS electrospray ionization mass spectrometry, LTQ-FT-ICR-MS/ MS linear ion trap Fourier transform ion cyclotron resonance mass spectrometry, db database

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2.Phosphorylation analysis of the mouse kidney proteome

Project leader Franz Theuring Coworkers Boris Neumann, Karima Schwab Funding University Research Funding Cooperation Dr. C. Scheler, Proteome Factory, Berlin

Phosphorylation is one of the most prominent post- or –peptides. While mass spectrometers for the analy- translational modifications of proteins. It regulates sis of proteins or peptides (i.e.: MALDI-MS, ESI-MS/ a wide range of metabolisms in living cells. Modern MS) permit an indirect detection of phosphorus due to methodology utilises mass spectrometry (MS) for the mass shift and neutral loss, the usage of element mass detection and localisation of phosphorylation sites. spectrometers (ICP-MS) allows the direct detection on However the detection and site-specific characteriza- atomic level. ICP-MS has unrivalled detection limits but tion is a challenging task in life science. While immuno- discards structural information of the analyte. logic techniques are highly specific (often limited to a Current focus of the project is the evaluation of single site and protein) and have a low detection limit common phosphopeptide or –protein enrichment the undirected analysis via mass spectrometry is often schemes for the mouse kidney proteome by ICP-MS. hindered by the low abundance and poor ionization of Further the project focusses the promising combina- the phosphorylated analyte in common methodology. tion of nanoLC-ESI-MS/MS with ICP-MS in a parallel Essential for the detection of phosphorylation sites in fashion to produce merged data of structural data, “real-life” samples via common mass spectrometers proteins phosphorylation and its absolute abundance. is an antecedent enrichment of the phospho-proteins

2D-PAGE of 120 mouse kidney proteins. Left: initial sample, mid: flow-through, right: MOAC-eluate. Horizon- tal: isoelectric focussing (pH 4-10), vertical: SDS-PAGE (approx. 250 kDa - 8 kDa)

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3. Endothelin-1 transgenic/eNOS Knockout Mice: A new genetic mouse model to study gender and age effects in cardiovascular diseases

Project leader Franz Theuring Co-workers Nicolas Vignon-Zellweger, Karima Schwab Funding Marie Curie Host Fellowship for ES-Training (CARDIOVASC, to N. V-Z and F.T), and DFG (TH 466/7-1) Cooperation Prof. B. Hocher, CCR; Dr. J.P. Stasch, Bayer Schering Pharma, Wuppertal

The intact endothelium produces a variety of vasoac- ET‐1 on an eNOS‐/‐ background could be beneficial tive substances. Important among those is endothe- for diastolic functions. lin-1 (ET-1), a potent vasoconstrictor and mitogen in In order to identify the underlying mechanisms leading vivo and in vitro. The nitric oxide (NO) system is con- to this phenotype, molecular, histological, physiologi- sidered as the natural functional counterpart of the cal and protein chemical methods were employed. ET system, thus contributing to the subtle balance of In particular to analyze for the complete cardiac pro- vascular tone. The clinical relevance of this delicate teome of three months old animals high resolution interplay has been acknowledged because of its impli- two Dimensional Gel Electrophoresis coupled to mass cation in many cardiovascular diseases such as pul- spectrometry was performed. We could demonstrate monary arterial hypertension, systemic hypertension, that the cardiac proteome of the three different geno- and coronary artery disease. However, the underlying types compared to WT animals resulted in prominent molecular mechanisms remain to be fully clarified. changes of the cardiac protein abundance. ET-1 overexpressing transgenic mice develop a scle- The proteomics study revealed that transgenic over- rotic and/or fibrotic renal, pulmonary and myocardial expression of ET‐1, with or without eNOS, led to a phenotype. Surprisingly the ET+/+ mice remain nor- higher abundance of proteins regulating oxidative motensive. This lack of hypertension is believed to be stress indicating that, in contrast to eNOS‐/‐ animals, the consequence of a compensatory effect of the nitric ET+/+ and ET+/+eNOS‐/‐ mice developed molecu- oxide system. To disrupt this compensatory effect we lar mechanisms limiting oxidative damages. Moreover, decided to crossbreed ET+/+ mice and eNOS knock- diastolic dysfunction observed in eNOS‐/‐ mice may out mice to generate the four different genotypes: be explained by the differential abundance of proteins ET+/+, eNOS-/-, ET+/+eNOS-/-, and WT. involved in the contractile machinery. Overexpression The crossbred animals (ET+/+eNOS-/-) develop sig- of ET‐1 in eNOS‐/‐ mice restored these changes nificantly high systolic blood pressure compared to WT and may have thereby benefited the cardiac functions. mice and eNOS-/- mice. Furthermore, at the age of Finally, this study indicated that a shift from fatty acid to nine month , the eNOS-/-, but not the ET+/+eNOS-/- glucose metabolism, considered as cardioprotective, mice, are characterized by diastolic dysfunction. These may have occurred to a greater extent in crossbred findings suggested that transgenic overexpression of animals than in eNOS‐/‐ mice.

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4. Characterization of signalling properties of the Endothelin system in mouse mammary gland physiology

Project leader Franz Theuring, Andreas Fischer Coworkers Nadir Gül Funding Charite-Stipend

Endothelin 1 (ET-1) is a small vasoactive peptide having to mammary gland physiology and pathophysiological wide physiological effects on vascular homeostasis processes. This is achieved by the detailed molecular and a variety of physiological and pathophysiological and histological characterization of female transgenic processes unrelated to cardiovascular physiology. It mice overexpressing ET-1. These mice were found exerts its effect by binding to two distinct G Protein to exhibit a lactational incompetence while lactating Coupled Receptors (GPCR). In addition to the classi- newborn animals. In parallel, an in vitro system was cal GPCR signaling pathways, these receptors are also established employing mammary epithelial cell lines to able to activate structurally unrelated receptors such further study relevant cell biological processes in these as those belonging to the receptor tyrosine kinase specific cell types. First results suggest that endothelin (RTK) family in what is termed receptor transactivation. is able to activate various signaling pathways in vivo Deregulation of this transactivation as induced by and in vitro and that this activation is accompanied by overexpression, amplification or mutation of critical impaired lactational competence in ET-1 transgenic pathway elements and autocrine stimulation through animals. To further characterize the molecular events aberrant growth factor loops is frequently linked to involved in this project, functional experiments employ- hyperproliferative diseases. ing various inhibitor compounds will be performed in The aim of this project is to further characterize the order to identify the signaling pathways mediating the crosstalk between these two systems in the mam- observed effects. mary epithelium in order to elucidate its contribution

Whole mount preparations of wild type and ET-1 transgenic mammary glands from lactation day 3 demonstrating lactational incompetence in the ET-1 transgenic mammary glands.

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5. Pharmacological Modulation of the Intestinal Barrier

Project leader Andreas Fischer, Franz Theuring Coworkers Markus Gluth, Cornelia Tanneberger Funding University Research Funding, Industry funding External cooperations Prof. D.C. Baumgart, Dr. U.F. Pape – Division of Hepatology and Gastroenterology CVK

The intestinal barrier constitutes the largest mucosal characterizing the mechanisms regulating paracellu- surface of the human body, separating the highly lar permeability in the intestine, these studies might antigenic environment of the intestinal lumen from provide a basis for developing new pharmacological the milieu intérieur. Perturbations of this barrier have approaches to modulate barrier function. long been recognized as key features in inflammatory bowel diseases such as Crohn’s disease and ulcera- tive colitis, but have also been found in celiac disease, graft-versus-host disease and food allergies. In addition, structural components of the epithelial tight junctions have been identified as primary targets for various pathogenic bacteria-derived toxins. Pharma- cological strategies to modulate the permeability of intestinal tight junctions therefore represent an attractive approach to improve the management of these disorders. Using Caco-2 and T-84 monolayers cultivated on semipermeable filter supports as a model system of the intestinal epithelial barrier, our work focuses on the analysis of the impact of proinflammatory cytokines such as TNFα or IFNγ on barrier function. Using specific inhibitor compounds, we aim at characterizing signaling pathways responsible for the deleterious effects of these cytokines in order to identify potential targets for the treatment of inflammatory bowel dis- Adalimumab prevents barrier disruption by the proinflamma- eases. In addition, compounds well established in the tory cytokines TNFα and IFNγ. T-84 intestinal epithelial cells treatment of these disorders such as glucocorticoid were treated with TNFα and IFNγ or a combination of both cytokines and the therapeutic anti-TNFα antibody adalimu- hormones and anti-TNFα antibodies are evaluated mab. The tight junction components occludin and ZO-1 were in terms of their effect on barrier function. By further visualized by immuno-staining.

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6. Characterization of the Rho Signaling Pathway and its Role in the Regulation of Tight Junction Permeability

Project leader Andreas Fischer, Franz Theuring Coworkers Markus Gluth, Cornelia Tanneberger Funding University Research Funding External cooperations Prof. J.R. Turner, University of Chicago

Among signaling pathways associated with the tight involved in the RhoA-mediated regulation of epithelial junction, the small GTPase RhoA has been shown to tight junction permeability. be of pivotal importance to the control of cell prolif- By employing an inducible expression system as an in eration, migration and differentiation. Previous stud- vitro barrier model, the effects of RhoA and PKN can be ies have demonstrated that expression of either con- studied at various developmental stages, enabling us stitutively active or dominant negative RhoA results to delineate their contribution to the regulation of tight in reduced tight junction barrier function in cultured junction function. Functional observations are com- MDCK monolayers. We have shown that modulation plemented by the analysis of changes in the expres- of signaling pathways downstream of RhoA regulates sion and localization of key tight junction components tight junction function in the mammary epithelium, and the characterization of the molecular cross-talk to both in vivo and in vitro. However, the precise mecha- other signaling pathways. As Rho GTPases are pivotal nisms by which RhoA controls barrier function have in the pathogenesis of a variety of diseases includ- not been defined. ing arterial and pulmonary hypertension, myocardial Therefore, our work focuses on the characterization hypertrophy and vasospastic angina as well as tum- of RhoA and one of its downstream targets, the pro- origenesis and metastasis, our work might contribute tein kinase PKN in order to elucidate molecular events to a better understanding of the signaling pathways downstream of these molecules.

A) Dose- and time-dependend induction of RhoA expression. B) Immunofluorescent staining of RhoA expression (red) and MLC phosphorylation (green) after doxycycline withdrawal in Caco-2 intestinal epithelial cells.

189 Franz Theuring – Molecular Pharmacology

7. Alzheimer’s Disease: Testing of new drug candidates in tau-transgenic mice

Project leader Franz Theuring Coworkers Silke Dietze, Claudia Zabke, Mandy Magbagbeolu, Bettina Seelhorst, Valeria Melis, Karima Schwab, Anna Thoma Funding TauRx Therapeutics, WisTa Laboratories, Singapore Cooperation Prof. C.M. Wischik, University of Aberdeen, Scotland

Tau transgenic mouse lines had been established to in the memory-critical brain regions where the density further analyse the functional role this protein and its of Alzheimer tangles is greatest. In the control group, aggregates, the so-called tau tangles play in clinical there was a significant decline from the starting score dementia, i.e. Alzheimer’s Disease and to test puta- in cognitive testing and on brain scans. tive new drug candidates in a preclinical setting to fight By employing our transgenic mice a group of second- this neurodegenerative and terminal brain disease. In a generation rember® derivatives had been discovered most fruitful scientific collaboration with Prof. Wischik’s and successfully tested. These compounds have the group from the University of Aberdeen for the last 12 same mechanism of action as rember® acting as Tau years these mice had been pivotal in establishing a rel- Aggregation Inhibitors, with potential utility in the treat- evant transgenic tau-based Alzheimer mouse model. ment of Alzheimer’s disease and other neurodegenera- Several dozens of newly discovered and synthesized tive disorders. TauRx has now initiated preparations for drug candidates had been tested in vivo for their activ- Phase 3 studies in mild and moderate AD, and also in ity in reducing tau pathology and to enhance cognitive orphan indications such as Fronto-Temporal Dementia behaviour and motor skills in the drug-treated animals. and provisionally Progressive Supranuclear Palsy. By teaming up with TauRx Pharma - a Singapore-based These data underline the importance of generating company spun out of the University of Aberdeen and relevant transgenic mouse models and their use in the Charité - it is now our great pleasure to reveal a identification and validation of new drug candidates major breakthrough in the treatment of Alzheimer’s dis- for human diseases, which then are planned to enter ease. A total of 321 patients with mild and moderate into clinical testing. Alzheimer’s Disease were treated in a phase II clinical trial with Rember® a novel form of methylthioninium chloride (MTC). Patients receiving the study treatment experienced an 81% reduction in cognitive decline over one year, and did not experience a significant decline in their mental function over 24 months. In addition patients had repeated brain scans at the start of the study and after 25 weeks. These An increase of tau aggregates in cortical sections of transgenic mice is showed that the treatment effect was greatest observed with increasing age (left: 6 mo old, right 12 mo old).

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8. Parkinson’s Disease: Testing of new drug candidates in α-synuclein transgenic mice

Project leader Franz Theuring Coworkers Silke Dietze, Mandy Magbagbeolu, Bettina Seelhorst, Karima Schwab, Anna Thoma, Claudia Zabke Funding WisTa Laboratories, Singapore Cooperation Prof. C.M. Wischik, University of Aberdeen, Scotland

Parkinson’s disease (PD) is a common human neuro- tion or reversal of synuclein aggregation is believed to degenerative movement disorder and affects 1% of the be of therapeutic benefit. elderly population. PD is neuropathologically charac- The development of drugs that prevent this aggre- terized by a marked and progressive degeneration of gation form the basis for the scientific rationale dopaminergic neurons and by the presence of fibrillar for this project. The aim is to model the molecular cytoplasmic inclusions (Lewy bodies [LBs]) and dys- processes of α-synuclein aggregation in an animal trophic neurites (Lewy neurites [LNs]) in the substantia model to test and evaluate new therapeutic drug nigra and other regions of the brain. Although the loss of candidates for PD. dopamine neurons is certainly related to the major clini- Consequently α-synuclein transgenic mice had been cal symptoms of PD, the causes and the pathogenesis generated carrying different transgenic constructs. of this multifactorial disease as well as that of related These mice are currently being characterized in more “synucleinopathies” are still largely unknown. detail and will be used to study basic mechanisms The major components of both LBs and LNs are of the pathogenesis underlying PD. Most importantly, fibrillar aggregates of α-synuclein. α Synuclein is a these mice will then be used to screen for α-synuclein widely expressed, neuronal presynaptic protein that aggregation inhibitors to facilitate the development of a appears to play a role in membrane-associated proc- new therapeutic intervention for this disease. esses and synaptic plasticity and has been linked to learning and development processes. While the mechanism(s) of formation of LBs and LNs and their association with PD are yet not understood, several lines of evidence suggest that α synuclein fibrillization is associated with PD and that α-synuclein fibrillization Hippocampal sections of wildtype (left) and transgenic mouse (right) brains exhibiting causes toxicity. Thus the inhibi- prominent α-synuclein staining

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Publications (2006-2010) Fischer A, Stuckas H, Gluth M, Russell TD, Rudolph MC, Beeman NE, Bachmann S, Umemura S, Ohashi Y, Neville Bert B, Fink H, Hörtnagl H, Veh RW, Davies B, Theuring F, MC,* Theuring F* (2007): *both authors contributed equally. Kusserow H (2006): Mice over-expressing the 5-HT1A recep- Impaired Tight Junction Sealing and Precocious Involution in tor in cortex and dentate gyrus display exaggerated locomo- Mammary Glands of PKN Transgenic Mice. J Cell Sci 120: tor and hypothermic response to 8-OH-DPAT. Behav Brain 272-2283 Res 167: 328-41. Quaschning T, Voss F, Herzfeld S, Relle K, Kalk P, Godes Davies B, Behnen M, Cappallo-Obermann H, Spiess AN, M, Pfab T, Kraemer-Guth A, Bonz AW, Theuring F, Galle J, Theuring F, KirchhoffC.(2006): Novel epididymis-specific Hocher B (2008): Lack of iNOS impairs endothelial function in mRNAs down-regulated by HE6/Gpr64 receptor gene disrup- endothelin-1 transgenic mice. Kidney Blood Press Res 31: tion. Mol Reprod Dev 74: 539-553 127-134 Epub 2008 Apr 7

Quaschning T, Voss F, Relle K, Kalk P, Vignon-Zellweger Heiden S, Pfab T, von Websky K, Vignon-Zellweger N, Godes N, Pfab T, Bauer C, Theilig F, Bachmann S, Kraemer-Guth M, Relle K, Kalk P, Theuring F, Zidek W, Hocher B (2008): A, Wanner C, Theuring F, Galle J, Hocher B (2007): Lack Tissue specific activation of the endothelin system in severe of Endothelial Nitric Oxide Synthase Promotes Endothelin- acute liver failure. Eur J Med Res 13: 327-329 Induced Hypertension: Lessons from Endothelin-1 Trans- genic/Endothelial Nitric Oxide Synthase Knockout Mice. J Bert B, Voigt JP, Kusserow H, Theuring F, Rex A, Fink H Am Soc Nephrol 18: 730-740 (2009): Increasing the number of 5-HT1A-receptors in cortex and hippocampus does not induce mnemonic deficits in Slowinski T*, Kalk P*, Christian M, Schmager F, Relle K, mice. Pharmacol Biochem Behav 92: 76-81. Epub 2008 Godes M, Funke-Kaiser H, Neumayer NN, Bauer C, Theuring Oct 31 F*, Hocher B* (2007): *authors contributed equally. Cell-type specific interaction of Endothelin- and Nitric Oxide System Küster K, Grötzinger C, Koschel A, Fischer A, Wiedenmann - Pattern of prepro-ET-1 expression in kidneys of L-NAME B, Anders M (2010): Sodium butyrate increases expression of treated prepro ET-1 promoter-LacZ-transgenic mice. J. the Coxsackie and Adenovirus Receptor in colon cancer cells. Physiol 581: 1173-1181 Epub 2007 Mar 29 Cancer Invest 28: 268-274

Russell TD, Palmer CA, Orlicky DJ et al. (2007): Cytoplasmic Küster K, Koschel A, Rohwer N, Fischer A, Wiedenmann lipid droplet accumulation in developing mammary epithelial B, Anders M (2010): Downregulation of the coxsackie and cells: roles of adipophilin and lipid metabolism. J Lipid Res adenovirus receptor in cancer cells by hypoxia depends on 48:1463-75 HIF-1alpha. Cancer Gene Ther 17: 141-146

Baumgart DC, Fischer A (2007): Virchow’s node. Lancet Schwab K, Neumann B, Scheler C, Jungblut PR, Theuring 370:1568 F (2010). Adaptation of proteomic techniques for the identification and characterization of protein species from murine Baumann C, Davies B, Peters-Kottig M, Kaufmann U, Lessl heart. Amino Acids: Jul 6. [Epub ahead of print]. M, Theuring F (2007): AKR 1B7 (Mouse Vas Deferens Protein) is dispensible for mouse development and reproductive success. Reproduction 134:97-109

Deng DR, Djalali S, Ahnert-Hilger G, Große G, Stroh T, Voigt, I, Kusserow H, Theuring F, Hen, R, Hörtnagl H (2007): Embry- onic and postnatal development of the serotonergic raphe system and its target regions in the brains of 5-HT1A receptor knockout and transgenic mice. Neuroscience 147: 388-402. Epub 2007 Jun 1

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General information Third party funding (2006-2010)

Project leader Project titel Sponsor Period Schwab K. Sex, age and diet influences on the Hypathia and Charite PhD pro- 2006-2009 Theuring F. cardiac proteome grams Gül N. Characterisation of the mouse mam- Charite PhD program 2006-2008 Theuring F. mary gland Theuring F. Tau and a-synuclein transgenic mice TauRxTherapeutics/WisTa Labo- 2006-2010 ratories Theuring F. Endothelin and NO interaction in car- EU-EST Program CARDIOVASC 2006-2008 diac remodelling Theuring F. Cardiac proteome analysis DFG, TH 466/7-1 2009-2010 Fischer A. Analysis of PKN1 DFG FI 1718/1-1 2010-2012 Theuring F. Applied for: 01.06.2010

Awards

2008 United European Gastroenterology Federation Travel Grant Award: (Adalimumab Prevents TNFα Induced Barrier Disruption in an In vitro Model) Andreas Fischer

193

Thomas Unger – Pharmacology

Head of the Group Pharmacology

Prof. Dr. med. Thomas Unger

Curriculum Vitae: Professor Thomas Unger holds the Chair of Pharmacology and is Director of the Institute of Pharmacology at the Charité – Universitätsmedizin Ber- lin. He is also the Director of the Center for Cardiovascular Research (CCR) at the Charité, Berlin and the Chairman of the German Institute for High Blood Pressure Research in Berlin (DIB). Between 1994 and 2001, he was Director of the Institute of Pharmacology at the University of Kiel, Germany. Professor Unger studied medicine in Germany and the UK, and gained his MD from the University of Heidelberg, Germany. He then carried out postdoctoral research at the Clinical Research Institute of Montreal, Canada, and the Department of Pharmacol- ogy in Heidelberg, where he received his PhD in Pharmacology. Until 1994, Professor Unger held professorships in pharmacology and hypertension research at the University of Heidelberg. In recognition of his work, Profes- sor Unger has received the German Hypertension Society´s Franz Gross Award for Hypertension Research, the Meilahti Lecture Award of the Medical Faculty, University of Helsinki, Finland, the Björn Folkow Award of the European Society of Hypertension, and the Robert Tigerstedt Award of the Finnish Hypertension Society. He is a member of the German Societies of Pharmacology, Cardiology and Hypertension (Council Member 1995–2001), the International Society of Hypertension, the European Society of Hypertension (Council Member 1989–97), the European Council for Blood Pressure and Cardiovascular Research (President, 2000–2), the Inter-American Society of Hypertension, He is Honorary Member of the British, Finnish and Italian Hypertension Society and a Fellow of the European Society of Cardiology and the American Heart Association. Professor Unger has authored more than 700 scientific publications. He is or has been a member of the Editorial Boards of the American Journal of Physiology, American Journal of Hypertension, Biochemical Pharmacology, Blood Pressure, Cardiovascular Drugs and Therapy, Clinical and Experimental Hypertension, Hypertension, Hyper- tension Research, Journal of Hypertension, Fundamental and Clinical Pharmacology, Physiological Genomics, Regulatory Peptides, High Blood Pressure & Cardiovascular Prevention.

195 Thomas Unger – Pharmacology

Members of the group

Administrative Assistant Workshop Schröder, Miranda Röder , Bettina

Groups of the Institute of Pharmacology in the CCR

196 HEIKO FUNKE-KAISER – Pharmacology

Head of the group

PD Dr. med. Heiko Funke-Kaiser

Curriculum Vitae: He studied medicine at the universities of Düsseldorf and Berlin. He obtained his medical approbation (3rd state examination) in 1998 and received his MD degree - based on an experimental work at the Institute of Clinical Pharma- cology and Toxicology of the Freie Universität Berlin in 1999. After a specialization (“Facharzt”) in pharmacology and toxicology in 2004, he built up his research group in the Institute of Pharmacology at the Center for Cardiovascular Research (CCR). In 2008 he passed his habilitation in pharmacology and toxicology. His research focus is on eukaryotic gene regulation and drug development

Members of the group

Scientists Zollmann, Frank Dr. med. Goldin-Lang, Petra Dr. rer. nat. Schmitz, Jennifer Dr. rer. nat. Li, Yaosi Physician Schmerbach, Kristin Dipl.-Biol. Seidel, Kerstin Dr. rer. nat.

Technicians Klare, Sabrina Dipl.-Biol. Kroh, Melanie

Students Kirsch, Sebastian Dipl.-Biochem., PhD student Zaade, Daniela M.Sc., PhD student Bernhard, Sarah MD student Hope, Antonia MD student Schrezenmeier, Eva MD student

197 HEIKO FUNKE-KAISER – Pharmacology

Summary

Drug development Eukaryotic gene regulation Cardiac, renal and ophthalmological end-organ Eukaryotic gene regulation - especially on the pro- damage due to hypertension and/ or diabetes is moter level - is a further major focus of our group. currently one of the major medical challenges. They Promoters are key regulatory elements in our contribute to approximately 60-80% of all heart genome that transform genomic information into failure and 70% of all renal failure cases. In addi- structure. They determine when (e.g. regarding tion, up to 30% of blindness in western countries developmental biology), where (i.e, cell and tissue is caused by these diseases. Current therapeutic specificity) and under which (patho)physiological strategies, such as ACE inhibitors, angiotensin AT1 conditions a gene is transcribed. Furthermore, they receptor blockers (ARBs), b-adrenergic receptor can increase the complexity of the transcriptome antagonists or antidiabetic agents only ameliorate relative to the genome, since, e.g., multiple mRNA but do not abolish cardiovascular end-organ dam- isoforms can be generated from one gene through ages. Therefore, cardiovascular end-organ dam- the use of alternative promoters. This mechanism is age represents an unmet medical need. of importance in the “postgenomic era” which has Worsening this situation, the pharmaceutical to explain the physiology and pathophysiology of the industry faces a productivity crisis. While research human species with its relative low gene number. and development (R&D) spend has risen annually The (patho)physiological importance of promoters is ~7% over the last decade, investigational new demonstrated by effects of genetic polymorphisms drug applications (IND) and new molecular entity within promoter regions which can be associated (NME) output largely remained unaltered. This with psychological traits, physiological traits and the insufficient transfer of scientific concepts into susceptibility for certain diseases. Furthermore, pro- approved drugs opens interesting opportunities moter polymorphisms are involved in the frequently for small and flexible biotech companies based discussed gene-environment interactions as well as on an academic backbone. pharmacokinetic and pharmacodynamic aspects. Supported by the BMBF GO-Bio initiative [http:// Our past and current projects center around the fol- www.bmbf.de/de/6868.php] the aim - as described lowing topics: below in greater detail - of an interdisciplinary project (1) Basic mechanisms of transcriptional regulation team is to develop a novel drug class, which inhibits including epigenetics, the renin/ prorenin receptor, for the indication renal (2) functional characterisation of polymorphisms in and cardiac end-organ damage. Within this GO-Bio regulatory regions, program a start-up company called CCR Pharma (3) altered gene regulation in cardiovascular and has been founded as a spin-off from the Charité to neurodegenerative diseases, develop scientific ideas up to clinical phase I and to (4) and transcriptional regulation in developmental license compounds in return for payments ensuring biology. the growth of CCR Pharma´s own pipeline.

198 HEIKO FUNKE-KAISER – Pharmacology

Methods used by our group to explore these top- scription factors to regulatory regions is analysed ics include e.g. yeast-two-hybrid screening and by DNA affinity chromatography and mass spec- coimmunoprecipitation (CoIP) for protein-protein trometry. interactions, electromobility shift assay (EMSA) and Furthermore, we consider promoters as therapeutic chromatin-immunoprecipitation (ChIP) for protein- targets which can be influenced by classical drugs (e.g. DNA interactions as well as promoter reporter gene steroids and glitazones) and gene therapy (e.g. decoy assays, 5´-RACE and RNase protection assay (RPA) oligonucleotides and triple helix forming substances), for promoter analysis. The binding of unknown tran- driving synergy with our drug development focus.

Zusammenfassung

Medikamentenentwicklung Wie weiter unten ausführlich beschrieben besteht Kardiale, renale und ophthalmologische Endorgan- das Ziel eines durch die GO-Bio-Initiative des BMBF schäden bedingt durch arterielle Hypertonie und/ oder [http://www.bmbf.de/de/6868.php] geförderten, Diabetes mellitus stellen derzeit eines der relevantesten interdisziplinären Projektteams darin, eine neue medizinischen Probleme dar. Sie sind u.a. verantwort- Medikamtenklasse, welche den Renin-/ Prorenin- lich für ca. 60-80% aller Herzinsuffizienzen, ca. 70% Rezeptor inhibiert, für die Indikation renaler und aller dialysepflichtigen Niereninsuffizienzen und bis zu kardialer Endorganschäden zu entwickeln. Innerhalb 30% der Erblindungen in den Industrienationen. Die dieses GO-Bio-Programms wurde ein Unternehmen bisherigen Therapiestrategien (u.a. ACE-Hemmer, mit dem Namen "CCR Pharma" aus der Charité aus- Angiotensin-AT1-Rezeptorblocker (ARBs), b-Blocker gegründet, welches präklinische Medikamentenent- und orale Antidiabetika) können allesamt die Entwick- wicklungen bis hin zur klinischen Phase I durchfüh- lung von Endorganschäden nur verlangsamen, jedoch ren soll. Dabei sollen u.a. im Rahmen der derzeitigen nicht verhindern. Endorganschäden stellen somit einen Substanzentwicklung generierte Lizenzeinnahmen sogenannten "unmet medical need" dar. dazu genutzt werden, eine solide, finanzielle Basis Erschwerend kommt hinzu, dass die pharmazeuti- für "CCR Pharma" bereitzustellen, um die Entwick- sche Industrie derzeit eine Produktivitätskrise erlebt, lung von innovativen Pharmaka zu gewährleisten. da die Ausgaben für Forschung und Entwicklung in der letzten Dekade um ca. 7% jährlich gestie- Eukaryontische Genregulation gen sind, während die Anzahl an Neuzulassungen Eukaryontische Genregulation, insbesondere auf nahezu konstant geblieben ist. Promotorebene, stellt einen weiteren Fokus unserer Dies spiegelt unter anderem einen insuffizienten Forschungsarbeit dar. Promotoren sind regulative Transfer von wissenschaftlichen Ideen in die Ent- Schlüsselelemente in unserem Genom und bedin- wicklung von Pharmaka wider, ermöglicht jedoch gen die Transformation von genetischer Information kleinen und flexiblen Biotech-Unternehmen mit aka- in Struktur. Sie bestimmen, wann (z.B. im Rahmen demischem Hintergrund Erfolgsmöglichkeiten. der Embryonalentwicklung), wo (d.h. Gewebe- bzw.

199 HEIKO FUNKE-KAISER – Pharmacology

Zellspezifität) und unter welchen physiologischen (2) funktionelle Charakterisierung von Promotorpo- und pathophysiologischen Bedingungen ein Gen lymorphismen, transkribiert wird. Sie tragen des Weiteren zur Erhö- (3) Genregulation im Rahmen von kardiovaskulären hung der Komplexität des Transkriptoms relativ zum und neurodegenerativen Erkrankungen, Genom bei, da mittels alternativer Promotoren eines (4) sowie transkriptionelle Regulation bei entwick- Gens multiple mRNA-Isoformen aus diesem gene- lungsbiologischen Prozessen. riert werden können. Promotoren sind daher in der Dabei werden u.a. „yeast-two-hybrid screening“ Postgenom-Ära von besonderem Interesse, welche und Coimmunopräzipitation (CoIP) zur Analyse die Physiologie und Pathophysiologie der humanen von Protein-Protein-Interaktionen, „electromobility Spezies mit seiner relativ geringen Genanzahl erklä- shift assay“ (EMSA) und Chromatin-Immunopräzi- ren muss. pitation (ChIP) zur Untersuchung von Protein-DNA- Die (patho)physiologische Bedeutung von Promoto- Interaktionen sowie Promotor- Reportergen-Assays, ren zeigt sich anhand des Effektes von genetischen 5´-RACE und „RNase protection assay“ (RPA) zur Polymorphismen in Promotorbereichen, die mit psy- Promotoranalyse verwendet. Die Bindung von mole- chologischen Merkmalen, physiologischen Merk- kular nicht identifizierten Transkriptionsfaktoren an malen und der Anfälligkeit für bestimmte Erkrankun- Promotorregionen wird mittels DNA-Affinitätschro- gen assoziiert sein können. Des Weiteren spielen matographie und Massenspektrometrie analysiert. Promotorpolymorphismen bei den vieldiskutierten Schließlich betrachten wir die Promotorebene als Gen-Umwelt-Interaktionen, sowie bei pharmakoki- therapeutische Zielstruktur, welche durch klassische netischen und pharmakodynamischen Fragestellun- Pharmaka (z.B. Steroide oder Glitazone) und gen- gen eine Rolle. therapeutische Ansätze (z.B. “decoy”-Oligonukleo- Unsere Forschungsprojekte weisen folgende tide oder “triple helix”-Bildner) beeinflusst werden Schwerpunkte auf: kann, wodurch sich Synergien mit unserem “Drug (1) Basale Mechanismen der transkriptionellen development”-Schwerpunkt ergeben. Regulation inklusive Epigenetik,

200 HEIKO FUNKE-KAISER – Pharmacology

1. Drug development - the renin/ prorenin receptor as a pharmacological target

Project leader Heiko Funke-Kaiser Coworkers Jennifer Schmitz, Petra Goldin-Lang, Sebastian Kirsch, Kristin Schmerbach, Daniela Zaade, Sabrina Klare, Kerstin Seidel, Melanie Kroh, Sarah Bernhard, Eva Schrezenmeier, Yaosi Li, Jan H. Schefe, Frank Zollmann Funding BMBF GO-Bio IBB ProFIT (EU/ EFRE) Stiftung Charité External Cooperations Dr. Jens Peter von Kries (Leibniz-Institut für Molekulare Pharmakologie (FMP), Berlin), Evotec AG, Hamburg, Prof. Dr. Achim Kramer, Dr. Rudolf Volkmer-Engert (Department of Immunology, Charité)

Cardiac, renal and ophthalmological end-organ dam- of a decoy peptide can prevent or even abolish the age due to hypertension and/ or diabetes is currently development of cardiac fibrosis and diabetic neph- one of the major medical challenges. They contribute ropathy via angiotensin II-independent mechanisms. to approximately 60-80% of all heart failure and 70% Therefore, the RER is a promising therapeutic drug of all renal failure cases. In addition, up to 30% of blind- target for cardiovascular disease. In addition, recent ness in western countries is caused by these diseases. publications indicate that the RER might also be a drug Current therapeutic strategies, such as ACE inhibitors, target in oncology, e. g. based on the direct physical angiotensin AT1 receptor blockers (ARBs), b-adren- interaction between this protein and Wnt receptors ergic receptor antagonists or antidiabetic agents only which is crucial for Wnt signaling. ameliorate but do not abolish cardiovascular end- Our group revealed a novel signal transduction organ damages. Therefore, cardiovascular end-organ pathway involving direct protein–protein interaction damage represents an unmet medical need. between the RER and the transcription factor pro- A human renin/ prorenin receptor (RER, also called (P) myelocytic zinc finger protein (PLZF) as well as the RR), which can specifically bind prorenin and renin, nuclear translocation of PLZF upon renin and prorenin has been cloned in 2002. Binding of renin to this recep- stimulation. Downstream effects of a RER activation by tor increases renin´s catalytic activity about 4- to 5-fold. renin and prorenin include repression of the RER gene Furthermore, the receptor is able to unmask the enzy- itself, induction of the p85alpha subunit of the phos- matic activity of prorenin. phatidylinositol-3 kinase (PI3K-p85alpha) and - con- The RER plays a crucial role in cardiac and renal end- sistently - an increase in proliferation and a decrease organ damage. Various animal models from independ- in apoptosis. ent research groups demonstrated that inhibition of This (pro)renin-RER-PLZF-RER/ PI3K pathway was prorenin binding to the RER by parenteral delivery filed as a patent (disclosure EP 1 890 152 A1; PCT

201 HEIKO FUNKE-KAISER – Pharmacology filing PCT/EP2007/006100), since it can be used as PLZF, mRNA quantification or protein-protein/ protein- a read-out for RER activity within high-throughput DNA interactions were set up to yield so-called hits. screening (HTS) assays. In addition, a hit-to-lead program to filter down these The aim of an interdisciplinary project team is to multiple hits according to pharmacodynamic, phar- develop a novel drug class called renin/ prorenin macokinetic and toxicologic parameters has been receptor blockers (RERBs). RERBs will represent prepared. Accordant methods including Ames test, small molecules with oral bioavailability and the ability micronucleus test, PAMPA assay, cytochrome P450 to inhibit the renin/ prorenin receptor for the indication induction as well as the cloning/ expression of multi- hypertension- and diabetes-related renal and cardiac ple recombinant proteins of the (pro)renin-RER-PLZF end-organ damage. pathway for subsequent analysis by e.g. the Alpha During a complex assay development phase, two Screen technology or Biacore have been built up. HTS assays based on double stably transfected cell Finally, after complex spin-off negotiations with the lines and a luciferase read-out were established. Charité, which were finalised in Summer 2010 by Subsequently, two independent HTS campaigns of notarial signing, a start-up company called CCR about 100,000 and about 20,000 compounds (cpds), Pharma GmbH [http://www.ccr-pharma.de] will be respectively, have been successfully performed. After- founded in late 2010. Furthermore, senior advisers wards, the primary hits were subjected to hit confirma- regarding e.g. medicinal chemistry and a putative CFO tion and hit profiling phases, with the latter analysing have been recruited. CCR Pharma´s aim is to develop dose-response relationships. RERBs up to clinical phase I/ II and to license com- To validate the confirmed compounds generated by pounds in return for payments ensuring the growth of these primary screenings, multiple secondary screen- CCR Pharma´s own pipeline. ing assays e. g. based on nuclear translocation of ,

Function and signal transduction of the renin/ prorenin receptor (RER). RERBs (Renin/ Prorenin Receptor Blockers) designate a putative novel drug class which inhibits the RER (also abbreviated (P)RR).

202 HEIKO FUNKE-KAISER – Pharmacology

2. Role of PLZF in neuroprotection

Project leader Heiko Funke-Kaiser Coworkers Kerstin Seidel, Sebastian Kirsch, Kristin Lucht, Daniela Zaade, Jana Reinemund, Jennifer Schmitz, Sabrina Klare, Yaosi Li, Jan H. Schefe, Kristin Schmerbach, Petra Goldin-Lang, Frank Zollmann Funding IBB ProFIT (EU/ EFRE) EU Network of Excellence “InGenious HyperCare” External Cooperations Christa Thöne-Reineke (Charité)

Stroke is one of the major medical burdens in industrial- were downregulated on the ipsilateral side in a stroke ised countries. Animal experiments indicate that block- model in vivo, whereas the neurodetrimental PLZF tar- ade of the angiotensin AT1 receptor (AT1R) improves get genes cyclin A2 and BID were upregulated under neurological outcome after cerebral ischemia. These this condition. Further analyses indicated that the neu- protective effects are partially mediated by the angi- roprotective AT2R is upregulated upon stable PLZF otensin AT2 receptor (AT2R). The transcription factor overexpression in cultured neuronal cells. promyelocytic zinc finger protein (PLZF) was identified Finally, reporter gene assays demonstrated the func- as a direct adapter protein of the AT2R. Furthermore, tionality of (P)RR promoter polymorphisms regarding our group was able to demonstrate that PLZF also basal and PLZF-induced activity. directly binds and mediates the effects of the human Our data indicate that the transcription factor PLZF (pro)renin receptor ((P)RR, also called RER) which is could be a novel regulator in the pathophysiology of involved in brain development. Therefore, we hypoth- ischemic brain injury based on its neuroprotective esised that PLZF is involved in neuroprotection. role in vitro and its downregulation on the stroke side Here we show that PLZF and its receptors (P)RR and in vivo. AT2R exhibited an ubiquitous expression pattern in different brain regions. Furthermore, stable Mechanisms of PLZF-induced neuro- PLZF overexpres- protection. sion in human neu- The effects of PLZF on the neuroprotective factor AT2R, the pro- ronal cells was able proliferative protein cyclin A2 and the to mediate neuropro- pro-apoptotic protein BID are shown. tection in a glutamate The link between pro-proliferative toxicity model in vitro. signals and neuronal loss is based on the fact that activation of the cell cycle Consistently, PLZF causes cellular death in terminally dif- mRNA and protein ferentiated neurons.

203 HEIKO FUNKE-KAISER – Pharmacology

3. Role of promoter polymorphisms within cardiovascular genes in Alzheimer´s disease

Project leader Heiko Funke-Kaiser Coworkers Yaosi Li, Michael Klein, Antonia Hope, Peter Marschall, Jens Schacherl, Sylvia Scheele, Kristin Schmerbach, Kerstin Seidel, Petra Goldin-Lang, Jennifer Schmitz, Mario Menk, Jan H. Schefe, Jana Reinemund Cooperations Kompetenznetz Demenzen [PD Dr. Heike Kölsch, Prof. Dr. Wolfgang Maier (Department of Psychiatry, University Bonn), Dr. Oliver Peters (Department of Psychiatry, Charité), Dr. Christian Roos (Leibniz-Institut für Primatenforschung, Göttingen), Ulrich Kintscher (CCR, Charité), Prof. Dr. Achim Kramer, Dr. Rudolf Volkmer-Engert (Department of Immunology, Charité), Prof. Dr. Peter Walden, Dipl.-Biochem. Rebecca Hugel (Department of Dermatology, Charité) Funding Dr. Werner Jackstädt-Stiftung

Alzheimer’s disease (AD) is the most common form ological factor. Important cardiovascular gene prod- of dementia affecting approximately 5 million patients ucts such as angiotensin-converting enzyme (ACE), within the European Union. AD can be grouped into neprilysin (NEP) and endothelin-converting enzyme-1 seldom familial (monogenetic) and frequent sporadic (ECE-1) are capable of degrading amyloid b. ECE-1 forms. Regarding the pathophysiology of AD extracel- can degrade Ab40 and Ab42 in vitro. Furthermore, lular deposition of amyloid b (Ab) plaques, composed of cerebral Ab40/42 content is significantly increased in Ab40-42, as well as intraneuronal neurofibrillary tangles, ECE-1 knockout mice compared to wild type animals. consisting of hyperphosphorylated tau protein, seem Since sporadic Alzheimer´s disease has a strong genetic crucial. Cardiovascular risk factors such as hypertension, component of about 80%, this project examines the diabetes and hypercholesterolemia not only contribute role of promoter polymorphisms within endothelin sys- to cardiovascular morbidity but also increase the risk tem genes. Especially, we functionally analyse regula- for Alzheimer´s disease. Research in recent years sug- tory sequences which bind unidentified transcription gests that cholesterol modulates synthesis of amyloid ß factors (i.e., a positive signal in EMSA but with unknown which itself is a potent vasoconstrictor and can cause matrix (consensus seqeunce) according to bioinformat- endothelial dysfunction. Clinical data indicate that cer- ics) using DNA affinity chromatography, mass spec- ebral hypoperfusion precedes cognitive decline in AD. trometry and reporter gene assay. In addition we have In addition, cerebral vessels in AD can be affected not genotyped promoter polymorphisms of several hun- only by the amyloid angiopathy but also by alterations of dreds of patients with AD and respective controls in endothelial cells, VSMCs and the basement membrane. cooperation with the German Kompetenznetz Demen- In sporadic forms of AD the decreased degradation/ zen, a nationwide network which includes 14 university clearance of amyloid b is an important pathophysi- centers in the field of dementia research.

204 HEIKO FUNKE-KAISER – Pharmacology

Role of ECE-1 in Alzheimer´s disease. Objectives: As shown by other groups, cerebral Ab40/ 42 content is significantly increased in ECE-1 knockout mice compared to wild type animals. Consistently, induction of ECE-1 by transgenic overexpression of PKC strongly reduces plaque and Ab deposit area. In humans, single nucleotide polymorphisms (SNPs) in the ECE-1b isoform-specific promoter, which were initially described by our group in the context of arterial hypertension (Funke-Kaiser, H. et al. (2003) Hum. Mol. Genet.), affect prefrontal ECE-1 mRNA expression and are associated with the likelihood of developing AD to a similar degree as the APOE2 allele.

205

Kai Kappert – Pharmacology

Head of the group

Priv.-Doz. Dr. med. Kai Kappert

Curriculum Vitae: After a three-year post-doctoral position at the Karolinska Institute, Stockholm, Sweden, Kai Kappert, MD, Group Leader Molecular Pharma- cology, joined the Center for Cardiovascular Research (CCR), Institute of Pharma- cology, in 2006. He received the medical approbation (3rd state examination) and an MD degree in 2003, based on experimental work at the German Heart Center Berlin (DHZB). Educated as a medical doctor (Georg-August-University Göttingen; Humboldt-University Berlin; University of California Los Angeles, USA; Inselspital Bern, Switzerland) with clinical experience in the field of cardiology (University Clinic Cologne), his major focus of his research has been growth factor signalling, including the impact of endogenous inhibitory protein tyrosine phosphatases, in vascular cells and tissue remodelling. In 2009 he passed his habilitation in experimental pharmacology at the Charité- University Medicine Berlin.

Members of the group

Students Hackbusch, Daniel PhD student Häßle, Paul Medical student Krüger, Janine PhD student

Daniel Hackbusch Paul Häßle Janine Krüger

207 Kai Kappert – Pharmacology

Summary peutic approach altering tyrosine-signaling. Further- more, accumulating data on the structural basis and Protein phosphorylation on tyrosine residues rep- protein interaction of PTPs should pave the way for resents an important cell-signaling mechanism, considering PTPs as strategic therapeutic targets in controlled by the combined balanced actions of human diseases. tyrosine kinases and protein tyrosine phosphatases The research focus of our group is the unravelling of (PTPs). Regulatory mechanisms of classical PTPs, the significance of PTPs on distinct regulatory levels, a cysteine-based subclass of the PTP superfamily such as post-transcriptional oxidation, expression, that exclusively dephosphorylates phospho-tyrosine and activity in various experimental organ-based in proteins, include changes in expression, phos- and animal models. This includes analyses of tran- phorylation, and subcellular localization. Further- sient negative regulation of PTPs by oxidation under more, a novel negative regulatory mechanism is the conditions of ischemia/reperfusion, and the charac- reversible oxidation of the conserved catalytic-site terization of PTPs during cerebro-vascular positive cysteine. outward remodelling. An additional research focus is Differential regulation of PTPs has recently been to investigate the impact of PTPs in models of insulin demonstrated in vascular tissue remodelling, dia- resistance. Thus, we expect to further identify and betes, cell differentiation, and cancer development. establish PTPs as targets for treatment of cerebral Thus, targeting PTPs might serve as a novel thera- occlusive vascular disease and insulin resistance.

208 Kai Kappert – Pharmacology

Zusammenfassung änderung der Tyrosin-vermittelten Signaltransduk- tion in Frage kommen. Darüber hinaus sollten die Die Phosphorylierung von Tyrosinresten in Proteinen zunehmenden Kenntnisse der strukturellen Basis stellt einen wichtigen Mechanismus in der zellulären der Proteininteraktionen von PTPs den Weg für die Signaltransduktion dar, der durch die koordinierte Nutzung von PTPs als Zielmoleküle in der humanen Aktivität von Tyrosinkinasen und Protein-Tyrosin- Therapieforschung ebnen. Phosphatasen (PTPs) reguliert wird. Zu den Regu- Der Forschungsschwerpunkt unserer Gruppe liegt lationsmechanismen der klassischen PTPs, eine in der Aufklärung der Bedeutung von PTPs auf Cystein-basierte Untergruppe der PTP-Superfa- unterschiedlichen regulatorischen Ebenen, wie die milie, die ausschließlich Phosphotyrosin in Protei- der post-transskriptionellen Oxidation, der Expres- nen dephosphoryliert, gehören Expressionsunter- sion und der Aktivität in unterschiedlichen Organ- schiede, Veränderung der Phosphorylierung von und Tiermodellen. Dies beinhaltet die Analyse von PTPs selbst, sowie die subzelluläre Lokalisation von transienter negativer Regulation von PTPs unter PTPs. Ein neuartiger negativer Regulationsmecha- den Bedingungen von Ischämie/ Reperfusion und nismus ist die reversible Oxidation des konservierten die Charakterisierung von PTPs während positivem Cysteinrestes im aktiven katalytischen Zentrum von zerebrovaskulären Outward-Remodelling. Ein wei- PTPs. terer Forschungsschwerpunkt ist die Untersuchung Die differentielle Regulierung von PTPs konnte kürz- der Bedeutung von PTPs in Insulinresistenzmodel- lich in Umbauprozessen von Gefäßen, beim Diabe- len. Insgesamt verfolgen wir den Ansatz, PTPs als tes mellitus, in der Zelldifferenzierung und auch bei Zielstrukturen im Rahmen der Behandlung von zere- der Tumorigenese nachgewiesen werden, so dass bralen Gefäßerkrankungen und der Insulinresistenz PTPs als molekulare Strukturen für die gezielte Ver- zu etablieren.

209 Kai Kappert – Pharmacology

Research projects

1. Protein tyrosine phosphatases as negative regulators and novel targets in collateral growth

Project leader Kai Kappert Coworkers Daniel Hackbusch Funding - DFG KA 1820/4-1 - Deutsche Stiftung für Herzforschung F/04/08 External Cooperations Ivo Buschmann, Nora Gatzke, André Dülsner (Department of Cardiology/ ) CCR Markus Dagnell, Arne Östman (Department of Oncology-Pathology, Cancer Centrum Karolinska, Karolinska Institute, Stockholm, Sweden)

Protein tyrosine phosphatases (PTPs) are dephos- PTP-antagonists. We hypothesize that PTP-inhibition phorylating enzymes with primarily negative impact and thus disinhibition of tyrosine-phosphorylation in on tyrosine signal transduction. This project will char- receptor tyrosine kinases will lead to an increase of acterize the dynamic regulation and function of PTPs vascular proliferation and consecutively to enhanced during cerebral collateral growth/ arteriogenesis in a cerebral blood flow. Taken together, the project aims model of 3-vessel occlusion. Furthermore, in a proof- at demonstrating that PTPs represent novel targets of-concept approach the role of PTPs on arteriogenesis for treatment of cerebral occlusive vascular disease. is being tested by pharmacological intervention using

Schematic diagram of the potential regulation of arteriogenesis by receptor tyrosine kinase (RTK)-antagonizing PTPs, and the therapeutic approach of enhanced arteriogenesis through inhibition of inhibitory PTPs.

210 Kai Kappert – Pharmacology

2. Role of protein tyrosine phosphatases in high-fat diet induced insulin resistance

Project leader Kai Kappert Coworkers Janine Krüger Funding Personal funding of Charité-University Medicine Berlin Dissertation scholarship of Charité-University Medicine Berlin German Diabetes Society External cooperations Christian Böhm, Christiane Sprang, Ulrich Kintscher (Institute of Pharmacology/ CCR) Heike Meyborg, Philipp Stawowy (German Heart Center Berlin)

Diabetes and the metabolic syndrome are major risk lyzed in an animal model of high-fat diet induced insulin factors for the development and progression of car- resistance in vivo. Mice are being fed either low-fat diovascular disease, including atherosclerosis. On a (10% kcal from fat) or high-fat (60% kcal from fat) diet molecular level, obesity-linked insulin resistance has for 16 weeks with or without additional treatment with been attributed to a post-receptor deficiency. Recent PTP-inhibitors. In parallel, the precise characterization data suggest protein tyrosine phosphatases (PTPs) of PTPs will be performed in insulin-sensitive cell in significantly impacting on insulin receptor signal trans- vitro. Aim of the project is to establish PTPs as novel duction (see Figure). In this research project the tissue- targets in the treatment of insulin resistance. specific regulation and function of PTPs will be ana-

Recent evidence suggests that protein tyrosine phosphatases (PTPs) are impacting on the tyrosine status of the insulin receptor, and thus downstream signaling.

211

Elena Kaschina – Pharmacology

Head of the group

Dr. med. Elena Kaschina

Curriculum Vitae: Elena Kaschina studied medicine at the St.-Petersburg State I.P.Pavlov Medical University. After medical approbation she worked as an internist at the hospital. 1995 she obtained MD degree based on the experimental work at the Institute of Pharmacology, Medical Academy St. Petersburg. 1999 she received a Grant from Otto Benecke Stiftung in order to continue research at the University of Kiel. 2005 she built up her research group at the Institute of Pharmacology, the Center for Cardiovascular Research (CCR). From 2010 she is passing her habilitation in experimental pharmacology. Her research is focused on vascular and cardiac remodelling and drug development.

Members of the group

Technicians Sommerfeld, Manuela BTA Kemnitz, Ulrich Rudolf BTA

Students Akohov, Aleksej Medical student ManuelaSommerfeld SvetlanaSlavic DilyaraLauer Lauer, Dilyara PhD student ManuelaSommerfeld SvetlanaSlavic DilyaraLauer Slavic, Svetlana Medical student Schrader, Felix Medical student

UlrichRudolfKemnitz AleksejAkohov

213 Elena Kaschina – Pharmacology

Summary causative factor for aneurysm formation, and also in vascular wall homeostasis. Well established animal Pathophysiological mechanisms of aortic aneu- models and primary cell cultures from aorta allow us rysms and possible therapeutic implications to evaluate important signalling cascades and inves- Abdominal aortic aneurysm (AAA) is a common tigate new therapeutic targets. Human abdominal vascular disorder which causes significant mortal- aortic tissue from patients undergoing surgery is also ity. The treatment of AAA is restricted to surgical studied, and candidate genes for aneurysm disease interventions. Determination of specific genes and are analysed. Research is focused on the role of dif- pathomechanisms relevant for the development of ferent components of the renin-angiotensin system as aneurysms may identify target sites for potential phar- well as kininogen in vascular inflammation, proteolysis macologic interventions. and apoptosis as part of the aneurysmal disease. Aortic aneurysms are characterised by vascular The link of AAA with known risk factors for the inflammation, apoptosis and the degradation of extra- disease could help us in the understanding of the cellular matrix. Our aneurysm research focuses on pathomechanisms. Therefore, we are also interested the kallikrein-kinin- and renin-angiotensin systems, in vascular remodelling by obesity, insulin resistance which are known players in hemodynamic stress, a and uremia.

Zusammenfassung Gruppen als pathogenetisch äußerst wichtig für die Aneurysma-Entstehung erkannt wurden. Diese bei- Pathophysiologische Mechanismen von Aor den endokrinen Systeme betreffend weiß man inzwi- tenaneurysmen und mögliche therapeutische schen, dass Angiotensin II über den AT1-Rezeptor Implikationen eine fördernde, das KKS über Kininogen anderseits Das Aneurysma der Aorta abdominalis (AAA) ist eine hemmende Wirkung auf Krankheitsgeschehen eine weitverbreitete vaskuläre Erkrankung mit hoher ausübt. Gut etablierte Tiermodelle und Primärzellkul- Sterblichkeitsrate. Bis heute existiert keine therapeu- turen aus Gewebe der Aorta sowie humanes abdo- tische Alternative zur chirurgischen Intervention. Die minelles Aortengewebe erlauben uns, wichtige Sig- genaue Bestimmung von spezifischen Genen und nalkaskaden zu evaluieren und neue therapeutische Pathomechanismen, die für die Aneurysma-Entste- Strategien zu untersuchen. hung bedeutsam sind, könnte Angriffsmöglichkeiten Die aktuelle Forschung wird sich auf die Rolle des Kini- für eine pharmakologische Therapie dieser Erkran- nogens sowie verschiedener Komponenten des RAS kung identifizieren. für Inflammation, Proteolyse und Apoptose im Rahmen Die Entstehung von AAA ist durch Inflammation, der Pathogenesis von Aneurysmen konzentrieren. Apoptose und einen massiven Abbau der extra- Die Bedeutung des Adipositas und der Insulinresis- zellulären Matrix gekennzeichnet. Unsere Untersu- tenz sowie der Einfluss von Uremia auf Gefäßremo- chungen konzentrieren sich auf das Renin-Angio- deling werden auch in verschiedenen experimentel- tensin-System (RAS), sowie auf das Kallikrein-Kinin- len Modellen untersucht. System (KKS), die bereits von uns und anderen

214 Elena Kaschina – Pharmacology

Research projects

1. High molecular weight kininogen, a novel factor in the regulation of matrix proteinases

Project leader Elena Kaschina Coworkers Dilyara Lauer Manuela Sommerfeld, Svetlana Slavic, Ulrich Rudolf Kemnitz Funding Bayer Schering Pharma External cooperations Christa Thöne-Reineke, CCR Prof. Marjan Boerma, University of Arkansas, USA

Increased activity of matrix metalloproteinases tion of expression levels of the endogenous inhibitors (MMPs), leading to degradation of extracellular matrix of MMPs, the TIMPs, revealed that TIMP-1 mRNA components, is considered to play a crucial role in expression, already increased as a result of cytokine- the pathogenesis of cardiovascular diseases such as stimulation, was further enhanced by cleaved HK. heart failure and aneurysms. Thereby, a pivotal role Altogether, these findings indicate that the balance has been attributed to MMPs belonging to the sub- between MMPs and TIMPs was shifted towards less group of gelatinases, including MMP-2 (gelatinase A) net MMP activity by cleaved HK. and MMP-9 (gelatinase B). We previously reported that kininogen-deficient rats are predisposed to develop abdominal aortic aneurysms. Thereby, aneurysm formation was associated with enhanced elastolysis and increased expression of MMPs, thus Our current investigations are focused on the antiopro- indicating a role for kininogens in the regulation of teolytic effect of kininogen and its domains in primary MMPs. We investigated whether cleaved high molecu- cardiomyocytes. We found that cleaved HK prevented lar weight kininogen (cleaved HK) affects the regula- cytokine-induced release of MMP-9 and reduced tion of MMPs in primary vascular smooth muscle cells gelatinase/collagenase activity of cardiomyocytes. (VSMCs), cultured from the rat aorta. We found that In summary, high molecular weight kininogen is a cleaved HK reduced in a concentration dependent potential drug target for diseases with excessive manner cytokine-induced release of both MMP-9 and extracellular matrix turnover. Further research will be MMP-2 by VSMCs. Furthermore, cytokine-induced focused on therapeutic effects of kininogen in the MMP-9 mRNA expression was negatively regulated models of heart failure and aneurysm formation. by cleaved HK to almost the same extent. Determina- (published in Biochemical Pharmacology, 2010)

215 Elena Kaschina – Pharmacology

2. Aneurysm formation in mild uremia

Project leader Elena Kaschina Coworkers Aleksej Akohov, Manuela Sommerfeld, Ulrich Rudolf Kemnitz Funding internal resources External cooperations Harm Peters, CCR, Department of Nephrology, Charité Universitätsmedizin Berlin, Dr. Stephanie Kraemer, Deutsche Institut für Ernährungsforschung, Berlin

Mild renal failure is known to be an independent risk renal functional parameters and serum pro-inflamma- factor for cardiovascular disease. Mild uremia is fol- tory cytokines were investigated. lowed by fluid overload, inflammation and production Uremia induced aortic dilatation. Histological analy- of uremic toxins. All these factors may contribute to sis revealed an outward aortic remodelling, increased vascular remodelling. elastin fragmentation, cystic medial degradation, cal- The aim of this study was to investigate the effects of cification of tunica media and inflammatory infiltrates mild uremia on aneurysm size, as well as on matrix in the adventitia. After aneurysm induction, aortic remodelling, apoptosis, proteolysis and inflamma- diameter was further increased in the NE group as tion and their related gene- and protein expression compared to AAA rats without NE. Hydralazine treat- patterns in the rat. Investigation was performed in a ment significantly reduced blood pressure but did not combined model of experimental uremia followed by influence aortic diameter. elastase-induced aneurysm formation. Mild uremia was induced in rats by right uni- nephrectomy and removing 2/3 of the left kidney sur- gically (5/6 nephrectomy) (NE). 4 weeks later aneurysm (AAA) was induced via continuous infusion of an isolated aortic segment with elastase. One group of NE animals was additionally treated with hydralazine. Aortic diameter and heart function were measured by A: Ultrasound image of rat aorta. Arrows represent aortic diameter. B: Histological cross- ultrasound. Aortic tissues, sections of the rat abdominal aorta: Weigert’s stain, Sirius red-picric acid stain, x20, Lum indicates luminal side of aorta; adv indicates adventitial side.

216 Elena Kaschina – Pharmacology

Protein expression of NF-kB was strongly (3-fold) Alltogether, we could demonstrate that mild uremia up-regulated in aortic tissues from NE rats. MMP2, induces aortic outward remodeling independently of MMP9, cathepsin D and TGF-beta1 were significantly blood pressure elevation. Activation of NFkB by uremic increased after AAA and further up-regulated in the toxins may contribute to remodelling via inhibition of NE/AAA group. Heart function was also negatively elastin- and collagen gene transcription. affected by uremia: Ejection fraction and fractional shortening were significantly decreased whereas left ventricular volume was increased in the systole and in the diastole.

3. Regulation of cardiac and vascular functions by selective cannabinoid 1 receptor blocker rimonabant

Project leader Elena Kaschina Coworkers Svetlana Slavic, Manuela Sommerfeld, Ulrich Rudolf Kemnitz Funding Sanofi-Aventis GmbH Deutschland External cooperations Ulrich Kintscher, CCR PD Dr. med. Johannes Baulmann, University of Lübeck

The biological effects of endocannabinoids such as were studied in the model of myocardial infarction anandamide, are mediated by specific G protein-cou- (MI), which was induced by permanent ligation of the pled cannabinoid (CB) receptors. The CB1 receptor left coronary artery. Treatment with rimonabant was is present in heart and vascular tissues, fat tissue and started one week before MI and continued for 7 days or liver. CB1 receptor blockade by a selective antagonist 6 weeks. Hemodynamic parameters were measured rimonabant is known to be beneficial by obesity and via transthoracic Doppler echocardiography and int- metabolic syndrome in humans. racardiac Samba catheter. Arterial function was stud- Our study aimed to investigate whether cannabinoid-1 ied using pulse wave analysis. Cytokines, apoptotic receptor blockade may result in heart protection by and fibrotic markers were determined in heart tissue. metabolic syndrome and whether this protection is pri- Rimonabant improved systolic left ventricular func- mary and not dependent from metabolic corrections. tion, lowered left ventricular end-diastolic pressure We could show that rimonabant improves heart func- and increased heart contractility in the early and late tion in Obese Spontaneously Hypertensive Koletsky phase after myocardial infarction. Arterial function Rats. In non-obese rats, the effects of CB1-blockade parameters, such as pulse pressure, augmentation

217 Elena Kaschina – Pharmacology

pressure- and index, were decreased in the rimona- Thus, the cannabinoid-1 receptor antagonist rimonabant group mostly 6 weeks after MI compared to vehi- bant continually improves heart function in rats after cle. Analysis of locomotor behaviour demonstrated myocardial infarction. Reduced inflammation in the continuously increasing activity of treated rats. Up- heart and reduced pulse wave reflection may contrib- regulated IL-1beta-expression and TGF-beta expres- ute to cardiac protection by rimonabant. sion in postinfarcted myocardium was ameliorated by Since rimonabant improved vascular function by rimonabant treatment at both time points, whereas decreasing arterial stiffness as well as by diminishing caspase 3 was down-regulated only 7 days after MI. concentration of hydroxyprolin in aorta, our research will be further focused on the role of CB1 receptor in vascular diseases.

Representative M-mode echocardiograms (short axis parasternal view) of the LV from rats 6 weeks after myocardial infarction treated with rimonabant (A) and vehicle (B).

218 Jun Li – Pharmacology

Head of the group

Dr. med. Jun Li

Curriculum Vitae: Jun Li initially studied medicine in China and subsequently at the University of Kiel, where he earned his MD. He then pursued research in cellular immunology and molecular cardiology at the University of Mainz and the Free University of Berlin. Since July 2002, he has been working as a group leader dealing with adaptive protective mechanisms against ischemic injury in the brain and heart at the Center for Cardiovascular Research and Institute of Pharmacology at the Charité – Universitary Medicine in Berlin. In 2005, he won the Dieter-Klaus-Award for Hypertension Research of the German Hypertension Society for his innovative work exploring the interactions of angiotensin AT2 receptor and T lymphocytes in the heart. A main focus of his current research is the elucidation of the adaptive protective mechanisms of cell biology and the impact of these cellular mechanisms on the outcomes of cardiovascular disease.

Members of the group

Technicians Timm, Melanie

Students Adjemian, Sandy Master student Altarche-Xifró, Wassim PhD student Brinckmann, Marie Medical student Curato, Caterina PhD student Miteva, Kapka Master student Skorska, Anna PhD student Slavic, Svetlana Medical student (physician)

219 Jun Li – Pharmacology

Summary

Acute myocardial infarction (MI) leads to loss of of information indicates that the renin-angiotensin cardiomyocytes and impaired pump function of the system and endogenous estrogen can interfere with heart. When the remaining cardiomyocytes are una- the cardiac injury/repair process after MI. Given the ble to reconstitute with time, heart failure results. A recent evidence that both angiotensin AT2 receptor central question in the cardiac cell biology is what are and estrogen receptor a are involved in cardiopro- the cellular mechanisms that mediate the generation tective actions, we are particularly interested in the and maintenance of cardiac performance. The focus elucidation of cellular mechanisms by which cells of our research is the adaptive interaction of cardiac of the periinfarct myocardium exert cardioprotec- various cell populations including c-kit+ precursor tion via the activation of angiotensin AT2 receptor or cells, T lymphocytes, and cardiomyocytes. A wealth estrogen receptor a.

Zusammenfassung

Angiotensin II, das Haupteffektorprotein des Renin- Rezeptor Antiinflammation, Differenzierung, sowie Angiotensin-System (RAS), ist an der Entwicklung Regeneration vermittelt. Weitere Forschungen von kardiovaskulären Erkrankungen beteiligt. Der befassten sich mit der Rolle des AT2-Rezeptors AT2-Rezeptor ist im fetalen Leben der dominierende beim Entzündungs- und Regenerationsprozess ATR-Subtyp. Später wird er nach Verletzungen bzw. nach einem Herzinfarkt. Ein anderer Schwerpunkt im Verlauf pathosphysiologischer Prozesse, wie zum lag auf Untersuchungen zum Einfluss von Östrogen Beispiel Schämie im Herz bzw. Gehirn, exprimiert. auf das kardiale Remodeling nach Myokardinfarkt. Dieses spezifische Expressionsmuster weist darauf Insbesondere wird die Rolle von Östrogenrezepto- hin, dass der AT2-Rezeptor bei der Gewebsadap- ren, wie ERa und ERb auf die Selbsterneuerung und tation und -regeneration eine wichtige Rolle spielt. Differenzierung von Stammzellen im Tiermodel mit In Vorarbeiten konnten wir zeigen, dass der AT2- experimentellem Myokardinfarkt untersucht.

220 Jun Li – Pharmacology

Research projects

1. Angiotensin AT2 receptor in cardiac regeneration

Project leader Jun Li Coworkers Wassim Altarche-Xifró, Caterina Curato, Svetlana Slavic, Melanie Timm, Elena Kaschina, Aleksandra Grzesiak Funding - BMBF (BCRT-kick off grant and 1st. BCRT grant) External cooperations Institute of Cell Biology, University of Bern, Switzerland (Prof. H. Imboden); Reference and Translation Center for Cardiac Stem Cell Therapy, University of Rostock (Prof. Dr. G. Steinhoff)

The expression pattern of AT2 receptors with pre- receptor activation on the performance of adult car- dominance during fetal life and upregulation under diomyocytes co-cultured with post-infarct cardiac pathological conditions during tissue injury/repair c-kit+AT2+ cells. To extrapolate the in vivo role of points towards the fact that AT2 receptors may exert post-infarct cardiac c-kit+AT2+ cell population, we an important action in injury/repair adaptive mecha- studied whether selective AT2 receptor activation may nisms, which may be entirely different from the known enhance adaptive cellular regeneration, especially in effects of angiotensin II mediated through the AT1 post-infarct cardiac c-kit+AT2+ cell population and receptor. Indeed, recent experimental evidence from cardiomyocytes following acute ischemic injury in rats. our group supports that angiotensin II, through its AT2 Thus far we have shown that cardiac c-kit+AT2+ cell receptor subtype, promotes cellular differentiation and population exists and increases after acute ischemic tissue regeneration. In this project, we aimed to under- injury. AT2 receptor activation supports cardiomyocyte stand the adaptive cellular mechanisms underlying performance, hence contributing to cardioprotection angiotensin AT2 receptor-mediated cardioprotection. via cardiac c-kit+AT2+ cell population. First of all, we examined the cellular regulation of cardiac AT2 receptor in response to acute myocardial infarction in Representative images of rats. Further, we isolated and stained cardiac free-floating characterized post-infarct sections showed that specific cardiac c-kit+AT2+ cell popu- signals for AT2 receptor were detected in accumulating lation. To explore a potential c-kit+ cells distributing in role of post-infarct cardiac the peri-infarct myocardium. c-kit+AT2+ cell population in Arrows are pointing at the c-kit+AT2+ cell clusters under cardioprotection, we exam- a Leica LSM confocal micro- ined the in vitro effect of AT2 scope. Scale bar, 10 mm

221 Jun Li – Pharmacology

2. Angiotensin AT2 receptor in cardiac inflammation

Project leader Jun Li Coworkers Caterina Curato, Anna Skorska, Svetlana Slavic, Melanie Timm, Kapka Miteva, Elena Kaschina Funding - EU EST- CARDIOVASC-(TP 3.2) External cooperations German Rheumatism Research Centre and Berlin-Brandenburg Center for Regenerative Therapies (Dr. J. Dong, Prof. Dr. A. Thiel); 2nd Affliated Hospital of Zhejiang University, China (Prof. Dr. J. Wang); Institute of Cell Biology, University of Bern, Switzerland (Prof. H. Imboden); Reference and Translation Center for Cardiac Stem Cell Therapy, University of Rostock (Prof. Dr. G. Steinhoff); Department of Cardiology, Charité-Universitätsmedizin Berlin, Campus Virchow – Klinikum (Dr. S. von Haehling)

Emerging evidence suggests a cardioprotective role CD8+AT2R+ T cells and IL-10 production, likely con- of the angiotensin AT2R, albeit the underlying cellular tributing to reduced infarct size and improved cardiac mechanisms are poorly understood. Here, we defined function as shown recently by our group. In addition, the CD8+AT2R+ T cell population, which increased intramyocardial transplantation of CD8+AT2R+ T cells following acute myocardial infarction (MI). Further, (vs. CD8+AT2R-) led to reduced ischemic heart injury. we developed a method to selectively isolate these These findings advance our understanding of the func- cardiac CD8+AT2R+ T cells, which exhibited down- tional role of AT2R in the heart and provide a novel regulated expression of pro-inflammatory cytokines cellular mechanism via cardioprotective CD8+AT2R+ and noncytotoxicity to cardiomyocytes. AT2R activa- T cells. tion engendered a significant increment in cardiac

222 Jun Li – Pharmacology

3. Cardioprotective role of estrogen receptor a via c-kit+ precursor cells

Project leader Jun Li Coworkers Marie Brinckmann, Svetlana Slavic, Anna Skorska, Wassim Altarche-Xifró, Caterina Curato, Melanie Timm, Elena Kaschina, Aleksandra Grzesiak Funding - DFG (GK 754-III, TP7b) - FERMENTATION – BIOTEC GmbH External cooperations Institute of Gender in Medicine and Center for Cardiovascular Research (CCR), Charité University Medicine Berlin (Prof. Dr. V. Regitz-Zagrosek); Reference and Translation Center for Cardiac Stem Cell Therapy, University of Rostock (Prof. Dr. G. Steinhoff); Department of Cardiology, Charité-Universitätsmedizin Berlin, Campus Virchow – Klinikum (Dr. S. von Haehling)

A growing body of clinical evidence demonstrates the regulation of estrogen receptor (ER) in post-infarct that endogenous estrogen may protect cardiovas- cardiac c-kit+ cells and their functional relevance in cular health in both female and male patients. Given response to acute ischemic injury in male rats. The the recent observations that estrogen exerts car- current results have demonstrated that ERa is mainly dioprotective effects via augmented mobilization of induced in post-infarct cardiac c-kit+ cells in response bone marrow-derived endothelial progenitor cells to acute myocardial infarction in rats and that ERa and enhanced angiogenesis, it is also possible that stimulation promotes the proliferation of undifferenti- estrogen receptors may be involvoed in regulating ated myoblast cells. In addition, ERa activation sup- cardiac performance via c-kit+ precursor cells. To ports survival of adult cardiomyocytes via post-infarct understand the potential cellular mechanisms underly- cardiac c-kit+ cells which affords a direct explanation ing estrogen-mediated cardioprotection, we examined for protective actions of ERa against cardiac injury.

223 Jun Li – Pharmacology

Awards

2009 PhD Student Award of the Berlin-Brandenburg School for Regenerative Therapies Wassim Altarche-Xifró

224 Ulrike Steckelings – Pharmacology

Head of the group

Dr. med. Ulrike Steckelings

Curriculum Vitae: Ulrike Steckelings is a dermatologist and has longterm and profound experience in pharmacological teaching and research. She gained her MD at the University of Heidelberg and did her specialisation in dermatology at the Charité Universitary Medicine in Berlin. She worked as a postdoc at the Institute for Pharmacology in Heidelberg, in the Department for Internal Medicine at the University of Melbourne, Australia and in the Department for Cardiovascular Research at Ciba Geigy in Basel, Switzerland. Her research foci are the AT2 receptor as well as the renin angiotensin system of the skin. At the CCR she coordinates preclinical research of compound 21, an oral, non peptidogenic AT2 agonist.

Members of the group

Scientists Villela, Daniel PhD student (guest scientist, Paulis, Ludovit MD, PhD (EU Marie Curie Fellow) University of Belo Horizonte, Brazil) Santi, Francecsca MD (visiting scientist from Wardat, Sami PhD student (student on joint Bologna, Italy) project with Prof. Kintscher’s group) Wieland, Anja MD student Technicians Lucht, Kristin

Students Becker, Sophie MD student Hosenfeld, Ann-K. Diploma student Horlbeck, Marie MD student Kummert, Maxi MD student Leonhardt, Julia MD student Namsolleck, Pawel PhD student Reichenbach, Anne MD student Rompe, Franziska PhD student Sadegh pour Saleh, H. Diploma student Schwengel, Katja PhD student Ströder, Katja PhD student Valero, Veronica PhD student

225 Ulrike Steckelings – Pharmacology

Summary through the AT2R (Rompe et al., Hypertension 2010, 55: 924-931; see project 1 below for more details). The peptide hormone angiotensin II exerts its actions A first in vivo study in rats in cooperation with Elena mainly via two receptor subtypes, the AT1- and the Kaschina (AG Unger, CCR) showed the in vivo activ- AT2-receptor. Actions mediated via the AT1-receptor ity of the compound in general and specifically a are well defined and mainly associated with cardio- favourable therapeutic effect of AT2R stimulation vascular physiology and disease. In contrast, AT2R after myocardial infarction resulting in improved scar research has been hampered for almost 2 decades formation and cardiac function (Kaschina et al., Cir- by the lack of an in vivo active and stable AT2R- culation 2008, 118: 2523-2532). At present, approx. agonist. In November 2004, design and synthesis 30 studies are going on – many with cooperations of the first specific and selective non-peptide AT2R- inside and outside of CCR - testing AT2R function agonist, Compound 21 (C21), have been published and the therapeutic potential of AT2R-stimulation in by the group around Anders Hallberg from Uppsala a broad spectrum of experimental models mainly University. Our group started working with C21 in covering cardiovascular, chronic inflammatory, neu- mid 2005 first confirming in vitro the agonistic prop- rological and dermatological diseases. erties of this molecule and the specificity of its effects

Zusammenfassung bestätigten (Rompe et al., Hypertension 2010, 55: 924-931; für weitere Details siehe Projekt 1 unten). Das Peptidhormon Angiotensin II wirkt im wesent- Eine erste in vivo Studie in Kooperation mit Elena lichen über zwei Rezeptor-Subtypen, den AT1- und Kaschina (AG Unger, CCR) belegte nicht nur prinzi- den AT2-Rezeptor. Die über den AT1-Rezeptor ver- piell die in vivo Wirksamkeit von C21, sondern zeigte mittelten Wirkungen sind gut charakterisiert und auch im Speziellen einen günstigen, therapeutischen betreffen im Wesentlichen physiologische und Effekt auf die Narbenbildung und die Herzfunktion patho-physiologische Wirkungen im Bereich des im infarzierten Herzen in Ratten. (Kaschina et al., kardiovaskulären Systems. Die Erforschung des Circulation 2008, 118: 2523-2532). Zur Zeit werden AT2-Rezeptors dagegen wurde jahrelang dadurch ca. 30 Projekte unter Verwendung von Compound erschwert, dass kein in vivo stabiler und wirksamer 21 durchgeführt - viele davon mit Kooperationen Agonist verfügbar war. Im November 2004 publi- innerhalb und außerhalb des CCR – in denen die zierte die Gruppe um Anders Hallberg von der Uni- Funktion des AT2R sowie das therapeutische Poten- versität Uppsala Design und Synthese des ersten tial einer AT2R-Stimulation in einem breiten Spekt- spezifischen und selektiven, nicht peptidischen rum experimenteller Modelle getestet werden, wobei AT2R-Agonisten, Compound 21. Unsere Gruppe der Schwerpunkt auf kardiovaskulären, chronisch arbeitet seit Mitte 2005 mit dieser neuen Substanz, inflammatorischen, neurologischen und dermatolo- zunächst mit in vitro Arbeiten, die die agonistische gischen Erkrankungen liegt. und AT2R-spezifische Wirkweise von Compound 21

226 Ulrike Steckelings – Pharmacology

Research projects

This group performs a whole series of projects – either at CCR or in cooperation with external partners - studying direct AT2-receptor stimulation with the novel AT2-receptor agonist Compound 21 (C21) in various in vivo and in vitro models and testing AT2-receptor stimulation as therapeutic approach for a range of potential indications focusing on cardiovascular, chronic-inflammatory and neurological diseases. In the following, three of these studies are described in more detail:

1. AT2-receptor stimulation as anti-inflammatory therapeutic approach

Project leader Ulrike Steckelings Coworkers Franziska Rompe, Pawel Namsolleck, Kristin Lucht Funding internal resources External cooperations Prof. Wolf-Hagen Schunck, MDC Berlin, Prof. Michael Bader, MDC Berlin, Dr. Metin Artuc, Dept. of Dermatology, Charité, Prof. Anders Hallberg, Uppsala University, Sweden, Prof. Björn Dahlöf, Göteborg University, Sweden

Angiotensin AT2-receptors can be regarded as an deficient cells. AT2-receptor coupled signalling lead- endogenous repair system because the AT2-receptor ing to reduced interleukin-6 levels involved inhibition is upregulated in tissue damage and mediates tissue- of NF-kB, activation of protein-phosphatases, and protection. A potential therapeutic utilisation of this synthesis of epoxyeicosatrienoic acid (EET) – the lat- system has only recently come within reach through ter being a signalling pathway described for the first synthesis of the first selective, orally active, non-pep- time for AT2R-mediated anti-inflammation. Compound tide AT2-receptor agonist, Compound 21. This study 21 also reduced monocyte-chemoattractant-protein tested AT2-receptor stimulation by Compound 21 (MCP)-1 and TNFa levels in vitro and in bleomycin- (C21) as a potential future therapeutic approach for the induced toxic cutaneous inflammation in vivo. inhibition of pro-inflammatory cytokines and of NF-kB. Since inhibition of NF-kB activity is a mechanisms Using primary human and murine dermal fibroblasts, of action shared by the AT2R and glucocorticoids, we found that C21 dose-dependently (1nM-1µM) we compared the efficiency of equal doses of C21 reduced tumor-necrosis-factor (TNF)a-induced inter- and hydrocortisone with respect to Inhibition of interleukin-6 levels. AT2-receptor specificity was control- leukin-6 promoter activity. We found that inhibition of led for by inhibition with the AT2-receptor antagonist, promoter activity by C21 was comparable in strength PD123319, and by absence of effects in AT2-receptor to inhibition by hydrocortisone.

227 Ulrike Steckelings – Pharmacology

Data of this study suggest that pharmacological AT2-receptor stimulation may be an orally applicable future therapeutic approach in pathological settings requiring the reduction of interleukin-6 or inhibition of NF-kB. (published 2010: Hypertension 55: 924-931)

228 Ulrike Steckelings – Pharmacology

2. AT2-receptor stimulation in spinal cord injury

Project leader Ulrike Steckelings Coworkers Pawel Namsolleck, Katja Schwengel, Christa Thöne-Reineke, Kristin Lucht Funding - Vicore Pharma External cooperations Prof. Sven Hendrix, Hasselt University, Belgium Francesco Boato, PhD student, Dept. of Neuroanatomy, Charité Prof. Anders Hallberg, Uppsala University, Sweden Prof. Björn Dahlöf, Göteborg University, Sweden

One of the first physiological effects ever described for vehicle for 4 weeks. Organotypic co-culture of GFP- the AT2R was neuroprotection. In this study we test positive entorhinal cortices with hippocampal target pharmacological AT2R-stimulation as a therapeutic tissue serves to evaluate the impact of C21 (1µM) on approach in spinal cord injury in mice using the novel reinnervation. Neuronal differentiation, apoptosis and non-peptide AT2R-agonist, Compound 21 (C21). expression of neurotrophins are investigated in pri- Complementary experiments in primary neurons and mary murine neuronal cells. organotypic cultures serve to identify underlying pro- Preliminary data indeed indicate that AT2-receptor regenerative mechanisms. Functional recovery and stimulation improves functional recovery in experi- plasticity of corticospinal tract (CST) fibers following mental spinal cord injury through promotion of axonal spinal cord injury (SCI) by compression in mice is mon- outgrowth and through neuroprotective and anti- itored after application of C21 (0.3 mg/kg/day i.p.) or apoptotic mechanisms.

229 Ulrike Steckelings – Pharmacology

3. AT2-receptor stimulation in experimental stroke

Project leader Ulrike Steckelings Coworkers Katja Schwengel, Pawel Namsolleck, Christa Thöne-Reineke Kristin Lucht Funding - Vicore Pharma External cooperations Prof. Masatsugu Horiuchi, Ehime University, Japan Francesco Boato, PhD student, Dept. of Neuroanatomy, Charité Prof. Anders Hallberg, Uppsala University, Sweden Prof. Björn Dahlöf, Göteborg University, Sweden

The aim of this study is to investigate the effect of direct performed daily to assess the severity of neurological AT2-receptor (AT2R) stimulation with the novel specific deficits. Infarct volumes are measured in vivo 96 hours and selective non-peptide AT2R agonist Compound post-stroke by MRI. To get a first impression about 21 (C21) on infarct size, survival and neurological out- potential molecular mechanisms of AT2R stimulation come after middle cerebral artery occlusion (MCAO) in stroke, mRNA-levels of neurotrophins and markers in mice. For this purpose, C57/BL-6 or AT2R knock- for neuronal sprouting are measured in brain samples out mice (on C57/BL-6 background) are forwarded to by quantitative RT-PCR. MCAO for 30 minutes followed by reperfusion. Starting Preliminary results indicate that C21 improves neuro- 45 minutes after MCAO, mice are treated daily with logical outcome and survival without affecting blood either vehicle (0,9% NaCl i.p.) or C21 (0,03mg/kg i.p.) pressure. for a period of 4 days. Garcia neurological score is

230 Ludovit Paulis – Pharmacology

Head of the group

Dr. med. Ludovit Paulis, PhD.

Curriculum Vitae: Ludovit Paulis, MD, PhD is currently holder of an EU Fellow- ship (Marie Curie) at the CCR under the supervision of Ulrike Steckelings and Prof. Thomas Unger. Dr. Paulis graduated in biomedical physics in 2004 and in general medicine 2006 at the University in Bratislava. He undertook his postgraduate studies in Prague, Berlin and Montpellier and again in Bratislava, where he received his PhD in 2008. He continued his post-doctoral research in Montpellier and later in Berlin.

Members of the group

Students Becker, Sophie Medical student

231 Ludovit Paulis – Pharmacology

Summary

The group represents a young researcher team in damage in experimental hypertension induced by the frame of the Steckelings workgroup. The main NO-synthase inhibition in rats. This project is sup- interest of the group is directed towards the preven- ported by the 7th EU FP project “COME-in-CARE”. tion and modulation of pathological cardiovascular The aim is to observe, whether and to what extent conditions, in particular arterial hypertension, myo- the investigated substances are able to prevent the cardial fibrosis and arterial remodeling. The target- development of target-organ damage. Furthermore, organ damage is investigated in several animal the aim is to asses, whether their effect can be addi- models, including L-NAME-induced hypertension tive and what mechanisms might contribute to this or experimental autoimmune myocarditis. expected protection. Special attention is devoted The main project of this group will investigate the to the regulation of collagen turn-over and immune effects of the AT2 receptor agonist, compound 21, modulating effects of the AT2 receptor agonist. and the pineal hormone, melatonin, on target-organ

Zusammenfassung

Die Arbeitsgruppe ist eine Nachwuchswissen- Hypertonie durch eine Inhibierung der NO-Synthase schaftlergruppe innerhalb der AG Steckelings, mit in der Ratte induziert wird. Dieses Projekt wird durch der sie eng zusammenarbeitet. Das Hauptinteresse das EU 7th FP Projekt „COME-in-CARE“ gefördert. der Gruppe richtet sich auf die Prävention und das Ziel dieses Projektes ist es, zu beobachten, ob und Modulieren von pathologischen Veränderungen im welchem Ausmaß die genannten Substanzen im Herzkreislaufsystem wie arterieller Hypertonie, die Entstehung der Endorganschäden verhindern Herzfibrose und arteriellem Remodeling. Die Organ- können. Darüber hinaus soll festgestellt werden, ob schäden werden an verschiedenen Tiermodellen die Effekte dieser zwei Substanzen additiv sind und untersucht wie L-NAME induzierte Hypertonie,oder welche zellulären Mechanismen den Wirkungen der experimentelle Autoimmunmyokarditis. Substanzen zugrunde liegen. Besondere Aufmerk- Im Hauptprojekt der Gruppe werden die Effekte des samkeit wird der Regulation des Kollagenmetabolis- AT2 Rezeptor Agonisten Compound 21 und des mus und den immunmodulierenden Eigenschaften Pinealhormons Melatonin auf Endorganschaden bei des AT2 Agonisten gewidmet. experimenteller Hypertonie untersucht, wobei die

232 Ludovit Paulis – Pharmacology

Research project

COmpound 21 and MElatonin in CArdiovascular REmodeling (COME IN CARE)

Project leader Ludovit Paulis, Thomas Unger, Ulrike M. Steckelings Coworkers Sophie Becker Funding EU: 7. FP-PIEF-237834 External cooperations Dr. Johannes Baulmann, Medizinische Klinik und Poliklinik I, Universitätsklinikum Würzburg, Würzburg, Prof. Fedor Simko, Faculty of Medicine, Comenius University, Bratislava

The most effective current therapeutic strategies for including cardiac hypertrophy, fibrosis, renal damage the treatment of hypertension including the use of and arterial remodelling. These alterations are followed ACE inhibitors, AT1 receptor blockers (and partially by echocardiographic, hemodynamic and biochemi- also β-blockers and renin inhibitors) are aimed to cal means. The aim is to observe, whether and to what reduce the deleterious stimulation of AT1 receptors. extent the investigated substances are able to prevent However, only the recent discovery of the non-peptide the development of target-organ damage. Further- AT2 receptor agonist, compound 21, unleashed the more, the aim is to asses, whether their effect can be possibility of the investigation of the role of the AT2 additive and what mechanisms might contribute to this receptor and whether its stimulation could counteract expected protection. the undesired effects by the AT1 receptor. Melatonin The preliminary results suggest that while mela- is a pineal hormone with antioxidant, blood pressure tonin shows antifibrotic effects in the left ventricle, lowering and cardioprotective effects, but the mecha- compound 21 prevents arterial remodelling and the nisms of these protective actions are not completely increase in arterial stiffness, which is an independ- elucidated. ent cardiovascular risk factor. Further experiments are In this project, adult male Wistar rats are made performed to tackle the anti-stiffening mechanisms of hypertensive and NO deficient by administration of compound 21 with special attention to the regulation an NO-synthase inhibitor, L-NAME. The subsequent of collagen turn-over and immune modulating effects hypertension is associated with target-organ damage of the AT2 receptor agonist.

233 Pharmacology

Thomas Unger’s Group Schupp M, Lee LD, Frost N, Umbreen S, Schmidt B, Unger Publications 2006-2010 T, Kintscher U (2006): Regulation of peroxisome proliferator- activated receptor gamma activity by losartan metabolites. Sandmann S, Li J, Fritzenkoetter C, Spormann J, Tiede K, Hypertension 47: 586-589 Fischer JW, Unger T (2006): Differential effects of olmesartan and ramipril on inflammatory response after myocardial infarc- Thoene-Reineke C, Kalk P, Dorn M, Klaus S, Simon K, Pfab T, tion in rats. Blood Press 15: 116-128 Godes M, Persson P, Unger T, Hocher B (2006): High-protein nutrition during pregnancy and lactation programs blood Schefe JH, Lehmann KE, Buschmann IR, Unger T, Funke- pressure, food efficiency, and body weight of the offspring in Kaiser H (2006): Quantitative real-time RT-PCR data analy- a sex-dependent manner. Am J Physiol Regul Integr Comp sis: current concepts and the novel “gene expression’s C (T) Physiol 291: R1025-1030 difference” formula. J Mol Med 84: 901-910 Rutschow S, Li J, Schultheiss HP, Pauschinger M (2006): Schefe JH, Menk M, Reinemund J, Effertz K, Hobbs RM, Myocardial proteases and matrix remodeling in inflammatory Pandolfi PP, Ruiz P, Unger T, Funke-Kaiser H. (2006): A heart disease. Cardiovasc Res 69: 646-656 Novel Signal Transduction Cascade Involving Direct Physical Interaction of the Renin/Prorenin Receptor With the Transcrip- Clemenz M, Kintscher U, Unger T (2006): The metabolic tion Factor Promyelocytic Zinc Finger Protein. syndrome: cluster with a self-fulfilling loop? J Hypertens 24: Circ Res 99: 1355-1366 257-258

Kappert K, Caglayan E, Huntgeburth M, Bäumer AT, Sparwel Xia QG, Reinecke A, Dorenkamp M, Daemen MJ, Simon R, J, Uebel M, Rosenkranz S (2006): 17-beta estradiol attenu- Unger T (2006): Effects of endothelin ET(A) receptor blocker ates PDGF signaling in vascular smooth muscle cells at the LU 135252 on cardiac remodeling and survival in a hyperten- post-receptor level. Am J Physiol Heart Circ Physiol 290: sive rat model of chronic heart failure. Acta Pharmacol Sin H538-46 27: 1417-1422

Kappert K*, Leppänen O*, Paulsson J, Furuhashi M, Carls- Hoheisel U, Unger T, Mense S (2007): Sensitization of rat son MA, Heldin CH, Fätkenheuer G, Rosenkranz S, Östman dorsal horn neurons by NGF-induced subthreshold potentials A (2006): Highly Active Antiretroviral Therapy Attenuates Re- and low-frequency activation. A study employing intracellular Endothelialization and Alters Neointima Formation in the Rat recordings in vivo. Brain Res 1169: 34-43 Carotid Artery After Balloon Injury. J Acquir Immune Defic Syndr 43: 383-921 (*: contributed equally) Kappert K, Paulsson J, Sparwel J, Leppänen O, Hellberg C, Östman A, Micke P (2007): Dynamic changes in the expres- Kappert K, Sparwel J, Sandin Å, Seiler A, Siebolts U, Lep- sion of DEP-1 and other PDGF receptor-antagonizing PTPs pänen O, Rosenkranz S, Östman A (2006): during onset and termination of neointima formation Antioxidants relieve phosphatase inhibition and reduce PDGF FASEB J 21: 523-34 signaling in cultured VSMCs and in restenosis Arterioscler Thromb Vasc Biol 26: 2644-51 Weibrecht I, Böhmer SA, Dagnell M, Kappert K, Östman A, Böhmer FD (2007): Oxidation sensitivity of the catalytic Schupp M, Kintscher U, Fielitz J, Thomas J, Pregla R, Hetzer cysteine of the protein-tyrosine phosphatases SHP-1 and R, Unger T, Regitz-Zagrosek V (2006): Cardiac PPARalpha SHP-2.Free Radic Biol Med 43: 100-10 expression in patients with dilated cardiomyopathy. Eur J Heart Fail 8: 290-294 Micke P*, Kappert K*, Ohshima M, Sundquist C, Scheidl S, Lindahl P, Heldin CH, Ponten F, Östman A (2007): In situ Pechanova O, Matuskova J, Capikova D, Jendekova L, Paulis identification of genes regulated specifically in fibroblasts of L, Simko F (2006): Effect of spironolactone and captopril on human basal cell carcinoma. J Invest Dermatol 27: 1516- nitric oxide and S-nitrosothiol formation in kidney of L-NAME- 231 (*: contributed equally) treated rats. Kidney Int 70: 170–176

234 Pharmacology

Pechanova O, Zicha J, Paulis L, Zenebe W, Dobesova Z, Unger T, Stoppelhaar M (2007): Rationale for double renin- Kojsova S, Jendekova L, Sladkova M, Dovinova I, Simko F, angiotensin-aldosterone system blockade. Am J Cardiol Kunes J (2007): The effect of N-acetylcysteine and melatonin 100: 25J-31J in adult spontaneously hypertensive rats with established hypertension. Eur J Pharmacol 561: 129-136 Westermann D, Rutschow S, Jaeger S, Linderer A, Anker S, Riad A, Unger T, Schultheiss HP, Pauschinger M, Tschoepe Paulis L, Liskova S, Pinterova M, Dobesova Z, Kunes J, Zicha C (2007): Contributions of inflammation and cardiac matrix J (2007): Nifedipine-sensitive noradrenergic vasoconstriction metalloproteinase activity to cardiac failure in diabetic cardio- is enhanced in spontaneously hypertensive rats: the influ- myopathy: the role of angiotensin type 1 receptor antagonism. ence of chronic captopril treatment. Acta Physiol (Oxf) 191: Diabetes 56: 641-646 255-266 Li J, Leschka S, Rutschow S, Schwimmbeck PL, Husmann L, Paulis L, Važan R, Simko F, Pechánˇová O, Styk J, Babál P, Noutsias M, Westermann D, Poller W, Zeichhardt H, Klingel K, Janega P (2007): Morphological Alterations and NO-Synthase Tschöpe C, Schultheiss HP, Pauschinger M (2007): Immu- Expression in the Heart after Continuous Light Exposure of nomodulation by interleukin-4 suppresses matrix metallopro- Rats.Physiol Res 56: S71-S76 teinases and improves cardiac function in murine myocarditis. Eur J Pharmacol 554: 60-68 Simko F, Matuskova J, Luptak I, Pincikova T, Krajcirovicova K, Stvrtina S, Pomsar J, Pelouch V, Paulis L, Pechanova O Bakris G, Boehm M, Dagenais G, Diener HC, Fujita T, Gorelick (2007): Spironolactone Differently Influences Remodeling of P, Kjeldsen SE, Laakso M, Mancia G, Pitt B, Sharma A, Sleight the Left Ventricle and Aorta in L-NAME-Induced Hypertension. P, Teo K, Unger T, Weber M, Williams B, Zannad F (2008): Car- Physiol Res 56: S25-S32 diovascular protection for all individuals at high risk: evidence- based best practice. Clin Res Cardiol 97: 713-725 Simko F, Potacova A, Pelouch V, Paulis L, Matuskova J, Krajcirovicova K, Pechanova O, Adamcova M (2007): Spon- Kappert K, Meyborg H, Clemenz M, Graf K, Fleck E, taneous, L-Arginine-Induced and Spironolactone-Induced Kintscher U, Stawowy P (2008): Insulin facilitates monocyte Regression of Protein Remodeling of the Left Ventricle in migration: a possible link to tissue inflammation in insulin- L-NAME-Induced Hypertension. Physiol Res 56: S55-S62 resistance. Biochem Biophys Res Commun 365: 503-8

Vazan R, Janega P, Hojna S, Zicha J, Simko F, Pechanova O, Bäumer AT, Ten Freyhaus H, Sauer H, Wartenberg M, Styk J, Paulis L (2007): The Effect of Continuous Light Expo- Kappert K, Schnabel P, Konkol C, Hescheler J, Vantler M, sure of Rats on Cardiac Response to Ischemia-Reperfusion Rosenkranz S (2008): Pi3 kinase-dependent membrane and NO-Synthase Activity. Physiol Res 56: S63-S69 recruitment of rac-1 and p47phox is critical for alpha PDGF receptor-induced production of reactive oxygen species. J Scholze J, Grimm E, Herrmann D, Unger T, Kintscher U Biol Chem 283: 7864-76 (2007): Optimal treatment of obesity-related hypertension: the Hypertension-Obesity-Sibutramine (HOS) study. Circulation Tabet F, Schiffrin EL, Callera G, Yao G, Östman A, Kappert 115: 1991-1998 K, Tonks NK, Touyz RM (2008): Redox-sensitive Signaling by Angiotensin II Involves Oxidative Inactivation and Blunted Phos- Slowinski T, Kalk, P, Christian, M, Schmager, F, Relle, K., phorylation of Protein Tyrosine Phosphatase SHP-2 in Vascular Godes M, Funke-Kaiser H, Neumayer H-H, Bauer C, Smooth Muscle Cells from SHR. Circ Res 103: 149-158 Theuring F, Hocher B (2007): Cell-type specific interaction of endothelin- and nitric oxide system - pattern of prepro- Clemenz M, Frost N, Schupp M, Caron S, Foryst-Ludwig A, ET-1 expression in kidneys of L-NAME treated prepro ET-1 Boehm C, Hartge M, Gust R, Staels B, Unger T, Kintscher U promoter-lacZ-transgenic mice. J Physiol 581: 1173-1181 (2008): Liver-specific peroxisome proliferator-activated receptor alpha target gene regulation by the angiotensin type 1 receptor blocker telmisartan. Diabetes 57: 1405-1413

235 Pharmacology

Foryst-Ludwig A, Clemenz M, Hohmann S, Hartge M, Sprang Van Linthout S, Spillmann F, Riad A, Trimpert C, Lievens J, C, Frost N, Krikov M, Bhanot S, Barros R, Morani A, Gustafs- Meloni M, Escher F, Filenberg E, Demir O, Li J, Shakibaei M, son JA, Unger T, Kintscher U (2008): Metabolic actions of Schimke I, Staudt A, Felix SB, Schultheiss HP, De Geest B, estrogen receptor beta (ERbeta) are mediated by a negative Tschöpe C (2008): Human apolipoprotein A-I gene transfer cross-talk with PPARgamma. PLoS Genet 4: 1-16 reduces the development of experimental diabetic cardiomyopathy. Circulation 117: 1563-1573 Kaschina E, Schrader F, Sommerfeld M, Kemnitz UR, Grze- siak A, Krikov M, Unger T (2008): Telmisartan prevents aneu- Schmerbach K, Schefe JH, Krikov M, Muller S, Villringer A, rysm progression in the rat by inhibiting proteolysis, apoptosis Kintscher U, Unger T, Thoene-Reineke C (2008): Comparison and inflammation. J Hypertens 26: 2361-2373 between single and combined treatment with candesartan and pioglitazone following transient focal ischemia in rat brain. Kaschina E, Grzesiak A, Li J, Foryst-Ludwig A, Timm M, Brain Res 1208: 225-233 Rompe F, Sommerfeld M, Kemnitz UR, Curato C, Namsolleck P, Tschoepe C, Hallberg A, Alterman M, Hucko T, Paetsch I, Schefe JH, Neumann C, Goebel M, Danser J, Kirsch S, Gust Dietrich T, Schnackenburg B, Graf K, Dahloef B, Kintscher U, R, Kintscher U, Unger T, Funke-Kaiser H (2008): Prorenin Unger T, c (2008): Angiotensin II type 2 receptor stimulation: engages the (pro)renin receptor like renin and both ligand a novel option of therapeutic interference with the renin- activities are unopposed by aliskiren. J Hypertens 26: 1787- angiotensin system in myocardial infarction? Circulation 118: 1794 2523-2532 Paulis L, Zicha J, Kunes J, Hojna S, Behuliak M, Celec P, Kintscher U, Hartge M, Hess K, Foryst-Ludwig A, Clemenz Kojsova S, Pechanova O, Simko F (2008): Regression of M, Wabitsch M, Fischer-Posovszky P, Barth TF, Dragun D, L-NAME-induced hypertension: The role of NO and endothe- Skurk T, Hauner H, Blueher M, Unger T, Wolf AM, Knippschild lium-derived constricting factor. Hypertens Res 31: 793-804 U, Hombach V, Marx N (2008): T-lymphocyte infiltration in visceral adipose tissue: a primary event in adipose tissue Paulis L, Matuskova J, Adamcova M, Pelouch V, Simko J, inflammation and the development of obesity-mediated insulin Krajcirovicova K, Potacova A, Hulin I, Janega P, Pechanova O, resistance. Arterioscler Thromb Vasc Biol 28: 1304-1310 Simko F (2008): Regression of left ventricular hypertrophy and aortic remodelling in NO-deficient hypertensive rats: effect of Krikov M, Thoene-Reineke C, Muller S, Villringer A, Unger T l-arginine and spironolactone. Acta Physiol (Oxf) 194: 45-55 (2008): Candesartan but not ramipril pretreatment improves outcome after stroke and stimulates neurotrophin BNDF/TrkB Telmisartan Randomised AssessmeNt Study in ACE iNtoler- system in rats. J Hypertens 26: 544-552 ant subjects with cardiovascular Disease (TRANSCEND) Investigators (2008): Effects of the angiotensin-receptor Lueders S, Schrader J, Berger J, Unger T, Zidek W, Boehm blocker telmisartan on cardiovascular events in high-risk M, Middeke M, Motz W, Luebcke C, Gansz A, Brokamp L, patients intolerant to angiotensin-converting enzyme inhibi- Schmieder RE, Trenkwalder P, Haller H, Dominiak P (2008): tors: a randomised controlled trial. Lancet 372: 1174-1183 The PHARAO study: prevention of hypertension with the angiotensin-converting enzyme inhibitor ramipril in patients Thoene-Reineke C, Neumann C, Namsolleck P, Schmerbach with high-normal blood pressure: a prospective, randomized, K, Krikov M, Schefe JH, Lucht K, Hoertnagl H, Godes M, controlled prevention trial of the German Hypertension Muller S, Rumschuessel K, Funke-Kaiser H, Villringer A, League. J Hypertens 26: 1487-1496 Steckelings UM, Unger T (2008): The beta-lactam antibiotic, ceftriaxone, dramatically improves survival, increases gluta- The ONTARGET Investigators (2008): Telmisartan, ramipril, mate uptake and induces neurotrophins in stroke. J Hyper or both in patients at high risk for vascular events. N Engl J tens 26: 2426-2435 Med 358: 1547-1559

236 Pharmacology

Westermann D, Mersmann J, Melchior A, Freudenberger T, Paulis L, Pechanova O, Zicha J, Krajcirovicova K, Barta A, Petrik C, Schaefer L, Lullmann-Rauch R, Lettau O, Jacoby C, Pelouch V, Adamcova M, Simko F (2009): Melatonin prevents Schrader J, Brand-Herrmann SM, Young MF, Schultheiss HP, fibrosis but not hypertrophy development in the left ventri- Levkau B, Baba HA, Unger T, Zacharowski K, Tschoepe C, cle of NG-nitro-L-arginine-methyl ester hypertensive rats. J Fischer JW (2008): Biglycan is required for adaptive remod- Hypertens 27: S11-S16 eling after myocardial infarction. Circulation 117: 1269-1276 Simko F, Pechanova O, Pelouch V, Krajcirovicova K, Mullerova Altarche-Xifro W, Curato C, Kaschina E, Grzesiak A, Slavic M, Bednarova K, Adamcova M, Paulis L (2009):Effect of S, Dong J, Kappert K, Steckelings UM, Imboden H, melatonin, captopril, spironolactone and simvastatin on blood Unger T, Li J (2009): Cardiac c-kit+AT2+ Cell Population is pressure and left ventricular remodelling in spontaneously Increased in Response to Ischemic Injury and Supports Car- hypertensive rats. J Hypertens 27: S5-S10 diomyocyte Performance. Stem Cells 27: 2488-2497 Vrankova S, Jendekova L, Paulis L, Sladkova M, Simko F, Brinckmann M, Kaschina E, Altarche-Xifro W, Curato C, Pechanova O, (2009): Comparison of the effects of inda- Timm M, Grzesiak A, Dong J, Kappert K, Kintscher U, Unger pamide and captopril on the development of spontaneous T, Li J (2009): Estrogen receptor alpha supports cardiomyo- hypertension. J Hypertens 27: S42-S46 cytes indirectly through post-infarct cardiac c-kit+ cells. J Mol Cell Cardiol 47: 66-75 Benova M, Stebelova K, Paulis L, Simko F, Zeman M (2009): Effect of L-NAME-induced hypertension on melatonin recep- Goebel M, Staels B, Unger T, Kintscher U, Gust R (2009): tors and melatonin levels in the pineal gland and the periph- Characterization of new PPARgamma agonists: benzimida- eral organs of rats. Hypertens Res 32: 242-247 zole derivatives - the importance of position 2. ChemMed Chem 4: 1136-42 Kappert K, Tsuprykov O, Kaufmann J, Fritzsche J, Ott I, Goebel M, Bahr IN, Hassle PL, Gust R, Fleck E, Unger T, Sparwel J, Vantler M, Caglayan E, Kappert K, Fries JW, Dietrich Stawowy P, Kintscher U (2009): Chronic Treatment With H, Böhm M, Erdmann E, Rosenkranz S (2009):Differential effects Losartan Results in Sufficient Serum Levels of the Metabolite of red and white wines on inhibition of the PDGF receptor: EXP3179 for PPAR{gamma} Activation. Hypertension 54: Impact of the mash fermentation. Cardiovasc Res 81: 758-70 738-743

Kappert K, Meyborg H, Baumann B, Furundzija V, Kaufmann Kaschina E, Scholz H, Steckelings UM, Sommerfeld M, J, Graf K, Stibenz D, Fleck E, Stawowy P (2009): Integrin Kemnitz UR, Artuc M, Schmidt S, Unger T (2009): Transition cleavage facilitates cell surface-associated proteolysis from atherosclerosis to aortic aneurysm in humans coincides required for vascular smooth muscle cell invasion. Int J Bio with an increased expression of RAS components. Athero chem Cell Biol 41: 1511-7 sclerosis 205: 396-403

Ohshima M, Yamaguchi Y, Kappert K, Micke P, Otsuka K (2009): Micke P, Hackbusch D, Mercan S, Stawowy P, Tsuprykov O, bFGF rescues imatinib/STI571-induced apoptosis of sis-NIH3T3 Unger T, Ostman A, Kappert K (2009): Regulation of tyrosine fibroblasts. Biochem Biophys Res Commun 381: 165-70 phosphatases in the adventitia during vascular remodelling. Biochem Biophys Res Commun 382: 678-684 Goebel M, Clemenz M, Staels B, Unger T, Kintscher U, Gust R (2009): Characterization of new PPARgamma agonists: Reinemund J, Seidel K, Steckelings UM, Zaade D, Klare S, analysis of telmisartan’s structural components. ChemMed Rompe F, Katerbaum M, Schacherl J, Li Y, Menk M, Schefe Chem 4: 445-456 JH, Goldin-Lang P, Szabo C, Olah G, Unger T, Funke-Kaiser H (2009): Poly(ADP-ribose) polymerase-1 (PARP-1) transcrip- Bal MS, Paulis L, Zicha J, Kunes J (2009): Effect of protein tionally regulates angiotensin AT2 receptor (AT2R) and AT2R kinase C and protein kinase A inhibitors on contraction of binding protein (ATBP) genes. Biochem Pharmacol 77: isolated femoral arteries of SHR and Wistar rats. Physiol Res 1795-1805 58: 793-798

237 Pharmacology

Vosgerau U, Lauer D, Unger T, Kaschina E (2010): Cleaved Furundzija V, Fritzsche J, Kaufmann J, Meyborg H, Fleck E, High Molecular Weight Kininogen, a Novel Factor in the Regu- Kappert K, Stawowy P (2010): IGF-1 increases macrophage lation of Matrix Metalloproteinases in Vascular Smooth Muscle motility via PKC/p38-dependent alphavbeta3-integrin inside- Cells. Biochem Pharmacol 79: 172-179 out signaling. Biochem Biophys Res Commun 394(3):786-9

Kappert K, Furundzija V, Fritzsche J, Margeta C, Krüger J, Kintscher U, Marx N, Martus P, Stoppelhaar M, Schimkus Meyborg H, Fleck E, Stawowy P (2010): Integrin cleavage J, Schneider A, Walcher D, Kümmel A, Winkler R, Kappert regulates bidirectional signaling in vascular smooth muscle K, Dörffel Y, Scholze J, Unger T (2010): Effect of high-dose cells. Thromb Haemost 103(3):556-63 valsartan on inflammatory and lipid parameters in patients with Type 2 diabetes and hypertension. Diabetes Res Clin Rompe F, Artuc M, Hallberg A, Alterman M, Ströder K, Thöne- Pract 89(3):209-215 Reineke K, Reichenbach A, Schacherl J, Dahlöf B, Bader M, Alenina N, Schwaninger M, Zuberbier T, Funke-Kaiser H, Zimmermann M, Kappert K, Stan ACU373-MG cells express Schmidt C, Schunck W-H, Unger T, Steckelings UM (2010): PepT2 and accumulate the fluorescently tagged dipeptide- Direct angiotensin AT2-receptor stimulation acts antiinflamma- derivative β-Ala-Lys-N(ε)-AMCA. Neurosci Lett [in press] tory through epoxyeicosatrienoic acid and inhibition of NF-κB. Hypertension 55: 924-931 Ohshima M, Yamaguchi Y, Matsumoto N, Micke P, Tak- enouchi Y, Nishida T, Kato M, Komiyama K, Abiko Y, Ito K, Paulis L, Pechanova O, Zicha J, Liskova S, Celec P, Mullerova Otsuka K, Kappert K (2010): TGF-β Signaling in Gingival M, Kollar J, Behuliak M, Kunes J, Adamcova M, Simko F Fibroblast-Epithelial Interaction. J Dent Res [in press] (2010. Melatonin improves the restoration of EDCF-signalling and inner diameter in the rat femoral artery after cessation of Caglayan E, Romeo G, Kappert K, Odenthal M, Südkamp M, L-NAME treatment. J Hypertens 28: S19-S24 Body S, Sherman S, Hackbusch D, Vantler M, Kazlauskas A, Rosenkranz (2010): Profilin-1 is expressed in human athero- Menk M, von Haefen C, Funke-Kaiser H, Sifringer M, Schefe sclerotic plaques and induces atherogenic effects on vascular JH, Kirsch S, Seidel K, Reinemund J, Steckelings UM, smooth muscle cells. PloS ONE [in press] Unger T, Spies CD (2010): Ethanol-induced downregulation of the angiotensin AT2 receptor in murine fibroblasts is mediated Curato C, Slavic S, Dong J, Skorska A, Altarche-Xifró W, by PARP-1. Alcohol 44: 495-506 Miteva K, Kaschina E, Thiel A, Imboden H, Wang J, Steck- elings UM, Steinhoff G, Unger T, Li J (2010): Identification of Seidel K, Kirsch S, Lucht K, Zaade D, Reinemund J, Schmitz non-cytotoxic and IL-10-producing CD8+AT2R+ T cell popula- J, Klare S, Li Y, Schefe JH, Schmerbach K, Goldin-Lang P, tion in response to ischemic heart injury. J Immunol 185(10) Zollmann FS, Thöne-Reineke C, Unger T, Funke-Kaiser H [Epub ahead of print] (2010): The promyelocytic leukemia zinc finger (PLZF) protein exerts neuroprotective effects in neuronal cells and is dys- Koon K T, Sleight P, Dagenais G, Probstfield J, Anderson C, regulated in experimental stroke. Brain Pathol (in press) Gao P, Diaz R, Dans A, Levine M, Unger T, Fagard R, Yusuf S, for the ONTARGET (Ongoing Telmisartan Alone and in Simko F, Pechanova O, Pelouch V, Krajcirovicova K, Celec Combination with Ramipril Global Endpoint Trial) and TRAN- P, Palffy R, Bednarova K, Vrankova S, Adamcova M, Paulis L SCEND (Telmisartan Randomized Assessment Study in ACE (2010): Continuous light and L-NAME-induced left ventricular Intolerant Subjects with Cardiovascular Disease) Trials (2010): remodelling: different protection with melatonin and captopril. Effect of Ramipril, Telmisartan or Combination Therapy on the J Hypertens 28: S13-S18 Risk of Cancer in 31,000 Individuals. under review

Paulis L, Pechanova O, Zicha J, Barta A, Gardlik R, Celec P, Reviews & book chapters (2006-2010) Kunes J, Simko F (2010): Melatonin interactions with blood pressure and vascular function during L-NAME-induced Hartge MM, Kintscher U, Unger T (2006): Endothelial dys- hypertension. J Pineal Res 48:102-108 function and its role in diabetic vascular disease. Endocrinol Metab Clin North Am 35: 551-560

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Steckelings UM, Funke-Kaiser H, Unger T (2006): Mtus1. Kappert K (2007): Folsäure als Sekundärprophylaxe bei AfCS-Nature Molecule Pages (doi:101038/mpa00394801) kardiovaskulären Erkrankungen. Der Kardiologe 1: 120–122

Kappert K (2006) Weitere atherosklerotische Risikofak- Li J, Doerffel Y, Hocher B, Unger T (2007): Inflammation in toren In: Prävention atherosklerotischer Erkrankungen. the genesis of hypertension and its complications - the role of Rosenkranz, Schneider, Erdmann (Editors) Georg Thieme angiotensin II. Nephrol Dial Transpl 22: 3107-3109 Verlag Östman A, Kappert K (2007) Protein Tyrosine Phosphatases Kappert K, Fätkenheuer G, Rosenkranz S (2006): Beein- In: Endothelial Biomedicine William Aird (Editor) Cambridge flussung des Atherosklerose-Risikos durch medikamentöse University Press Therapien In: Prävention atherosklerotischer Erkrankun gen Rosenkranz, Schneider, Erdmann (Editors) Georg Micke P, Moustakas A, Ohshima M, Kappert K (2007): Thieme Verlag Transforming Growth Factor-b in Cancer Therapy: Cancer associated fibroblast and the role of TGFβ In: Transforming Peters H, Unger T (2006): Mast cells and the power of local Growth Factor-ß in Cancer Therapy; Volume 2: Cancer RAS activation. Nephrol Dial Transplant 22: 40-42 Treatment and Therapy Sonia B. Jakowlew (Editor) The Humana Press Clemenz M, Steckelings UM, Unger T (2006): Regulations- mechanismen des Renin-Angiotensin-Systems im kardio- Kaschina E, Doerfel N, Unger T (2007): Hypertonie In: vaskulären System. In: Ganten, Detlev; Köhrle, Josef und Schwandt P, Parhofer KG, eds. Handbuch der Fettst Ruckpaul, Klaus (Hrsg.) Molekularmedizinische Grundla offwechselstörungen Stuttgart: Schattauer 538-550 gen von para- und autokrinen Regulationsstörungen. Springer-Verlag Berlin Heidelberg 377-407 Funke-Kaiser H, Schefe JH, Unger T (2007): Signaltrans- duktion des Renin-/ Prorenin-Rezeptors. MedReport 31: 8 Funke Kaiser H, Steckelings UM, Unger T (2006): Stimula- tion of AT2 receptors: role in the effect of angiotensin II recep- Steckelings UM, Unger T (2007): Angiotensin in the kidney: tor antagonists. In: Mancia G, ed. Angiotensin II Receptor a key to understanding hypertension? Cell Metab 5: 7-8 Antagonists: Informa Healthcare; 31-46 Schacherl J, Menard J, Unger T (2007): Towards an under- Thoene-Reineke C, Steckelings UM, Unger T (2006): Angi- standing of the renin-angiotensin-aldosterone system: an otensin receptor blockers and cerebral protection in stroke. J historical review In: Sever P, ed. New Perspectives on Hypertens Suppl 24: S115-121 Hypertension. Direct Renin Inhibition. Birmingham, UK: Sherborne Gibbs Limited 8-19 Unger T (2006): [Innovation or pseudo-innovation: that is the question Med Klin (Munich) 101: 257-262 Unger T (2008): Comparing the cerebroprotective properties of antihypertensive drugs in terms of their effects on angi- Unger T (2006): Laudatio. Zur Verleihung des Robert Koch otensin. Nat Clin Pract Nephrol 4: 12-13 Award 2006 an MSD für das Medikament Alendronat (FOSA- MAX/ FOSAVANCE) Med Klin (Munich) 101: 25-26 Kappert K, Unger T, Kintscher U (2008): [Aliskiren hemifu- marate] Dtsch Med Wochenschr 133: 1308-1312 Unger T (2006): Stellungnahme. Antwort des Autors auf den Leserbrief von M.H. Freitag und W.A. Wohlgemuth Med Klin Kappert K (2008): Pharmakologie der antihypertensiven (Munich) 101: 590-593 Therapie bei metabolischem Syndrom In: Antihypertensiva bei metabolischem Syndrom und Typ 2 Diabetes Ulrich Unger T (2006): Stellungnahme. Antwort des Autors auf den Kintscher, Nikolaus Marx (Editors) UNI-MED Verlag AG Leserbrief von Herrn Becker-Brüser Med Klin (Munich) 101: 512-515 Schefe JH, Unger T, Funke-Kaiser H (2008): PLZF and the (pro)renin receptor. J Mol Med 86: 623-627

239 Pharmacology

Steckelings UM, Unger T (2008): The renin-angiotensin- Steckelings UM (2009): Direct stimulation of Angiotensin aldosterone system. In: Mancia G, Grassi G, Kjeldsen S, eds (AT)2-receptors: a therapeutic option of the future? Diabetes, Manual of Hypertension of the European Society of Stoffwechsel und Herz 17: 518-519 Hypertension London, U.K.: informa healthcare 110-116 Unger T, Dahlöf B (2009): Compound 21, the first orally active, Unger T (2008): Gegenwart und Zukunft der Hypertoniefor- selective agonist of the angiotensin II type 2 (AT2) receptor: schung. In: Lenz T, eds. Hypertonie in Klinik und Praxis. implications for AT2 receptor research and therapeutic poten- Stuttgart, New York: Schattauer Verlag; 461-467 tial. J Renin Angio Aldo Syst [Epub ahead of print]

Kintscher U, Foryst-Ludwig A, Unger T (2008): Inhibiting Angi- Funke Kaiser H, Reinemund J, Steckelings UM, Unger T otensin Type 1 Receptors as a Target for Diabetes. Expert (2009): Adapter proteins and promoter regulation of the angi- Opinion on Therapeutic Targets 12: 1257-1263 otensin AT2 receptor – implications for cardiac pathophysiology. J Renin Angio Aldo Syst 11: 7-17 Werner C, Baumhaekel M, Teo KK, Schmieder R, Mann J, Unger T, Yusuf S, Boehm M (2008): RAS blockade with ARB Steckelings UM, Rompe F, Kaschina E, Namsolleck P, and ACE inhibitors: current perspective on rationale and Grzesiak A, Funke-Kaiser H, Bader M, Unger T (2009): patient selection. Clin Res Cardiol 97: 418-431 The past, present and future of angiotensin II type 2 receptor stimulation. J Renin Angio Aldo Syst 11: 67-73 Kappert K, Kusserow H, Unger T (2008): The pharmacological rationale behind polypharmacy in heart failure. Heart Unger T (2009): Antihypertensives as over-the-counter drugs? Failure Monitor 6(1):20-7 Dtsch Med Wochenschr 134: 2302-2304

Kolloch R, Unger T (2009): Aliskiren Stuttgart: Ligatur Verlag Häßle P, Fätkenheuer G, Rosenkranz S, Kappert K (2009): für Klinik und Praxis Direkte und indirekte atherogene Effekte der HIV-Infektion und antiretroviralen Therapie. Der Kardiologe 3: 24-34 Mancia G, Unger T, Zanchetti A (2009): Introduction: Reduc- ing cardiovascular risk: ONTARGET-a new standard in cardio- Unger T (2009): The rationale for choosing telmisartan and vascular protection. J Hypertens Suppl 27(Suppl 5): S1 ramipril in the ONTARGET programme. Eur Heart J Suppl 11(Suppl F):F3-F8 Unger T, Kintscher U, Kappert K, Steckelings UM (2009):The ONTARGET Trial Programme: Facts and Lessons. Funke-Kaiser H, Zollmann FS, Schefe JH, Unger T (2010): Current Hypertension Reviews 5: 202-209 Signal transduction of the (pro)renin receptor as a novel therapeutic target for preventing end-organ damage. Hyperten Steckelings UM, Unger T (2009): Angiotensin receptors and sion Research 33: 98-104 autophagy: live and let die. Hypertension 53: 898-899 Kaschina E and Unger T (2010). Pathophysiologic link Steckelings UM, Rompe F, Kaschina E, Unger T (2009): between atherosclerosis and nephrosclerosis. In: Cardiore The evolving story of RAAS in hypertension, diabetes and nal Syndrome (Berbari AE, Mancia G ed.), 396p, Springer cardiovascular disease- moving from macrovascular to micro- Italia, Milan 245-253 vascular targets. Fund Clin Pharmacol 23: 693-703 Steckelings UM, Th.Unger (2010): Kurzbewertung Aliskiren. Kaschina E, Steckelings UM, Unger T (2009): RAS activa- Internistische Praxis 50: 371-374 tion contributes to transition from atherosclerosis to aneurysm. International Atherosclerosis Society, IAS website, Ströder K, Unger Th, Steckelings UM (2010) The renin-angi- Commentary., 4 December. otensin-system and the eye. Frontiers in Diabetes: Diabetic retinopathy - an update; Eds.: M.Porta, H.-P. Hammes, Karger, Basel, Vol.20: 142-157

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Rompe F, Unger T, Steckelings UM (2010): The AT2-recep- Widdop RE, Steckelings UM, McCarthy CA, Callaway JC tor in inflammation. Drug News Perspectives 23: 104-111 (2010): Angiotensin receptors in neuroprotection. In: ‘Neu- ropeptides in Neuroprotection and Neuroregeneration’. F Steckelings UM, Widdop RE, Paulis L, Unger Th (2010): The Nyberg (Editor); Taylor & Francis (publisher); in press Angiotensin AT2-Receptor in left ventricular hypertrophy. J Hypertens 28 (suppl 1): S50–S55 Patents (2006-2009)

Kjeldsen SE, Schmieder RE, Unger T, Mancia G (2010): Com- DETERMINATION OF RENIN/ PRORENIN RECEPTOR bination therapy with telmisartan and hydrochlorothiazide: a ACTIVITY (EP 1890152A International Application No.: PCT/ strategy for improved blood pressure control. Curr Med Res EP2007/006100, Publication Date: 20.02.2008, International Opin (in press) Filing Date: 14.08.2006) Funke-Kaiser H, Zollmann F, Schefe JH, Unger T

Thomas Unger’s Group Third party funding (2006-2010)

Project leader Project title Sponsor Period Unger T. TP7: Der AT2-Rezeptor bei Myokard- DFG GK-754-III: Geschlechts- 2006-2010 Funke-Kaiser H. Hypertrophie. spezifische Mechanismen bei Steckelings UM. Myokard-hypertrophie Funke-Kaiser H. Effects of renin inhibitors on the renin/ Actelion Pharmaceuticals 2007-2008 Unger T. prorenin receptor-PLZF signal transduction cascade. Thöne-Reineke C. Neuroprotective and neuroregenerative Novartis 2008-2009 Funke-Kaiser H. effects of aliskiren in double transgenic Unger T. rats expressing human renin and angiotensinogen genes. Funke-Kaiser H. Identifizierung und molekulare, epi- DFG-Einzelantrag 2006-2007 genetische und klinische Charakter- (FU 463/2-1) isierung eines CpG-Mikrosatelliten- bindenden Transkriptionsfaktors. Funke-Kaiser H. JRP B1 (Genetics and genomics of EU: Network of Excellence 2006-2010 Unger T. hypertensive patients with early stroke) “InGenious HyperCare” - P15 (no. 037093) Funke-Kaiser H. Der Renin-/ Prorenin-Rezeptor als Investitionsbank Berlin (IBB) 2008-2010 Schefe J.H. neue pharmakologische Zielstruktur. - ProFIT-Programm Zollmann F. (Antragsnummer Unger T. 10138510)

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Project leader Project title Sponsor Period Funke-Kaiser H. Entwicklung von RERBs, einer neuen BMBF -GO-Bio-Programm 2008-2010 Medikamentenklasse zur Endorgan- (GO-Bio 0315092) protektion. Funke-Kaiser H. Der Renin/ Prorenin-Rezeptor als Stiftung Charité 2008-2010 Schefe J. pharmakologische Zielstruktur. Zollmann F. Unger T. Funke-Kaiser H Molekulare und klinische Charak- Dr. Werner Jackstädt-Stiftung 2009-2010 Unger T. terisierung eines Transkriptionsfaktor bindenden CpG-Promotor-Mikrosatel- liten bei M. Alzheimer. Li J. Unger T. Angiotensin receptors in apoptosis and EU: CardioVasc-3.2 (contract 2006-2009 inflammation after myocardial infarc- MEST-CT-2005-020268) tion Li J. Unger T. Angiotensin AT2 receptor in cardiac BMBF: BCRT-kick off grant 2007-2008 regeneration Li J. Functional relevance of AT2+c-kit+ BMBF: 1st. BCRT grant 2008-2010 Unger T. cells in cardiac ischemic injury Bader M. Li J. Unger T. Einfluss von Östrogen auf die Umge- DFG: GK-754-III 2006-2010 staltung der myocardialen Matrix nach Myokardinfarkt Li J. Einfluß von Östrogenrezeptor auf die FERMENTATION – BIOTEC GmbH 2008-2010 Unger T. Plastizität und Regenerationsfähigkeit hämatopoetischer Stammzellen Unger T. Aliskiren im Schlaganfall in DTGR Novartis Pharma GmbH 2006-2010 Thöne-Reineke C. Unger T. Schlaganfall Intervention Solvay Pharmaceuticals GmbH 2006-2009 Thöne-Reineke C. u.Präventation Unger T. Regulation of tissue-specific endothe- Nippon Boehringer Ingelheim Co., 2006-2009 Kintscher U. lial dysfunction by telmisartan: The role Ldt. of PPARg activation Unger T. Stammzellen von Patienten mit Herzin- Praxis Dr. Pesic 2008-2011 Li J. suffizienz Kaschina E. Genetics of aneurysm National Genome Research Net- 2004-2008 Unger T. work (NGFN -2), BMBF Kaschina E. Rimonabant in heart failure Sanofi-Aventis 2006-2009 Unger T.

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Project leader Project title Sponsor Period Kaschina E. AT2 receptor agonist Compound 21 in EU: Marie Curie Early Stage 2006-2009 Unger T. myocardial infarction Research Training Program CARDI- OVASC MEST-CT-2005-020268 Kaschina E. Telmisartan in aneurysm Bayer Schering Pharma 2006-2008 Unger T. Kaschina E. Kininogen in heart failure Bayer Schering Pharma 2010 Unger T. Unger T COME-in-CARE (COmpound 21 EU: 7. FP-PIEF-237834 8/2009- Paulis L. and MElatonin in CArdiovascular 1/2011 REmodeling) Unger T. Gender specific mechanisms in myo- DFG GK 754-III 2006-2010 Funke-Kaiser H. cardial hypertrophy Steckelings UM. Steckelings UM. Bedeutung des Renin-Angiotensin- Deutsche Stiftung Sklerodermie 2006-2007 Systems bei Pathogenese und Thera- pie der Sklerodermie Steckelings UM. Candesartan in diabetic retinopathy Takeda Pharma 2006-2009 Thöne-Reineke C. Unger T. Steckelings UM. Preclinical development of an orally Vicore Pharma 2008-2010 Unger T. active AT2-receptor agonist Valero V. Evaluation of neuroprotective and anti- Berlin-Padua-Gdansk Graduate 2009-2011 Steckelings UM. inflammatory effects of direct AT2 Program/DIB receptor stimulation in multiple sclerosis Steckelings UM. Preclinical development of an orally Eurostars, HEARTSAVE, EU 2010-2011 Unger T. active AT2-receptor agonist Kappert K. Return Scholarship German Research Foundation 2006-2007 (no. KA1820/2-1) Kappert K. Role of protein tyrosine phosphatases European Commission 2007-2009 in metabolic and cardiovascular disease Kintscher U. PPARγ activation by Losartan in Hyper- Merck Sharp & Dohme (MSD) 2007-2008 Kappert K. tensive Patients: The Importance of Losartan-Metabolites Kappert K. Oxidation of the catalytic site cysteine Charité-University Medicine Berlin 2007-2007 of protein tyrosine phosphatases in Research Scholarship cardiac ischemia/reperfusion

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Project leader Project title Sponsor Period Kappert K. Transfer of the hemodynamic 3-vessel Charité-University Medicine Berlin 2007-2008 occlusion model into the genetically Habilitation Scholarship modified mouse – Role of the protein tyrosine phosphatase SHP-1 in cerebral arteriogenesis Kappert K. Protein Tyrosine Phosphatases in German Research Foundation 2008-2010 Arteriogenesis (no. KA1820/4-1) Kappert K. SHP-1 in cerebral arteriogenesis in Deutsche Stiftung für Herzforsc- 2008-2010 mice hung (F/04/08) Kappert K. Targeting protein tyrosine phos- Charité-University Medicine Berlin 2008-2010 phatases in high fat diet induced Personal funding insulin resistance Kappert K. Protein Tyrosine Phosphatase DEP-1 German Diabetes Society 2010-2011 as molecular target in insulin resistance Krüger J. Protein Tyrosine Phosphatases (PTPs) Charité-University Medicine Berlin 2010-2011 as therapeutic targets treating high fat PhD scholarship diet induced insulin resistance

Awards (2006-2010)

2006 Charité Research Scholarship K. Kappert

2006 Travel Award, ESH Investigator’s Initiative Group F. Rompe

2006 Finalist, Young Investigator Award, Gordon Research Conference on Angiotensin F. Rompe

2006 Travel Grant, European Society of Hypertension, Madrid L. Paulis

2006 Education Award of Volksbank AG L. Paulis

2006 Jiri Widimsky Award for promising young scientists in hypertension research on the 16th European Society for Hypertension Meeting, Madrid L. Paulis

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2006 1st Prize winner in the 1st PhD Student Scientific Conference, School of Medicine, Bratislava L. Paulis

2007 Prize winner of the GO-Bio-competition of the BMBF H. Funke-Kaiser

2007 Young Investigator Travel Award, European Society of Cardiology K. Kappert

2007 Young Investigator Award, German Hypertension Society K. Kappert

2007 Travel Award, European Society of Hypertension F. Rompe

2007 Participant of the 57th Meeting of Nobel Laureates, Lindau, Germany J. Schefe

2007 Charité Habilitation Scholarship K. Kappert

2007 Travel Award, European Council for Cardiovascular Research P. Namsolleck

2007 1st Prize winner in the Physiology Section and 5th in the Zondek Price competition of the 18th European Student Conference, Berlin L. Paulis

2007 Travel Grant, European Society of Hypertension, Milan L. Paulis

2007 Poster award: 12th Annual Meeting of the European Council for Cardiovascular Research (ECCR) E. Kaschina

2008 Travel Grant, European Society of Hypertension, Berlin L. Paulis

2008 Young Investigator Travel Award, European Society of Cardiology K. Kappert

2008 Austin Doyle Award of the International Society of Hypertension (ISH) A. Grzesiak

2008 Poster Prize, European Council for Cardiovascular Research F. Rompe

2008 Research Scholarship, Deutsche German Hypertension Society J. Schefe

245 Pharmacology

2009 Charité-Promotionspreis J. Schefe

2009 Berlin-Brandenburg School for Regenerative Therapies (BSRT) PhD Student Award Wassim Altarche-Xifró

2009 Travel Award, European Society of Hypertension P. Namsolleck

2009 Young Investigator Award, Deutsche Hypertonie Liga S. Wardat

2009 Travel Award, European Society of Hypertension F. Santi

2009 Travel Award, European Council for Cardiovascular Research F. Rompe

2009 Honorary Member of the British Hypertension Society T. Unger

2009 1st Prize winner in competition for the best publication in experimental hypertension by Blood Pressure, NGO, Prague L. Paulis

2009 Acknowledgement for Scientist of the Year 2008, Min. Edu. SR, Bratislava, L. Paulis

2010 Honorary Member of the Italian Hypertension Society T. Unger

2010 Subsession Poster Prize, Frontiers in Cardiovascular Biology, European Society of Cardiology J. Krüger

2010 Young Investigator Travel Award, European Society of Cardiology D. Hackbusch

2010 Accommodation Grant, European Society of Hypertension, Oslo L. Paulis

2010 Travel Award, Heart Failure of the ESC, Berlin L. Paulis

2010 Travel Grant, European Society for Cardiology, Stockholm L. Paulis

2010 1st prize winner in the Young Investigator Award in Life Sciences of the Slovak Academy of Sciences, Bratislava L. Paulis

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2010 Travelling Award for the 20th European Meeting of Hypertension, Oslo S. Slavic

2010 Scholarship for the Promotion from Charité Universitätsmedizin S. Slavic

Genbank entries

Schefe, J.H., Neumann, C., Funke-Kaiser, H., Thone-Reineke, C., Unger, T. (2006): Rattus norvegicus glutamate transporter 1 gene, promoter region and partial cds. Genbank accesion DQ489741, GI:94958152

247

PONTUS B. PERSSON – VEGETATIVE PHYSIOLOGY

Head of the Group

Prof. Dr. Pontus B. Persson

Curriculum Vitae: Pontus B. Persson, born in 1962 in Lund Sweden received his education in the USA, Germany and Italy. He graduated from the Ruprecht-Karls- University in Heidelberg, Germany in 1987 and subsequently received his training in physiology at the same University from 1987-1993. In 1991 he finished his Habilitation (an extensive PhD-like thesis). After his appointment of full professor of Physiology 1994 at the Charité-Universitätsmedizin Berlin, Germany he became Director of the Institute for Vegetative Physiology. Pontus B. Persson was awarded with the Ruprecht-Karls-Prize of the University Heidelberg (1990), the Research Prize of the German Research Foundation (Deutsche Forschungsgemeinschaft: Gerhard-Hess-Förderpro- gramm) (1991), the Young Investigator Award of the American Physiological Society (1998) and the Henry Pickering Bowditch Award of the American Physiological Society (2002). Pontus B. Persson was Editor-in-Chief of the American Journal of Physiology – Regulatory, Integrative and Comparative Physiology from 2001-2007 and is now Consulting Editor at the same Journal. He is Review Editor for the Journal Acta Physiologica. For Pflügers Archiv- European Journal of Physiology he works as Executive Editor. Prof. Persson’s research interests include kidney hemodynamics, the renin-angiotensin system and the reflex control of circulation.

249 PONTUS B. PERSSON – VEGETATIVE PHYSIOLOGY

Summary

The current focus of our department is kidney func- The various levels at which research takes place is tion and its importance for cardiovascular control. mirrored by the broad spectrum of Journals where The individual research groups act in a concerted the results of this department have been published effort to disentangle the complex relationship over the latest decade. between systemic hemodynamics and the kidney. For more information regarding research activities Moreover, light is shed on to developmental aspects. your are welcome to visit the individual research These efforts are supported by several federal group sites. grants, mainly by the German Research Foundation.

Zusammenfassung

Der derzeitige Forschungsschwerpunkt der Herz- sächlich durch die Deutsche Forschungsgemein- Kreislauf Physiologie liegt auf der Nierenfunktion schaft. Die Forschung findet auf unterschiedlichen und deren Bedeutung für die Kontrolle des Kreis- Ebenen statt. Während der letzten 10 Jahre wurden laufs. Die einzelnen Forschungsgruppen arbeiten die daraus resultierenden Ergebnisse in einem brei- eng zusammen, um die komplexen Beziehungen ten Spektrum von Journalen veröffentlicht. zwischen dem Blutkreislauf und der Niere zu unter- Weitere Informationen zu den Forschungstätigkeiten suchen. Des weiteren werden Aspekte der Entwick- können Sie auf den Seiten der Forschungsgruppen lungsphysiologie erforscht. Diese Versuche werden nachlesen. durch verschiedene Institutionen gefördert, haupt-

250 PONTUS B. PERSSON – VEGETATIVE PHYSIOLOGY

Publications 2006 – 2010 Bräutigam M, Persson PB (2006): Do iodinated contrast media interfere with renal tubular creatinine secretion? Lai EY, Patzak A, Steege A, Mrowka R, Brown R, Spielmann Radiol. 240: 615 N, Persson PB, Fredholm BB, Persson AEG (2006): Contribu- tion of adenosine receptors in the control of arteriolar tone Persson PB, Liss P, Hansell P (2007): Evaluation and compar- and adenosine-angiotensin II interaction. Kidney Intern. 70: ison between visipaque (Iodixanol) and Hexabrix (Ioxaglate) in 690-698 coronary angiography. J Am Coll Cardiol 49: 1668-1671

Stauss HM, Persson PB (2006): Cardiovascular variability and Persson PB, Liss P, Hansell P, Lagerqvist B (2007): Response the autonomic nervous system. J of Hyperten. 24: 1902- to “Iodixanol vs ioxaglate for preventing contrast nephropathy: 1905 Who is the winner?” Kidney Intern. 71: 828-829

Fähling M, Mrowka R, Steege A, Nebrich G, Perlewitz A, Mrowka R, Steege A, Kaps C, Herzel HP, Thiele BJ, Persson Persson PB, Thiele BJ (2006): Translational control of colla- PB, Blüthgen N (2007): Dissecting the action of an evolution- gen prolyl 4-hydroxylase-α (I) gene expression under hypoxia. ary conserved non-coding region on rennin promoter activity. J Biol Chem. 281: 26089-26101 Nucleic Acids Res. 35 (15): 5120-5129

Thone-Reineke C, Kalk P, Dorn M, Klaus S, Simon K, Pfab Gericke A, Martinka P, Nazarenko I, Persson PB, Patzak A

T, Godes M, Persson PB, Unger T, Hocher B (2006): High- (2007): Impact of α1 –adrenoreceptor expression on con- protein nutrition during pregnancy and lactation programs tractile properties of vascular smooth muscle cells. Am J blood pressure, food efficiency and body weight of the Physiol. 293: R1215-R1221 offspring in a gender dependent manner. Am J Physiol. 291: R1025-R1030 Seeliger E, Flemming B, Wronski T, Ladwig M, Arakelyan K, Godes M, Möckel M, Persson PB (2007): Viscosity of contrast Liss P, Persson PB, Hansell P, Lagerqvist B (2006): Renal media perturbs renal hemodynamics. J Am Soc Nephrol. failure in 57 925 patients undergoing coronary procedures 18: 2912-2920 using iso-osmolar or low-osmolar CM. Kidney Intern. 70 (10): 1811-1817 Patzak A, Lai EY, Fähling M, Sendeski M, Martinka P, Persson PB, Persson AEG (2007): Adenosine enhances long-term the Lai EY, Martinka P, Fähling M, Mrowka R, Steege A, Gericke contractile response to angiotensin II in afferent arterioles. Am A, Sendeski M, Persson PB, Persson AEG, Patzak A (2006): J Physiol. 293: R2232-2242 Adenosine restores angiotensin II-induced contractions by receptor-independent enhancement of calcium sensitivity in Patzak A, Steege A, Lai EY, Brinkmann JO, Kupsch E, Spiel- renal arterioles. Circ Res. 99 (10): 1117-1124 mann N, Gericke A, Skalweit A, Stegbauer J, Persson PB, Seeliger E (2008): Angiotensin II response in afferent arteri- Persson PB (2006): Guest editor appreciation. Am. J. oles of mice lacking either the endothelial or neuronal isoform Physiol. 290: R493 of nitric oxide synthase. Am J Physiol. 294:R429-R437

Persson PB (2006): From clinical insights to new therapies. Martinka P, Lai EY, Fähling M, Jankowski V, Jankowski J, Am. J. Physiol. 290: R124-R125 Schubert R, Gaestel M, Persson AEG, Persson PB, Pat- zak A (2008): Adenosine increases calcium sensitivity via Persson PB (2006): Where we stand: American Journal of receptor-independent activation of the P38/MK2 pathway in Physiology – Regulatory, Integrative and Comparative Physiol- mesenteric arteries. Acta Physiologica 193:37-46 ogy 2006. Am J Physiol. 291: R489-R490 Steege A, Fähling M, Paliege A, Bondke A, Kirschner KM, Persson PB (2007): A last look: American Journal of Phys- Martinka P, Kaps C, Patzak A, Persson PB, Thiele BJ, Scholz iology-Regulatory, Integrative and Comparative Physiology H, Mrowka R (2008): Wilms’ tumor protein (-KTS) modulates 2001-2007. Am J Physiol. 292: R665 3 renin gene transcription. Kidney Intern. 74: 458-466

251 PONTUS B. PERSSON – VEGETATIVE PHYSIOLOGY

Fähling M, Mrowka R, Steege A, Kirschner KM, Benko E, Perlewitz A, Nafz B, Skalweit A, Fähling M, Persson PB, Thiele Förstera B, Persson PB, Thiele BJ, Meier JC, Scholz H BJ (2010): Aldosterone and vasopressin affect α- and γ-ENaC (2009): Translational Regulation of the Human Achaete-scute mRNA translation. Nucleic Acids Res. 38: 5747-5760 Homologue-1 by Fragile X Mental Retardation Protein. J Biol Chem. 284 (7): 4255-4266 Seeliger E, Becker K, Ladwig M, Wronski T, Persson PB, Flemming B (2010): Up to 50-fold increase in urine viscos- Lüdemann L, Nafz B, Elsner F, Große-Siestrup C, Meissler ity with iso-osmolar contrast media in the rat. Radiol. 256: M, Kaufels N, Rehbein H, Persson PB, Michaely HJ, Lengs- 406-414 feld P, Voth M, Gutberlet M (2009): Absolute quantification of regional renal blood flow in swine by dynamic contrast- Schildroth J, Rettig-Zimmermann J, Kalk P, Steege A, Fähling enhanced magnetic resonance imaging using a blood pool M, Sendeski M, Paliege A, Lai EY, Bachmann S, Persson PB, contrast agent. Invest Radiol. 44(3):125-134 Hocher B, Patzak A (2010): Endothelin type A and B receptors in the control of afferent and efferent arterioles in mice. Sendeski M, Patzak A, Pallone TL, Cao C, Persson AEG, Nephrol Dial Transplant. E-pub ahead of print Sept 2nd, Persson PB (2009): Iodixanol, constriction of medullary 2010 descending vasa recta, and risk for contrast medium-induced nephropathy. Radiol. 251(3):697-704 Reviews & book chapters (2006-2010)

Seeliger E, Wronski T, Ladwig M, Dobrowolski L, Vogel Persson PB (2006): Pathophysiology of contrast-induced T, Godes M, Persson PB, Flemming B (2009): The renin- nephropathy. In: Contrast-Induced Nephropathy edited by A. angiotensin system and the third mechanism of renal blood L. Bartorelli and G. Marenzi. London: Taylor & Francis, p. 3-17 flow autoregulation. Am. J. Physiol. Renal Physiol. 296:F1334-F1345 Persson PB (2006): Temperature control: from molecular insights, regulation in king penguins and diving seals, to stud- Lai EY, Fähling M, Ma Z, Källskog Ö, Persson PB, Patzak A, ies in humans. Am. J. Physiol. 291: R512-R514 Persson AEG, Hultström M (2009): Norepinephrine increases calcium sensitivity of mouse afferent arteriole, thereby Persson PB (2007): The Magic Mountain or Death in Venice: enhancing angiotensin II-mediated vasoconstriction. Kidney Chronic Hypoxia may alleviate Oxidative Stress in the Kidney. Intern. 76:953-959 J Physiol. 582.1: p1

Sendeski M, Patzak A, Person PB (2010): Constriction of the vasa recta, the vessels supplying the area at risk for acute kidney injury, by four different iodinated contrast media, evaluating ionic, nonionic, monomeric and dimeric agents. Invest. Radiol. 45: 453-457

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Head of the group

Prof. Dr. med. Holger Scholz

Curriculum Vitae: Holger Scholz studied medicine at the University of Heidelberg. He received his medical degree in 1987 based on experimental studies on the electro- physiology of the renin secreting juxtaglomerular cells in the kidney. After a one-year practice at the Department of Pathology (University of Heidelberg) he worked from 1988 to 1991 as a post-doctoral fellow in the Institute of Physiology at the University of Zurich, Switzerland, on the oxygen-regulated synthesis of erythropoietin, the major humoral growth factor for red blood cell precursors. From 1991-1999 he was in the Institute of Physiology at the University of Regensburg where he received the Habilitation degree in 1995 for his work on the blood pressure-dependent control of renin secretion. Supported by fellowships from the Max-Kade-Foundation and the German Research Foundation (DFG), he joined the Developmental Biology Group at the Department of Nephrology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, USA (1995-1998). In 1998 Holger Scholz became a Professor in Physiology at the Medical Faculty Charité. The research projects of his group are at the interface of cancer and development. It is a major goal to elucidate the regulation and function of the Wilms’ tumor protein, Wt1, which is necessary for normal embryogenesis and additionally functions as a tumor suppressor molecule. Other scientific interests allude to the role of neurotrophins and their receptors in the cardiovascular system and the oxygen-dependent control of gene expression.

Members of the group Scientists Bondke Persson, Anja Dr. med. Kirschner, Karin Dr. rer. nat.

Technicians Grätsch, Inge Ing. (FH) Richter, Angelika Ing. (FH)

Students Braun, Julian MD student Hagen, Patricia MD student Jacobi, Charlotte PhD student Karadeniz, Serap Bachelor student (2009) Schiebel, Susann PhD student (2007-2008) From left to right, front row: H. Scholz, K. Kirschner, Sciesielski, Lina PhD student (2006-2009) L. Sciesielski, A. Richter, I. Grätsch Scientist (January to July 2010) Back row: C. Jacobi, S. Karadeniz, J. Braun, Trams, Mareike Diploma student (2008) A. Bondke Persson

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Summary get genes that are regulated by Wt1, we aim at analyzing important transcriptional pathways in Our research team is interested in the molecular cardiovascular development. Since tissue repair mechanisms that control the development of the in the heart and other organs frequently involves cardiovascular system. The focus of our work is reactivation of fetal genes, it is our expectation to on the regulation and function of the Wilms’ tumor discover novel candidate mechanisms for future gene, WT1. Another area of interest refers to the role regenerative therapies. of hypoxia as a physiological signal during devel- Besides acting as an oxidant, molecular oxygen is opment and a determining factor in many disease also important for the control of gene expression. processes. In this field we currently focus on the A variety of oxygen-regulated genes were identi- oxygen-regulated expression of the gene encoding fied since the initial discovery of the hematopoietic the neurotrophin receptor TrkB. growth factor Erythropoietin (Epo). A group of het- Normal organ formation is established by the tem- erodimeric molecules known as hypoxia-inducible porally and spatially coordinated gene expression, transcription factors (HIFs) play a key role in oxygen- which is controlled by a variety of transcription fac- dependent gene expression. Tissue hypoxia result- tors. Therefore, the identification and characteriza- ing from an imbalance between oxygen supply and tion of transcriptional regulators with essential func- oxygen demand is a frequent condition in rapidly tions during embryogenesis may allow one to deci- growing tumors. In many malignancies the hypoxic pher the key signaling pathways in cardiovascular microenvironment stimulates the formation of new development. The Wilms’ tumor transcription factor blood vessels (angiogenesis) thereby promoting WT1 was initially identified as a tumor suppres- tumor expansion. Recent studies indicate that local sor due to loss-of-function mutations in malignant tissue hypoxia can alter gene expression irrespective pediatric kidney tumors known as Wilms’ tumors of its proangiogenic effect in certain neuronal tumors or nephroblastomas. Targeted inactivation of Wt1 (neuroblastomas) towards an immature, i.e. malig- in mice caused intrauterine lethality of homozygous nant paradigm. By identification of the gene encod- embryos (Wt1-/-) with a failure of normal formation ing the neurotrophin receptor TrkB as a functional of the genitourinary system, mesothelium, sensory HIF target gene, we discovered a novel molecular neuropithelia, myocardial blood vessels, and the switch for the malignant transformation of neuro hematopoietic system. By the identification of tar- blastoma cells.

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Zusammenfassung Verständnis von Regenerationsprozessen und deren therapeutische Beeinflussung relevant. Das wissenschaftliche Interesse unserer Arbeits- Sauerstoff ist nicht nur als Oxidans wirksam, sondern gruppe gilt den molekularen Steuerungsprozessen spielt auch eine wichtige Rolle bei der Genexpres- bei der Entwicklung des kardiovaskulären Systems. sionskontrolle. Neben dem zunächst identifizierten Der Schwerpunkt der Untersuchungen liegt bei der hämatopoietischen Wachstumsfaktor Erythropoietin Regulation und Funktion des Wilmstumor Transkrip- ist inzwischen eine Vielzahl weiterer Gene bekannt, tionsfaktors WT1. Ein weiteres Arbeitsgebiet bein- deren Transkription durch Sauerstoffmangel (Hypo- haltet die Rolle von Sauerstoffmangel (Hypoxie) als xie) stimuliert wird. Hypoxieinduzierbare Transkrip- physiologisches Signal in der Entwicklung und als tionsfaktoren (HIFs) gelten als zentrale Mediatoren Determinante von Krankheitsprozessen. Gegenwär- einer bei Sauerstoffdefizit veränderten Genexpres- tig konzentrieren wir uns auf die sauerstoffabhängige sion. In Tumoren bewirkt ein Mißverhältnis von Regulation des Neurotrophinrezeptors TrkB. Sauerstoffangebot zu zellulärem Sauerstoffbedarf Die normale Organentwicklung erfordert eine durch eine lokale Gewebehypoxie, wodurch die Bildung Transkriptionsfaktoren in ihrer zeitlichen und räum- von Blutgefäßen (Angiogenese) stimuliert und das lichen Abfolge koordinierte Genexpression. Durch Tumorwachstum gefördert wird. Neueren Untersu- Identifizierung transkriptionaler Regulatoren, die für chungen zufolge kann Sauerstoffmangel, unabhän- eine normale Organogenese notwendig sind, kön- gig von einer proangiogenen Wirkung, die Genex- nen deshalb zentrale Signalwege in der Embryo- pression in neuronalen Tumoren (Neuroblastomen) nalentwicklung analysiert werden. Der Wilmstumor in Richtung auf ein entdifferenziertes, d.h. malignes Transkriptionsfaktor WT1 wurde ursprünglich auf- Muster verändern. Mit der Charakterisierung des grund von Mutationen in malignen Nierentumoren Neurotrophinrezeptors TrkB als ein HIF-reguliertes bei Kindern (Wilmstumoren, Nephroblastome) als Zielgen haben wir einen neuen molekularen Schalter ein Tumorsuppressor charakterisiert. Die gezielte für die maligne Transformation von Neuroblastom- Inaktivierung von Wt1 hatte bei Mäusen u. a. eine zellen entdeckt. Störung der Entwicklung von Nieren, Mesothel, Sinnesepithelien, Herzmuskelgefäßen und blutbil- dendem System zur Folge. Durch Ermittlung von Zielgenen, deren Expression durch den WT1 Tran- skriptionsfaktor kontrolliert wird, versucht unsere Arbeitsgruppe, wichtige molekulare Regulations- mechanismen bei der kardiovaskulären Entwick- lung aufzudecken. Die Untersuchungen erfolgen vor dem Hintergrund, daß Organschädigung häufig zur Reaktivierung eines fötalen Genexpressionspro- gramms führt. Detailkenntnisse über die molekularen Entwicklungsmechanismen sind deshalb für das Multiple roles of the WT1 zinc finger protein in development

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Research projects

1. The role of WT1 in blood vessel formation in the heart

Project leader Karin Kirschner Coworkers Anja Bondke Persson, Inge Grätsch, Angelika Richter Funding - SCHO 634/4-1 External cooperations Drs. Kay-Dietrich and Nicole Wagner, INSERM U907, University of Nice, France, Prof. Dr. Andreas Schedl, INSERM U636, Centre de Biochimie, University of Nice/Sophia-Antipolis, Nice, France

Recent studies demonstrate that blood vessel for- the injured tissue through promoting mesenchymal mation in the developing heart takes its origin in the transition of resident vascular cells. epicardium, a mesothelial tissue covering the outer The aim of this project is to identify Wt1-regulated heart surface. During cardiogenesis epicardial cells genes with a role in blood vessel formation in the heart. acquire a mesenchymal phenotype, loose their inter- In this regard we identified the gene encoding the TrkB cellular contacts and populate the subjacent myocar- neurotrophin receptor, Ntrk2, as a transcriptional Wt1 dium. Here, the epicardium-derived cells differenti- target. A Wt1 binding motif was identified in the TrkB ate to vascular endothelial and smooth muscle cells, promoter that was required for tissue-specific expres- amongst other cell types. Hence, normal formation sion of a reporter transgene in the developing epicar- of the epicardium is a critical precondition for blood dium of mice. TrkB-deficient embryos, like Wt1-/- fetal vessel development in the heart, and important infor- mice, had fewer blood vessels in their hearts. Addi- mation about myocardial vasculogenesis can be tional findings indicate that activation of the TrkB neu- expected from gene expression analysis in epicardial rotrophin receptor by its natural ligand, brain-derived cells. We reported for the first time that the Wilms’ neurotrophic factor (BDNF) prevents the newly formed tumor transcription factor Wt1, which is normally vascular cells in the heart from apoptosis. Another expressed in the epicardium, is necessary for normal downstream target of Wt1 with a presumed role in vascularization of the heart. Mouse embryos with Wt1 vascular development is vascular endothelial (VE) deficiency (Wt1-/-) exhibited a severely reduced blood cadherin. It is suggested that enhanced expression of vessel density in their hearts compared to wild-type VE-cadherin in response to Wt1 supports intercellular littermates. Remarkably, left coronary artery ligation contact formation. Overall our results qualify Wt1 as in rats resulted in de novo expression of Wt1 in blood an important transcriptional regulator for the appear- vessels adjacent to the infarcted tissue. It is therefore ance of the blood vessel system in the heart. Future conceivable that re-expression of Wt1 in response to studies shall address the role of Wt1 in tissue repair myocardial ischemia supports neovascularization of after myocardial damage.

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2. Gene transcription in the epicardium

Project leader Karin Kirschner Coworkers Inge Grätsch, Angelika Richter Funding - SCHO 634/4-1 External cooperation Drs. Kay-Dietrich and Nicole Wagner, INSERM U907, University of Nice, France

The epicardium is a mesothelial tissue on the outer sur- in the developing heart. In an effort to analyze transcrip- face of the heart. It arises during embryogenesis from tional signaling pathways in cardiovascular progenitor the proepicardial tissue which is formed by a cluster of cell formation we identified the gene encoding alpha-4 cells just dorsal to the developing heart tube. Integrity of integrin as a novel downstream target of Wt1. Alpha-4 the epicardium is a critical precondition for normal heart integrin is a heterodimeric cell adhesion molecule development. Thus, epicardial defects are frequently which is expressed on epidardial and hematopoietic associated with hypoplasia of the myocardium sug- progenitor cells. It binds to the extracellular matrix pro- gesting that normal heart formation requires the transfer teins fibronectin and vascular cell adhesion molecule-1 of growth promoting signals from the epicardium to the (VCAM-1) that are present on cardiomyocytes. Lack of cardiomyocytes. Recent findings indicate that epicar- alpha-4 integrin in mice resulted in a failure of proepi- dial cells contribute to the cardiomyocyte lineage and cardial cells to attach to the surface of the heart and, also provide the source for different vascular cell types consequently, myocardial growth defects. Other Wt1 in the embryonic heart. During cardiogenesis epicardial candidate targets are currently investigated by analy- cells undergo a process of epithelial-to-mesenchymal sis of differentially expressed genes in the hearts of transition (EMT), which allows them to become cardio- wild-type (Wt1+/+) vs. Wt1-deficient (Wt1-/-) mouse vascular progenitor cells. Epicardium-derived mesen- embryos. Hopefully, analysis of Wt1-regulated genes chymal cells migrate towards the myocardium, where may advance our understanding of the mechanisms of they form vascular endothelial and smooth muscle cells myocardial tissue repair. in addition to perivascular fibroblasts and cardiomyocytes. Important insights into the molecular mechanisms of heart development can be obtained from studies on the regulation and function of epicardial genes. The Wilms’ tumor transcription factor Wt1 is among the molecules that are necessary for normal development of the epicardium. Mice with homozygous Wt1 deletion (Wt1-/-) lack an intact epicardium Co-localization of Wt1 (red) and alpha-4 integrin (green) in the devel- and exhibit severe myocardial hypoplasia. oping epicardium of a mouse embryo by double immunofluorescence Wt1 is necessary for EMT of epicardial cells labeling. Nuclei were counterstained with Dapi.

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3. Identification of WT1 target genes in the developing kidney and other tissues

Project leader Anja Bondke Persson Coworkers Karin Kirschner, Julian Braun, Charlotte Jacobi, Inge Grätsch, Angelika Richter External cooperation Dr. Kai Schmidt-Ott, MDC Berlin, Prof. Dr. Christof Englert, Fritz-Lipmann-Institut, Jena

Loss-of-function mutations in the WT1 gene are regulatory sequences of these genes are currently responsible for approximately 10% of pediatric renal cloned and tested for Wt1 responsiveness in reporter tumors (Wilms’ tumors, nephroblastomas). Wilms’ assays. The respective cis-elements are mapped, and tumors can arise when pluripotent progenitor cells binding of Wt1 protein is assessed by electrophoretic in the developing kidney rather continue to prolifer- mobility shift assay and chromatin immunoprecipita- ate than differentiating to glomeruli and tubules. The tion (ChIP). Additional experiments include expression WT1 gene product is a zinc finger protein, whose analysis of candidate target genes by immunohisto- alternative splice variants predominantly function as chemistry and/or in situ mRNA hybridization of normal transcription factors. The most striking characteristic and Wt1-deficient murine embryos. Functional studies of mice with homozygous Wt1 deletion (Wt1-/-) con- will be performed on explant cultures of embryonic sists in a failure of normal formation of the kidneys and mouse kidneys. Eventually this project will allow for the gonads. Mammalian kidney development depends on discovery of novel candidate genes in kidney formation the reciprocal interaction of the metanephric mesen- and renal tissue repair. chyme and the invading ureteric bud. Epithelial differentiation and subsequent nephron formation does not occur in Wt1-deficient metanephric mesenchyme, which undergoes programmed cell death (apoptosis) instead. It is the goal of this project to discover molecular Wt1 downstream target genes in the developing kidney and other tissues. For this purpose we made use of the M15 cell line, which is derived from the murine mesonephros and expresses Wt1 at a robust level. Differentially expressed genes were identified by DNA microarray analysis of M15 cells with normal and low Wt1 expression. Wt1 levels in M15 cells were adjusted by transfection with gene-specific and non- targeting siRNAs, respectively. More than one dozen differentially expressed genes, encoding cell adhesion Organ culture of a mouse embryonic kidney explant. Devel- oping glomeruli were stained with anit-WT1 antibody (red). molecules, metalloproteinases, interleukins and other Labeling of cyterkeratin was used for visualization of the molecules, could be verified by real time RT-PCR. The branching ureteric bud (green).

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4. Nuclear import pathways for WT1

Project leader Holger Scholz Coworkers Anja Bondke Persson, Karin Kirschner, Inge Grätsch, Angelika Richter External cooperation Dr. Reinhard Depping, Institut für Physiologie, Universität Lübeck

More than two dozens protein variants which are cellular GFP is easy to detect by its emission of a green generated by alternative mRNA splicing, the use of fluorescence upon excitation at approximately 490 nm additional transcription start sites, RNA editing and wavelength. The basic construct was engineered such post-translational modification, contribute to the mul- that GFP is retained outside the nuclei in the cytoplasm tiple functions of the WT1 protein. A further regulatory of the transfected cells. Nuclear localization of GFP paradigm is provided by the recently discovered abil- molecules fused to different amino acid sequences ity of WT1 to shuttle between the nucleus and cyto- of the WT1 protein therefore suggests inclusion of plasm. Consistent with its established function as a potential NLS. Using this approach we could map a transcription factor WT1 is contained predominantly in sequence of 10 amino acids in the zinc finger domain the cell nuclei of embryonic tissues. However, a vari- of WT1 that was necessary for nuclear import. Co- ety of tumors express WT1 mainly in the cytoplasm immunoprecipitation experiments revealed that WT1 suggesting that cytoplasmic WT1 has a role in tum- protein interacts with importin alpha-1 and importin origenesis. Prompted by this possibility we under- beta in embryonic kidney cells. We intend to gener- took efforts to analyze the nuclear import pathways ate mutant WT1 proteins, which lack functional NLS for Wt1 protein. Bidirectional transport between the and are therefore expressed exclusively in the cell cell nucleus and cytoplasm occurs through nuclear cytoplasm. These proteins will be used to address pore complexes and involves a group of transport the question whether cytoplasmic WT1 can promote proteins known as karyopherins. Translocation of pro- malignant growth characteristics. teins into the nucleus is mediated by importins which bind to nuclear localization sequences (NLS) in their cytoplasmic car- gos. NLS frequently consist of clusters of basic amino acids that are sometimes separated by a spacer of approximately 10 amino acids (bipartite NLS). In order to pinpoint relevant NLS Intracellular localization of green fluorescent protein (GFP) GFP in human embryonic in the Wt1 protein we made use kidney cells. Transfection of empty expression vector causes GFP expression exclusively in the cytoplasm sparing the nuclei (A). Inclusion of a nuclear localization signal of plasmids expressing a green (NLS) from the zinc finger domain of the WT1 protein directs GFP expression into the fluorescent protein (GFP). Intra- cell nuclei (B).

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5. Regulation of the genes encoding Eryrthopoietin (Epo) and its receptor (EpoR) during hematopoiesis

Project leader Karin Kirschner Coworkers Patricia Hagen, Inge Grätsch, Angelika Richter Funding - DFG DA 484/2-1 External cooperation Prof. Dr. Christof Dame, Klinik für Pädiatrie m. S. Molekulare Neonatologie, Charité, Berlin, Dr. Matthias Ballmeier, Klinik für Pädiatrische Hämatologie und Onkologie, Medizinische Hochschule Hannover

The Wilms’ tumor transcription factor WT1 is expressed immunoprecipitation (ChIP) confirmed the interaction in CD34-positive hematopoietic progenitor cells, and of Wt1 protein with the Epo and EpoR promoters in high WT1 levels are detected in acute leukemias with their normal chromosomal configuration. Cellular co- unfavorable outcome. Previous studies showed that expression of Wt1 and Epo/EpoR was detected by murine hematopoietic progenitor cells with Wt1 defi- immunohistochemistry in fetal mouse liver, which is ciency (Wt1-/-) have a reduced colony forming capac- the major site of prenatal hematopoiesis. Consistently, ity compared to wild-type (Wt1+/+) and heterozygous Wt1-deficient fetal liver cells contained significantly (Wt1+/-) cells. It is the aim of this project to study the lower Epo and EpoR mRNA levels than cells of wild- role of Wt1 in normal hematopoiesis and to analyze the type and Wt1-heterozygous (Wt1+/-) embryos. The molecular signaling pathways involved. In particular, proliferative response of CD117-positive hematopoietic we investigate whether the expression of the major progenitor cells to recombinant human (rh) EPO was hematopoietic growth factor, Erythropoietin (Epo), and approximately 10-fold lower in the absence than in its cognate receptor (EpoR) are regulated by Wt1. the presence of Wt1. Further on, significantly fewer Electrophoretic mobility shift assays (EMSAs) hemoglobin-positive cells could be isolated from the revealed that Wt1 protein can bind to several con- livers of Wt1-/- embryos than of their wild-type litter- sensus motifs in the promoters of the Epo and EpoR mates. These results demonstrate that transcription genes. Reporter constructs carrying Epo and EpoR of the genes encoding Epo and its receptor is regu- promoter sequences were stimulated 20- and 7-fold, lated by Wt1. Our findings establish Wt1 as a novel respectively, by co-transfection of blood-derived transcriptional regulator during mouse hematopoi- cell lines with Wt1 expression constructs. Promoter esis. Future investigations shall address the question activation by Wt1 was abrogated upon site-directed whether abnormal EpoR expression contributes to the mutagenesis of the identified cis-elements. Chromatin malignancy of certain forms of human leukemia.

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6. Mechanisms and (patho)physiological consequences of an oxygen-dependent expression of the TrkB neurotrophin receptor

Project leader Lina Scisielski Coworkers Anja Bondke Persson, Karin Kirschner, Inge Grätsch, Angelika Richter Funding - SCHO 634/6-1 External cooperation Dr. Christina Warnecke, Med. Klinik IV, Universität Erlangen-Nürnberg

Neurotrophins are a family of secreted nerve growth encoding gene, NTRK2, in hypoxia. Using transwell factors with critical roles in the development and func- assays we could show that low oxygen stimulates the tion of the nervous system and other tissues. The cel- migration of cultured neuroblastoma cells. This effect lular effects of neurotrophins are mediated by three of hypoxia was abrogated by blockade of the BDNF- high-affinity tyrosine kinase receptors (Trks). Nerve TrkB signaling pathway. growth factor binds to TrkA, brain-derived neuro- Interestingly, highest TrkB expression under hypoxic trophic factor (BDNF) and neurotrophin-4 interact conditions in vivo was found in the airway epithelium. with TrkB, whereas neurotrophin-3 has high affinity Exposure of rats to 8% O2 increased TrkB transcripts for TrkC. The p75NTR membrane receptor binds to and protein in airway epithelial cells approximately all neurotrophins with low affinity. 15-fold. Consistently, the TrkB ligand, BDNF, signifi- High level expression of TrkB in certain types of cantly enhanced the contractile response to acetyl- neuronal neoplasias (neuroblastomas) correlates choline (ACh) of isolated tracheal segments from with aggressive tumor growth and poor prognosis. hypoxic (8% O2) but not from normoxic (21% O2) rats. Regional hypoxia due to insufficient supply with oxy- This effect of BDNF was prevented by pre-incuba- gen changed the gene expression pattern in neurob- tion of the tissue specimens with the tyrosine kinase lastomas towards an immature, more malignant pat- inhibitor K252a and by mechanical removal of the TrkB tern. We therefore reasoned that hypoxia favors tumor expressing airway epithelium. Likewise, the nitric oxide expansion by stimulating TrkB expression in neurob- (NO) synthase inhibitor L-NAME abrogated the influ- lastoma cells. Exposure of neuroblastoma-derived ence of BDNF on ACh-induced airway contractions. Kelly cells in a 1% oxygen atmosphere increased TrkB These findings indicate that activation of TrkB signal- mRNA levels more than 20-fold compared to incuba- ing by BDNF in hypoxia enhances mechanical airway tion at 21% O2. The rise in TrkB transcripts was associ- contractility to ACh through a mechanism that requires ated with increased receptor protein expression. With NO. Our results are in agreement with clinical obser- the combinatorial use of various cell and molecular vations that neurotrophins contribute to airway hyper- biology techniques we identified the hypoxia-inducible responsiveness in patients with bronchial asthma. factor-1 (HIF-1) as a transcriptional activator of the TrkB

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Publications 2006 - 2010

Dame C, Kirschner KM, Bartz KV, Wallach T, Hussels CS, Sciesielski LK, Paliege A, Martinka P, Scholz H (2009): Scholz H (2006): Wilms tumor suppressor, Wt1, is a transcrip- Enhanced pulmonary expression of the TrkB neurotrophin tional activator of the erythropoietin gene. Blood 107 (11): receptor in hypoxic rats is associated with increased acetyl- 4282-4290 choline-induced airway contractility. Acta Physiol 197(3): 253-264 Wagner N, Wagner KD, Scholz H, Kirschner KM, Schedl A (2006): Intermediate filament protein nestin is expressed in Paliege A, Rosenberger C, Bondke A, Sciesielski L, Shina developing kidney and heart and might be regulated by the A, Heyman SN, Flippin LA, Arend M, Klaus SJ, Bachmann S Wilms’ tumor suppressor Wt1. Am J Physiol Regul Integr (2010): Hypoxia-inducible factor-2alpha-expressing interstitial Comp Physiol 291(3): R779-787 fibroblasts are the only renal cells that express erythropoietin under hypoxia-inducible factor stabilization. Kidney Int 77(4): Kirschner KM, Wagner N, Wagner KD, Wellmann S, Scholz H 312-318 (2006): The Wilms tumor suppressor Wt1 promotes cell adhesion through transcriptional activation of the alpha4integrin Kirschner KM, Sciesielski LK, Scholz H (2010): Wilms’ tumour gene. J Biol Chem 281(42): 31930-31939 protein Wt1 stimulates transcription of the gene encoding vascular endothelial cadherin. Pflügers Arch PMID: 20811903, Martens LK, Kirschner KM, Warnecke C, Scholz H (2007): Sep 02 Hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator of the TrkB neurotrophin receptor gene. J Biol Chem 282(19): 14379-14388 Reviews & book chapters (2006-20010)

Steege A, Fähling M, Paliege A, Bondke A, Kirschner KM, Scholz H, Wagner KD, Wagner N (2009): Role of the Wilms’ Martinka P, Kaps C, Patzak A, Persson PB, Thiele BJ, Scholz tumour transcription factor, Wt1, in blood vessel formation. H, Mrowka R (2008): Wilms’ tumor protein WT1(-KTS) modu- Pflügers Arch 458(2): 315-323 lates renin gene transcription. Kidney Int 74(4): 458-466 Kamkin A, Scholz H, Kiseleva I (2010): Cardiac fibroblasts Kirschner KM, Hagen P, Hussels CS, Ballmaier M, Scholz H, in normal and diseased heart: Single mechanically-gated Dame C (2008): The Wilms’ tumor suppressor Wt1 activates ion channels, mechanosensitive currents and mechanically transcription of the erythropoietin receptor in hematopoietic induced potentials in isolated cells and tissue. In: The car progenitor cells. FASEB J 22(8): 2690-2701 diac fibroblast, Neil Turner (ed.), Research Signpost

Fähling M, Mrowka R, Steege A, Kirschner KM, Benko E, Förstera B, Persson PB, Thiele BJ, Meier JC, Scholz H (2009): Translational regulation of the human achaete-scute homologue-1 by fragile X mental retardation protein. J Biol Chem 284(7): 4255-4266

262 HOLGER SCHOLZ – DEVELOPMENTAL PHYSIOLOGY AND CELLULAR PATHOPHYSIOLOGY

General information Third party funding (2006-2010)

Project leader Project title Sponsor Period Scholz H. Characterizing the oxygen-dependent Deutsche Forschungsgemein- 2004-2007 Nafz B. expression of the Wilms’ tumor gene, WT1, schaft (DFG SCHO 634/5-1) in vivo and in vitro Nafz B. Subproject “Functional genomics of cardio- BMBF, Medical genome Research 2004-2007 Scholz H. vascular damage in hypertension” (NGFN), KBCV 1.2, 01GS0416 Scholz H. Mechanisms and (patho)physiological Deutsche Forschungsgemein- 2008-2011 consequences of an oxygen-dependent schaft (DFG SCHO 634/6-1) regulation of the gene encoding the neurotrophin receptor, TrkB Dame C. Regulation of the genes encoding eryth- Deutsche Forschungsgemein- 2005-2006 Scholz H. ropoietin and its receptor by the Wilms’ schaft (DFG DA 484/2-1) tumor transcription factor, Wt1 Fähling M. Post-transcriptional control of the basic Deutsche Forschungsgemein- 2008-2011 Scholz H. helixloop-helix transcription factor Mash1 schaft (DFG FA 845/2-1) Sciesielski, L. Oxygen-dependent expression of the neu- Studienstiftung des Deutschen 2006-2008 rotrophin receptor, TrkB. Volkes Jacobi C. Regulation and function of the metallopro- Sonnenfeld-Stiftung 2010-2011 Scholz H. teinase ADAMTS16

Awards

2007 Young Investigator Award: International Congress „Hypoxia, from Integrative Biology to Human Disease“, Monte Verità/ Ascona, Switzerland L. Martens

2008 Poster award: „The Wilms’ tumor transcription factor Wt1 regulates Expression of the VE-cadherin gene.” Annual Meeting of the German Physiological Society, Cologne Karin Kirschner

263

RALF MROWKA – SYSTEMS BIOLOGY – COMPUTATIONAL PHYSIOLOGY

Head of the group

Prof. Dr. med. Ralf Mrowka

Curriculum Vitae: Prof. Dr. med. Ralf Mrowka, received the ICID (Imperial College International Diploma) in “Engineering and Physical Science in Medicine” at Impe- rial College London (UK) in 1993 and finished Humboldt University medical school in 1996. He received his doctorate degree 1998 from Humboldt-University and his habilitation degree in Physiology in 2006 from Charité – Universitätsmedizin Berlin. In 2010 he became professor for Experimental Nephrology at Friedrich-Schiller- Universität Jena, Germany. His research interests are in the field of computational and experimental analysis of: Gene Regulation, Protein-Protein Interactions, Gene-Expression Data, DNA-sequences, Transcription Factor Binding Sites, miRNA function, drug based iPS. He took part in funded national collaborative networks such as the national genome network NGFN Herz-Kreislauf-Netzwerk. Currently, he coordinates a funded collaborative project within the BMBF initiative Medical Systems Biology dealing with drug induced generation of iPS.

Members of the group

Scientist Andreas Steege Dr. rer. nat. (Guest) Stephan Lorenzen Dr. rer. nat. Axel Göhring Dipl. Biol. Frank Wenke Dipl. Biochem.

Technicians Christine Reinhold

Students Thomas Brinkmeier MD Student

AG Systems Biology: From left to right: S. Lorenzen, F. Wenke, Ch. Reinhold, Th. Brinkmeier, A. Göhring, R. Mrowka, A. Steege

265 RALF MROWKA – SYSTEMS BIOLOGY – COMPUTATIONAL PHYSIOLOGY

Summary

We investigate questions of the physiology of the posttranscriptional level such as action of proteins cardiovascular system with theoretical approaches and miRNAs on gene regulation. Our work contrib- as well as by means of tools of molecular biology. utes to the elucidation of pathophysiological mecha- One research focus is the renin-angiotensin-aldos- nisms that lead to cardiovascular mortality which terone-system (RAAS) which plays a key role in the is the main cause of death in Europe. Furthermore blood pressure regulation and water and electro- we are interested in drug induced pluripotent stem lyte balance in humans. We investigate mechanisms cells (iPS). of gene regulation on the transcriptional as well as

Zusammenfassung

Wir untersuchen mit theoretischen und molekular- ressiert (drug induced iPS). Methodisch integrieren biologischen Ansätzen Fragestellungen auf dem wir dabei verschiedene biologische Datenentitäten Gebiet der Herz-Kreislauf-Physiologie. Der Fokus wie z. B. Gen-Expressionsdaten, DNA-Sequenzin- liegt dabei auf dem Renin-Angiotensin-Aldosteron formation, Protein-Protein-Wechselwirkungen und System (RAAS), das eine entscheidende Rolle für Ontologie-Information. Diese sind geeignet, um mit die Regulation des arteriellen Blutdrucks und den mathematischen Modellen Voraussagen zum Bei- Salz-Wasserhaushalt spielt. Eines unserer Schwer- spiel über die Wirkung von Transkriptionsfaktoren punktprojekte ist die Regulation von Genen im und deren Interaktion zu treffen, die wir dann mit RAAS. Von Interesse sind dabei Mechanismen der zellbiologischen Experimenten überprüfen. Die enge Genregulation auf transkriptioneller Ebene wie auch Kombination von mathematischer Modellierung fein modulierende Einflüsse, die z.B. über miRNA und experimentellen Ansätzen unterstützt damit die vermittelt werden. Darüber hinaus sind wir an subs- Erforschung von Herz-Kreislauf-Erkrankungen, die tanzinduzierter Reprogrammierung von Zellen inte- die Haupttodesursache in Europa darstellen.

266 RALF MROWKA – SYSTEMS BIOLOGY – COMPUTATIONAL PHYSIOLOGY

Research projects

1. Drug based induction of pluripotent human stem cells derived from human somatic cells

Project leader Ralf Mrowka Coworkers Stephan Lorenzen, Frank Wenke, Axel Göhring, Christine Reinhold Funding BMBF External cooperations Max-Delbrueck-Centre for Molecular Medicine Berlin, MDC Computational Systems Biology Group, Dr. Miguel Andrade Max-Planck-Institute for Molecular Genetics, Molecular Embryology Group, MPI-MEG, Dr. James Adjaye Humboldt-University Berlin, Prof. Edda Klipp University of Heidelberg, Institute for Pharmacy and Molecular Biotechnology, IPMB, Prof. Stefan Wölfl Genomatix Software GmbH, Munich, GENOMATIX, Dr. Alexander Hahn European Screening Port GmbH, Hamburg, ESP, Dr. Phil Gribbon

The topic relates to the field of gene regulation and This multidisciplinary project employs methods of stem cell research. The aim of the project is to exploit systems biology, such as mathematical modelling of large scale post genomic data entities by employing biological processes in an iterative manner with wet state of the art tools of systems biology to identify lab experiments. The project includes the exploitation optimize and commercialise small molecule drugs that of high throughput data that has been obtained previ- are capable to generate pluripotent human stem cells ously by projects that have been funded by the BMBF from adult tissue. The project addresses a highly rel- to reach a high synergism of the funding schemes evant issue of modern Biology and Medicine which of the BMBF. A main goal of the project is to obtain has a high potential for innovation. Pluripotent human chemical compounds that have the potential for com- stem cells are useful for multiple applications in trans- mercialization. plantation medicine and tissue engineering as well Because of its high economic, therapeutic and sci- as for the generation of new disease models and in entific impact, the production of induced pluripotent drug development. Induced pluripotent human stem stem cell lines (iPS) is a hot topic in current biomedical cells that are derived from adult tissue may serve as research. It has been shown recently that it is possi- a complementary alternative to inner cell mass (ICM)- ble to induce pluripotency in somatic mouse cells by derived embryonic pluripotent stem cells that are the activation of four genes. Human somatic fibrob- highly debated regarding ethical issues. Moreover, lasts have been converted to pluripotent cell lines by generation of pluripotent stem cells from an adult will the activation of the genes Sox2, Nanog, Oct4 and open completely new possibilities since auto trans- Lin28. Although it has been shown that it is in prin- plantation of engineered tissue will not be subjected ciple possible to induce pluripotency, current meth- to tissue rejection due to immune responses. ods however are of very limited use, since the genes

267 RALF MROWKA – SYSTEMS BIOLOGY – COMPUTATIONAL PHYSIOLOGY are activated by viral transfection which is the main is irreversible, which is another major limitation. There- hurdle for any therapeutic application and commer- fore it is highly desirable to have an alternative way to cial exploitation. The insertion of viral genetic material induce pluripotency. We suggest using chemical small into the human genome is highly problematic for a molecule compounds that would allow activating the number of reasons. First, the viral insertion of mate- mentioned human genes that convey pluripotency in rial is not directed to a specific locus in the genome. a reversible manner. This approach makes it possi- This is a major drawback, since it is unknown what ble to use pluripotent human stem cells in therapeutic genes and consequently what cellular functions are contexts and would allow translating that knowledge affected by this insertion. Second, the viral insertion into products.

2. Molecular basis of the regulation of the renin gene

Project leader Ralf Mrowka Coworkers Andreas Steege, Andreas Patzak, Christine Reinhold Funding Sonnenfeld Stiftung External cooperations Humboldt-University Berlin, Prof. Edda Klipp Institute for Pathology, Dr. Nils Blüthgen

Cardiovascular diseases account for approximately 40% Renin gene itself) as well as in its interactions, due of all deaths in European countries ranging from about to numerous positive and negative feedback loops at 30.6% in Spain up to 46.8% in Greece [OECD, 2007]. multiple levels. How precisely RAAS gene expression The renin-angiotensin-aldosterone-system (RAAS) is is regulated in healthy individuals, and dys-regulated in the most important system for long term regulation cases of hypertension has remained an enigma, for at of blood pressure and plays a key role in the water least two reasons: First, studies have tended to focus and electrolyte balance of the human body. Conse- on singular causes of the disease and there has been quently, Renin and other components of the RAAS little emphasis on studying the systems properties of have been the focus of cardiovascular and related RAAS in order to deal with the multifactorial essence clinical studies. Current agents targeting the RAAS of the disease. Second, the global importance of post- such as angiotensin-converting-enzyme (ACE) inhibi- translational gene regulation by micro-RNAs has only tors and angiotensin II receptor antagonists and renin been appreciated in the recent years and its impact on antagonists are not free of side effects, however exact RAAS remains essentially unexplored, although there numbers are sparse. is emerging evidence that this type of gene regula- Control of RAAS gene expression is determined simul- tion impacts the RAAS. For example, interactions of taneously at several levels, which are integrated and proteins with the renin 3’ untranslated region (3’ UTR) inter-connected inducing complexity on both in the of messenger RNA (mRNA) influences stability and individual control of gene expression (such as the translational efficiency.

268 RALF MROWKA – SYSTEMS BIOLOGY – COMPUTATIONAL PHYSIOLOGY

Integrative Systems Biology has developed as a dis- our means to predict and prevent RAAS-related dys- cipline that can deal with mutifactorial processes, functions at the clinical level: Identification of novel through an integration of quantitative experimentation regulatory elements or their targets could provide new and modelling. And, even more recently, quite a few focal points for genetic analysis in order to estimate methodologies have become available to assess the genetic risk factors for cardio-vascular, or even facili- role of miRNAs in the regulation of gene expression. tate the identification of potential new drug targets. Ultimately the progress in the understanding of RAAS that will be generated by the project is likely to impact

3. Posttranskriptional Control of the HIF-1α Gen

Project leader Ralf Mrowka Coworker Michael Fähling, Anja Bondke Persson, Andreas Patzak, Christine Reinhold Funding Intramural, funding application planned

HIF-1α is a key regulator in oxygen dependent gene Posttranscriptional processes of gene regulation in regulation. It is important in many cell functions as well the context of this factor are largely unexplored. This as in pathological situations such as in cancer. project is dedicated to fill this gap.

Figure: Oxygen dependent signalling of HIF1-alpha. Modified Figure: Genomic structure of HIF-1α. There is a high evo- from Schofield et al. lutionary conservation in a potential regulatory region. The figure was generated with Genome-Browser at UCSC.

269 RALF MROWKA – SYSTEMS BIOLOGY – COMPUTATIONAL PHYSIOLOGY

4. Systematic discovery of transcription factor target genes

Project leader Ralf Mrowka Coworker Michael Fähling, Stephan Lorenzen, Christine Reinhold Funding BMBF External cooperations Max-Planck-Institute for Molecular Genetics, Dr. Szymon Kiełbasa Institute for Pathology, Dr. Nils Blüthgen

Reliable prediction of specific transcription factor tar- genes. Additionally, the web site provides informa- get genes is a major challenge in systems biology and tion on a recovery/cross-validation test to check for functional genomics. Current sequence-based meth- consistency of the provided seed and the quality of ods yield many false predictions, due to the short and the ranking. Furthermore, the web site allows to ana- degenerated DNA-binding motifs. Here, we describe lyse affinities of a selected transcription factor to the a new systematic genome-wide approach, the seed- promoter regions of the top-ranked genes in order to distribution-distance method that searches large- select the best new candidate target genes for further scale genome-wide expression data for genes that experimental analysis. are similarly expressed as known targets. This method is used to identify genes that are likely targets, allow- ing sequence-based methods to focus on a subset of genes, giving rise to fewer false-positive predictions. We show by cross-validation that this method is robust in recovering specific target genes. Furthermore, this method identifies genes with typical functions and binding motifs of the seed. Targetfinder.org (http://targetfinder.org/) provides a web-based resource for finding genes that show a similar expression pattern to a group of user-selected genes. It is based on a large-scale gene expression compendium (>1200 experiments, >13 000 genes). The primary application of Targetfinder.org is to expand a list of known transcription factor targets by new candidate target genes. The user submits a group of genes (the ‘seed’), and as a result the web site provides a list of other genes ranked by similar- Figure: Free website of our transcription factor target finding ity of their expression to the expression of the seed algorithm.

270 RALF MROWKA – SYSTEMS BIOLOGY – COMPUTATIONAL PHYSIOLOGY

Publications 2006 - 2010

Kiełbasa SM, Blüthgen N, Fähling M and Mrowka R (2010): Mrowka R, Steege A, Kaps C, Herzel H, Thiele BJ., Pers- Targetfinder.org: a resource for systematic discovery of son PB and Blüthgen N (2007): Dissecting the action of an transcription factor target genes. Nucleic Acids Res 38, evolutionary conserved non-coding region on renin promoter W233-238 activity. Nucleic Acids Res 35, 5120-9

Fähling M, Mrowka R, Steege A, Kirschner KM, Benko E, Fähling M, Mrowka R, Steege A, Martinka P, Persson PB and Förstera B, Persson PB, Thiele BJ, Meier JC and Scholz H Thiele BJ (2006): Heterogeneous nuclear ribonucleoprotein- (2009): Translational regulation of the human achaete-scute A2/B1 modulate collagen prolyl 4-hydroxylase, alpha (I) homologue-1 by fragile X mental retardation protein. J Biol mRNA stability. J Biol Chem 281, 9279-86 Chem 284, 4255-66 Lai EY, Patzak A, Steege A, Mrowka R, Brown R, Spielmann Mrowka R, Blüthgen N and Fähling M (2008): Seed-based N, Persson PB, Fredholm BB and Persson AEG (2006): systematic discovery of specific transcription factor target Contribution of adenosine receptors in the control of arteriolar genes. FEBS J 275, 3178-92 tone and adenosine-angiotensin II interaction. Kidney Int 70, 690-8 Steege A, Fähling M, Paliege A, Bondke A, Kirschner KM, Martinka P, Kaps C, Patzak A, Persson PB, Thiele BJ et al. Fähling M, Mrowka R, Steege A, Nebrich G, Perlewitz A, (2008). Wilms’ tumor protein (-KTS) modulates renin gene Persson PB and Thiele BJ. (2006): Translational control of transcription. Kidney Int 74, 458-66 collagen prolyl 4-hydroxylase-alpha(I) gene expression under hypoxia. J Biol Chem 281, 26089-101 Kralemann B, Cimponeriu L, Rosenblum M, Pikovsky A and Mrowka R (2008): Phase dynamics of coupled oscillators Lai EY, Martinka P, Fähling M, Mrowka R, Steege A, Gericke reconstructed from data. Phys Rev E 77, 066205 A, Sendeski M, Persson PB, Persson AEG and Patzak A. (2006): Adenosine restores angiotensin II-induced contrac- Kralemann B, Cimponeriu L, Rosenblum M, Pikovsky A and tions by receptor-independent enhancement of calcium Mrowka R (2007): Uncovering interaction of coupled oscilla- sensitivity in renal arterioles. Circ Res 99, 1117-24 tors from data. Phys Rev E 76, 055201

General information Third party funding (2006-2010)

Project leader Project title Sponsor Period Mrowka R. Drug-iPS BMBF PTJ 2009-2011 Mrowka R. Regulation of the Renin Gene Sonnenfeld-Stiftung 2008-2012

271

ANDREAS PATZAK – VASCULAR PHYSIOLOGY

Head of the group

Prof. Dr. A. Patzak

Curriculum Vitae: He studied medicine at the university in Leipzig and at the Ernst- Moritz-Arndt-University in Greifswald, where he received the Diploma (Master) in Medicine in 1982. He was authorized as a physician in 1983 and was promoted to Dr. med. at the University of Greifswald in 1985. From 1983 he worked as a research fellow, and after the specialization in Physiology, as a senior scientist in the Institute of Physiology, Charité, Humboldt-University of Berlin. In 1998 he received the Habilita- tion in Physiology and the authorization in teaching. Andreas Patzak was guest scientist in the Department of Physiology, University Odense, Denmark in 1998 and at the Department of Medical Cell Biology, Biomedical Center, University Uppsala, Sweden in 2002, 2003, and 2004. He is professor in physiology since 2008. The main fields of his interest are vessel physiology and dynamics of respiratory and cardiovascular control.

Members of the group

Scientists Sendeski, Mauricio Dr.

Technicians Amoneit, Yvonne MTA (until 2009) Gerhardt, Andrea MTA (Ing., FH) Neumann, Ulrike BTA

Students Brinkmann, Ole MD student (degree 2008) Kaufmann, Jana MD student Liu, Zhizhao PhD student Moede, Olivia MD student Rettig-Zimmermann, Juliane MD student (degree 2009) Schildroth, Janice MD student Schmidt, Sebastian MD student (degree 2010) Da Silva, Ivana PhD student (2009) Viegas, Vinicius PhD student

273 ANDREAS PATZAK – VASCULAR PHYSIOLOGY

Summary

Our group works about the control of renal vessels The projects of our group focus on interactions of at the organ and cell level. The kidney significantly angiotensin II and adenosine in the control of the contributes to water and electrolyte balance of the arteriolar tone. Further, we investigate the function organism. Pre- and postglomerular arterioles are of endothelin receptors and the role of p38 MAPK effectors in control systems. They regulate renal and reactive oxygen species in the pathogenesis perfusion and pressure. Sympathetic nerve activ- acute renal injury in models of hypoxia/reperfusion ity, myogenic response, tubuloglomerular feedback, and contrast media induced nephropathy. Results and autocrine as well as paracrine substances con- of these studies improve our knowledge about the tribute to the adjustment of the arteriolar tone. Nore- function of small renal resistance vessel in renal pinephrine, angiotensin II, nitric oxide, adenosine physiology and pathophysiology. and endothelin are important players in this context.

Zusammenfassung

Unsere Arbeitsgruppe interessiert sich vor allem für einzelnen Projekten unserer Gruppe beschäftigen die Regulation der Nierengefäße auf Organ- und wir uns mit den Interaktionen von Angiotensin II Zellebene. Die Niere spielt eine zentrale Rolle für und Adenosin, mit Endothelinwirkungen und der den Wasser- und Salzhaushalt des Organismus. Funktion der Endothelinrezeptoren in glomerulären Wichtige Stellglieder renaler Regulationsmechanis- Arteriolen sowie pathophysiologischen Aspekten. men sind die prä- und postglomerulären Arteriolen, In Modellen der Nierenhypoxie/-reoxygenierung welche die Nierendurchblutung und die glomeruläre und der Wirkung von Röntgenkontrastmitteln auf Filtrationsrate über Änderungen ihres Durchmessers die Nierenfunktion wird die Rolle von p38 MAPK, bzw. Widerstandes beeinflussen. Der Tonus dieser Sauerstoffradikalen und der Stickstoffmonoxidver- Arteriolen wird durch die Aktivität sympathischer fügbarkeit bei der Pathogenese akuter Niereninsuf- Nerven, durch die myogene Antwort der Gefäße, fizienz untersucht. Diese Arbeiten erweitern unser den tubuloglomerulären Rückkopplungsmecha- Verständnis von der Regulation der Nierendurchblu- nismus sowie eine Reihe autokriner und parakriner tung und können weitere Einblicke in die Rolle großer Substanzen bestimmt. Noradrenalin, Angiotensin II, und kleiner Nierengefäße z.B. bei der Entstehung Stickstoffmonoxid, Adenosin und Endothelin gehö- von Bluthochdruck oder der kontrastmittelinduzier- ren zu den wichtigen vasoaktiven Stoffen. In den ten Nephropathie geben.

274 ANDREAS PATZAK – VASCULAR PHYSIOLOGY

Research projects

1. Endothelin receptors in the control of glomerular arterioles

Project leader A. Patzak Coworkers Janice Schildroth, Juliane Rettig-Zimmermann, Andrea Gerhardt, Ulrike Neumann, Mauricio Sendeski Funding - Charité

Endothelin is produced and released by endothelial out“). The study shows that endothelin type A recep- cells. Increased endothelin concentrations induce renal tors (ETA-R) mediates vasoconstriction in afferent damage via different signalling pathways. Endothelin arterioles and contribute to vasoconstriction in effer- also induce renal vascular smooth muscle cell con- ent arterioles. In contrast, ETB-R have no effect in traction. This leads to reduction of renal perfusion and afferent but mediates basal nitric oxide release and glomerular filtration, and to volume retention, which constriction in efferent arterioles. The stronger effect of may contribute to the development of hypertension. endothelin-1 on afferent arterioles compared to effer- We investigate the function and signalling of endothelin ent arterioles indicates an influence on glomerular fil- receptors in glomerular arterioles using mice lacking tration rate and a contribution to the pathogenesis of the endothelin type receptor („rescued ETB-knock kidney diseases.

Fig.1 The selective ETB-receptor agonist ALA-ET-1 constricts efferent arterioles of wild type mice. There is no effect in arterioles of ETB-deficient mice. A) Abso- lute diameter in µm. B) Relative changes in relation to the basal diameter, * indicates significant differences between both groups.

275 ANDREAS PATZAK – VASCULAR PHYSIOLOGY

2. Reactive oxygen species and vascular smooth muscle contraction – intracellular pathways

Project leader A. Patzak Coworkers Sebastian Schmidt, Mauricio Sendeski Olga Zavaritskaya, Ulrike Neumann, Ivana da Silva Funding - DAAD External cooperations Dr. Olaf Grisk, Dr. Torsten Günther, Ernst Moritz-Arndt-Universität Greifswald, Dr. Vera and Prof. Dr. Joachim Jankowski, Nephrology, Charité, Campus Benjamin Franklin

Oxidative stress plays a role in the development and In this project, we investigate the mechanisms and maintenance of cardiovascular dysfunction such as in intracellular pathways of such an effect. Pilot studies hyperlipidemia, ischemic heart disease, heart failure, demonstrate an increase of the contraction to angi- and probably hypertension. Reactive oxygen species otensin II in adult mice after stimulation of the NADPH- (ROS) affect vessel function by several mechanisms, oxidase (Fig. 2). This effect was completely prevented for example by decreasing the bioavailability of nitric by treatment with the p38 MAPK inhibitor SB 220025, oxygen („scavenger effect“). Oxidative stress may suggesting an important role of this enzyme in the also contribute to the process of aging. We hypoth- mediation of the NADPH-induced increase of the angi- esize that ROS increase the contractility of mesente- otensin II response. rial arteries and that this effect is dependent on age.

Fig. 2: Stimulation with NADPH, the substrate for the NADPH oxidase increases the contraction to angiotensin II (Ang II) in mesenteric vessels of adult mice (old). The selective p38 MAPK inhibitor SB 220025 prevents this effect. ** indicates significant differences in the course of the concentrations- response-curve of NADPH-treated compared to non-treated (NADPH) and SB 220025 treated vessels, respectively.

276 ANDREAS PATZAK – VASCULAR PHYSIOLOGY

3. Contrast media and function of descending vasa recta

Project leader A. Patzak Coworkers Mauricio Sendeski, Zhizhao Liu, P.B. Persson, Yvonne Amoneit, Andreas Gerhardt Funding - Dr. Werner Jackstädt-Stiftung (to M. Sendeski) - Else Kröner-Fresenius-Stiftung (to A. Patzak) - Government of People’s Republic of China External cooperations Prof. Dr. Tom Pallone, University of Maryland, Baltimore, USA Prof. Dr. A.E.G. Persson, University Uppsala, Sweden Dr. Vera and Prof. Dr. Joachim Jankowski, Nephrology, Charité, Campus Benjamin Franklin

The contrast media induced nephropathy (CIN) is constrict arterioles and vasa recta and reduce renal the third most common reason for hospital-acquired medullary perfusion. Our project focuses on the effect acute kidney injury. The renal damage goes along with of CM on the tone and vasoreactivity of small renal a reduction in renal medullary blood flow and oxy- vessels. CM probably induce oxidative stress in the gen tension. We hypothesize that iodinated contrast wall of the vessels and in tubular cells thereby reduc- media (CM) affect small renal vessels supplying the ing the bioavailability of the vessel dilator nitric oxygen renal medulla, namely the arterioles of juxtamedullary (Fig. 3). We assume that signalling pathways involving nephrons and the descending vasa recta. Contrast the p38 MAPK also mediate the increase in vascular media may also damage tubular epithelial cells. These tone and reactivity. cells then release vasoactive substances, which in turn

Fig. 3 Increase of the fluorescence of DAF‑FM, an indica- tor of nitric oxygen, after application of angiotensin II in an isolated perfused vas rectum of the rat (pseudo color presentation of fluorescence intensity).

277 ANDREAS PATZAK – VASCULAR PHYSIOLOGY

4. Influence of sex hormones on the reactivity of interlobar arteries in the mouse - role of nitric oxide

Project leader A. Patzak Coworkers Pontus B. Persson, Vinicius Viegas, Mauricio Sendeski, Ulrike Neumann, Andrea Gerhardt Funding - DFG GK754-III

Women have lower blood pressure until menopause Nitrite from food and from other sources may act as in comparison with men. This phenomenon is prob- substrate fort the generation of nitric oxide. Studies ably due to differences in the hormone status between show a reduction of blood pressure after uptake of women and men. It has been shown in animal experi- nitrate in form of vegetables. The mechanisms of ments that estrogens have protective effects on blood this effect are not completely known. In the present vessels. Also, mesenterial arteries of female mice project, we ask whether nitric oxide produced from express lower vascular tone compared to male ani- nitrite plays a role fort the differences of vascular tone mals. The differences in vascular tone disappear in seen in female compared to male mice. We also test superoxid dismutase deficient mice, suggesting an a possible protective effect of nitrite in vessels of male important role for reactive oxygen species and nitric animals. oxide in this context.

Fig. 4: Original traces of vessel tension (renal interlobar artery) in response to increasing doses of estrogen (upper trace) and testosterone (lower trace).

278 ANDREAS PATZAK – VASCULAR PHYSIOLOGY

Publications 2006 - 2010 Carlstrom M, Lai EY, Steege A, Sendeski M, Ma Z, Zabihi S, Eriksson UJ, Patzak A, Persson AE (2008): Nitric oxide Sendeski MM, Consolim-Colombo FM, Leite CC, Rubira MC, deficiency and increased adenosine response of afferent Lessa P, Krieger EM (2006): Increased sympathetic nerve arterioles in hydronephrotic mice with hypertension. Hyper activity correlates with neurovascular compression at the tension 51: 1386-1392 rostral ventrolateral medulla. Hypertension 47:988-995 Jankowski V, Patzak A, Herget-Rosenthal S, Tran TN, Lai EY, Lai EY, Martinka P, Fahling M, Mrowka R, Steege A, Gericke Gunthner T, Buschmann I, Zidek W, Jankowski J (2008): Uri- A, Sendeski M, Persson PB, Persson AE, Patzak A (2006): dine adenosine tetraphosphate acts as an autocrine hormone Adenosine restores angiotensin II-induced contractions by affecting glomerular filtration rate. J Mol Med 86: 333-340 receptor-independent enhancement of calcium sensitivity in renal arterioles. Circ Res 99: 1117-1124 Martinka P, Lai EY, Fahling M, Jankowski V, Jankowski J, Schubert R, Gaestel M, Persson AE, Persson PB, Pat- Lai EY, Patzak A, Steege A, Mrowka R, Brown R, Spielmann zak A (2008): Adenosine increases calcium sensitivity via N, Persson PB, Fredholm BB, Persson AE (2006): Contribu- receptor-independent activation of the p38/MK2 pathway in tion of adenosine receptors in the control of arteriolar tone mesenteric arteries. Acta Physiol (Oxf) 193: 37-46 and adenosine-angiotensin II interaction. Kidney Int 70: 690-698 Nordquist L, Lai EY, Sjoquist M, Patzak A, Persson AE (2008): Proinsulin C-peptide constricts glomerular afferent arterioles Gericke A, Martinka P, Nazarenko I, Persson PB, Patzak in diabetic mice. A potential renoprotective mechanism. Am J A (2007): Impact of alpha1-adrenoceptor expression on Physiol Regul Integr Comp Physiol 294: R836-R841 contractile properties of vascular smooth muscle cells. Am J Physiol Regul Integr Comp Physiol 293: R1215-R1221 Patzak A, Steege A, Lai EY, Brinkmann JO, Kupsch E, Spiel- mann N, Gericke A, Skalweit A, Stegbauer J, Persson PB, Hultstrom M, Lai EY, Ma Z, Kallskog O, Patzak A, Persson AE Seeliger E (2008): Angiotensin II response in afferent arteri- (2007): Adenosine triphosphate increases the reactivity of the oles of mice lacking either the endothelial or neuronal isoform afferent arteriole to low concentrations of norepinephrine. Am of nitric oxide synthase. Am J Physiol Regul Integr Comp J Physiol Regul Integr Comp Physiol 293: R2225-R2231 Physiol 294: R429-R437

Lai EY, Jansson L, Patzak A, Persson AE (2007): Vascular Steege A, Fahling M, Paliege A, Bondke A, Kirschner KM, reactivity in arterioles from normal and alloxan-diabetic mice: Martinka P, Kaps C, Patzak A, Persson PB, Thiele BJ, Scholz studies on single perfused islets. Diabetes 56: 107-112 H, Mrowka R (2008): Wilms’ tumor protein (-KTS) modulates renin gene transcription. Kidney Int 74: 458-466 Patzak A, Lai EY, Fahling M, Sendeski M, Martinka P, Persson PB, Persson AE (2007): Adenosine enhances long term the Stegbauer J, Kuczka Y, Vonend O, Quack I, Sellin L, Patzak contractile response to angiotensin II in afferent arterioles. Am A, Steege A, Langnaese K, Rump LC (2008): Endothelial J Physiol Regul Integr Comp Physiol 293: R2232-R2242 nitric oxide synthase is predominantly involved in angiotensin II modulation of renal vascular resistance and norepinephrine Petersson J, Schreiber O, Steege A, Patzak A, Hellsten A, release. Am J Physiol Regul Integr Comp Physiol 294: Phillipson M, Holm L (2007): eNOS involved in colitis-induced R421-R428 mucosal blood flow increase. Am J Physiol Gastrointest Liver Physiol 293: G1281-G1287 Carlstrom M, Lai EY, Ma Z, Patzak A, Brown RD, Persson AE (2009): Role of NOX2 in the regulation of afferent arteriole Petersson J, Phillipson M, Jansson EA, Patzak A, Lundberg responsiveness. Am J Physiol Regul Integr Comp Physiol JO, Holm L (2007): Dietary nitrate increases gastric mucosal 296: R72-R79 blood flow and mucosal defense. Am J Physiol Gastroin test Liver Physiol 292: G718-G724

279 ANDREAS PATZAK – VASCULAR PHYSIOLOGY

Gericke A, Mayer VG, Steege A, Patzak A, Neumann U, Cao C, Edwards A, Sendeski M, Lee-Kwon W, Cui L, Cai CY, Grus FH, Joachim SC, Choritz L, Wess J, Pfeiffer N (2009): Patzak A, Pallone TL (2010): Intrinsic nitric oxide and superox- Cholinergic responses of ophthalmic arteries in M3 and M5 ide production regulates descending vasa recta contraction. muscarinic acetylcholine receptor knockout mice. Invest Am J Physiol Renal Physiol (ahead of print) Ophthalmol Vis Sci 0: 4822-4827 Schildroth J, Rettig-Zimmermann J, Kalk P, Steege A, Fahling Kastner C, Pohl M, Sendeski M, Stange G, Wagner CA, M, Sendeski M, Paliege A, Lai EY, Bachmann S, Persson PB, Jensen B, Patzak A, Bachmann S, Theilig F (2009): Effects of Hocher B, Patzak A (2010): Endothelin type A and B recep- receptor-mediated endocytosis and tubular protein composi- tors in the control of afferent and efferent arterioles in mice. tion on volume retention in experimental glomerulonephritis. Nephrol Dial Transplant (ahead of print) Am J Physiol Renal Physiol 296: F902-F911 Sendeski M, Patzak A, Persson PB (2010): Constriction of Kovacs R, Rabanus A, Otahal J, Patzak A, Kardos J, Albus the vasa recta, the vessels supplying the area at risk for acute K, Heinemann U, Kann O (2009): Endogenous nitric oxide is kidney injury, by four different iodinated contrast media, evalu- a key promoting factor for initiation of seizure-like events in ating ionic, nonionic, monomeric and dimeric agents. Invest hippocampal and entorhinal cortex slices. J Neurosci 29: Radiol 45: 453-457 8565-8577 Carlstrom, M, Lai EY, Ma Z, Steege A, Patzak A, Eriksson U J, Lai EY, Fahling M, Ma Z, Kallskog O, Persson PB, Patzak A, Lundberg J O, Wilcox C S, and Persson A E G (2010): SOD1 Persson AE, Hultstrom M (2009): Norepinephrine increases limits renal microvascular remodeling and attenuates arteriole calcium sensitivity of mouse afferent arteriole, thereby and blood pressure responses to Ang II via modulation of NO enhancing angiotensin II-mediated vasoconstriction. Kidney bioavailability. Hypertension (accepted) Int 76: 953-959

Lai EY, Patzak A, Persson AE, Carlstrom M (2009): Angi- Reviews & book chapters (2006-20010) otensin II enhances the afferent arteriolar response to adenosine through increases in cytosolic calcium. Acta Physiol Patzak A, Persson AE (2007): Angiotensin II-nitric oxide (Oxf) 196: 435-445 interaction in the kidney. Curr Opin Nephrol Hypertens 16: 46-51 Sendeski M, Patzak A, Pallone TL, Cao C, Persson AE, Persson PB (2009): Iodixanol, constriction of medullary descending vasa recta, and risk for contrast medium-induced nephropathy. Radiology 251: 697-704

280 ANDREAS PATZAK – VASCULAR PHYSIOLOGY

General information Third party funding (2006-2010)

Project leader Project title Sponsor Period Patzak A. Sex- and Gender- Specific Mechanisms DFG GK 754-III, GK 754-II, 2006-2010 in Myocardial Hypertrophy Sendeski M. Influence of contrast media on the control Dr. Werner-Jackstädt-Stiftung 2007-2008 of outer medullary descending vasa recta Patzak A. Persson Contrast media and oxidative stress in the Else Kröner Fresenius-Stiftung 2010-2013 P.B. Jankowski V. renal medulla. Liu Z. Effect of contrast media on the function Government of People’ Republic 2009-2013 of the juxtaglomerular apparatus of China

Awards

2008 Poster award of the German Society for Sleep Medicine and Sleep Research Patzak A, Gesche H

2009 „Postdoctoral Novel Disease Model Award“, American Physiological Society Sendeski M

281

ERDMANN SEELIGER – INTEGRATIVE KIDNEY RESEARCH

Head of the group

Dr. med. Erdmann Seeliger

Curriculum Vitae: Erdmann Seeliger studied medicine at Berlin’s Charité, and obtained his license to practise medicine (Approbation) in 1988. He joined the Insti- tute of Physiology at the Charité for his postgraduate specialisation and received his board certification as a specialist in Physiology (Facharzt für Physiologie) in 1993. In his thesis (Dr. med.), he focussed on cardiovascular control in an animal model of cardiomyopathy. In 1993, he joined the Experimental Anaesthesia group at the Virchow Clinics of Berlin’s Free University; the group studied the interrelationship between control of sodium and water balance and long-term control of arterial blood pressure. Seeliger, who returned to the Institute of Physiology at the Charité in 1998, continued these studies until 2006. He then joined the Integrative Kidney Research group at the Institute of Vegetative Physiology; in 2009, he became head of the group. By use of integrative experimental methods, the group studies renal hemodynamics and oxygenation under physiological conditions, and pathophysiological mechanisms involved in kidney diseases like contrast media induced nephropathy, diabetic nephropathy, and renal ischemia/reperfusion injury.

Members of the group

Scientists Cantow, Kathleen Dr. med. vet. Flemming, Bert OA Dr. med. (guest scientist, head of the group till 2009) Ladwig, Mechthild Veterinarian Sargsyan, Lilit Dr. med. Wronski, Thomas Dr. rer. nat. (guest scientist)

Technicians Anger, Ariane BTA Gerhardt, Andrea MTA, Dipl.-Ing. (FH)

283 ERDMANN SEELIGER – INTEGRATIVE KIDNEY RESEARCH

Summary mechanism (3M) that has been discovered only recently. Both the TGF and the 3M seem to serve From a physiological point of view, the kidney is a the purpose of balancing oxygen delivery and, thus, special organ, not just because of the functional RBF with metabolic and oxygen demands arising role that it plays for the organism, but because of from tubular resorption. The outer medulla is particu- the unique and very complex interactions among larly prone to imbalance between oxygen delivery perfusion, glomerular filtration, tubular resorption, and demand, because, in this region, the oxygen and hormonal control. Unfortunately, such interac- demand is high while the pO2 is low. Imbalance tions also play pivotal roles in the pathophysiological between medullary oxygen delivery and oxygen development of various kidney diseases. demand is widely accepted to constitute a pivotal Renal hemodynamics offer striking differences to element in acute kidney injury (AKI) of various origins hemodynamics of other vascular beds. Whereas (e.g., contrast-medium induced nephropathy [CIN], metabolic necessities and oxygen consumptions ischemia-reperfusion injury [I/R-I]) as well as in some determine perfusion in other organs, in the kidney chronic kidney diseases, e.g., diabetic nephropathy it is the other way round: Renal oxygen consump- (DNP). tion is largely determined by perfusion, because an For a couple of years, our group’s focus has been increase in renal blood flow (RBF) that is accompa- on regulation and autoregulation of global (total nied, as a rule, by an increase in glomerular filtration renal blood flow) and regional (cortical and medul- rate (GFR) necessitates an increase in energy- and lary) hemodynamics and oxygenation under various oxygen-dependent tubular resorption. Although physiological and pathophysiological conditions. In whole-kidney blood flow is high and the arterio- this context, we developed a number of new meth- venous difference in oxygen content is low, there ods for integrative in vivo studies on the regulation exist certain regions in the kidney with low partial of kidney hemodynamics and oxygenation in rats. pressure of oxygen (pO2). Meagre pO2 in these This includes the simultaneous measurements of kidney areas is due to the particular architecture of local perfusion and oxygenation, and a number of intrarenal vasculature that enables shunt-diffusion of specific reversible tests, that allow us comprehen- oxygen both in the medulla and in the cortex. sive quantitative characterisation of regulation and The control of renal hemodynamics as well as of autoregulation. tubular resorption involves a number of factors, e.g. By use of these methods, we study 1) the contri- nitric oxide (NO), angiotensin II (Ang II), adenos- bution of certain regulatory elements like the NO- ine (Ado), among others. Furthermore, the kidney system, Ang II, and Ado to control of hemodynamics is equipped with highly efficient mechanisms of and oxygenation under physiological conditions, 2) autoregulation, i.e., the ability to maintain RBF and pathophysiological mechanisms involved in kidney GFR relatively constant in the face of changes in diseases like CIN, I/R-I, and DNP, and 3) pharma- renal perfusion pressure (RPP). Renal autoregula- cological and procedural measures that may enable tory mechanisms include the myogenic mechanism, prevention or treatment of these diseases. the tubulo-glomerular feedback (TGF), and a third

284 ERDMANN SEELIGER – INTEGRATIVE KIDNEY RESEARCH

Zusammenfassung neu entdeckter, langsamer Mechanismus (3M), der vermutlich eine metabolische Komponente darstellt. Was die Niere aus physiologischer Sicht zu einem Für TGF und 3M wird postuliert, dass sie vor einem besonderen Organ macht, ist nicht allein ihre Funk- Missverhältnis zwischen Resorptions-Notwendigkeit tion für den Organismus, sondern sind vor allem die und niedrigem O2-Angebot schützen sollen. Präde- einzigartigen und komplexen Wechselwirkungen stiniert für ein solches Missverhältnis ist der dicke zwischen Perfusion, glomerulärer Filtration, tubu- aufsteigende Teil der Henleschen Schleife, der mit lärer Resorption und hormoneller Regulation. Lei- seinem hohen O2-Bedarf in einer Region mit nied- der spielen solche Wechselwirkungen auch in der rigem pO2 lokalisiert ist. Ein solches Missverhältnis pathophysiologischen Entwicklung vieler Nierener- wird als zentrales Element in der Pathogenese ver- krankungen eine zentrale Rolle. schiedener Nierenerkrankungen wie der Röntgen- Die Durchblutung der Niere weist gegenüber anderen kontrastmittel-induzierten Nephropathie (CIN), der Organen bedeutende Unterschiede auf. Das Strom- Ischämie/Reperfusions-Schädigung (I/R-I) und der zeitvolumen ist gemessen am Sauerstoffverbrauch diabetischen Nephropathie (DNP) angesehen. sehr hoch. Während bei anderen Organen die aktu- Seit längerem befasst sich unsere Arbeitsgruppe mit elle Stoffwechsellage und damit der O2-Verbrauch der Regulation und Autoregulation der globalen (rena- die lokale Durchblutung bestimmt, bestimmt bei ler Blutfluss) und regionalen (kortikalen und medullären) der Niere umgekehrt die aktuelle Durchblutung den Hämodynamik und Oxygenierung unter physiologi- Stoffwechsel und damit den O2-Verbrauch, da ein schen wie auch pathophysiologischen Bedingungen. In erhöhter renaler Blutfluss (RBF) und eine i. d. R. damit diesem Rahmen haben wir auch eine Reihe von neuen verbundene erhöhte glomeruläre Filtrationsrate (GFR) Methoden für Untersuchungen zur Regulation von rena- eine vermehrte tubuläre Resorption erfordern. Trotz ler Hämodynamik und Oxygenierung an Ratten in vivo hoher Gesamtdurchblutung und niedriger arterio- etabliert. Dazu gehören u. a. die gekoppelte Messung venöser Differenz des O2-Gehaltes gibt es Nieren- von lokalen Perfusions- und Oxygenierungsgrößen und regionen mit sehr niedrigem O2-Partialdruck (pO2). eine Reihe spezifischer reversibler Testreize, die uns eine Ursache ist die besondere Gefäßarchitektur der umfassende quantitative Charakterisierung der Regula- Niere, die arterio-venöse O2-Shuntdiffusionen in der tion und Autoregulation erlauben. Nierenrinde und im Nierenmark zur Folge hat. Mittels dieser Methoden untersuchen wir zum einen An der Regulation der Nierenhämodynamik wie auch den Beitrag bestimmter Regulationselemente, wie der tubulären Resorption sind eine Reihe von Fakto- z.B. des NO-Systems, des Ang II und des Ado, zu ren beteiligt, u. a. Stickstoffmonoxid (NO), Angioten- Regulation und Autoregulation von renaler Hämo- sin II (Ang II), und Adenosin (Ado). Die Niere verfügt dynamik und Oxygenierung unter physiologischen darüber hinaus über Mechanismen der Autoregula- Bedingungen, zum anderen pathophysiologische tion, d. h. einer relativen Konstanz von RBF und GFR Mechanismen bestimmter Nieren-Erkrankungen wie bei Änderung des renalen Perfusionsdruckes. Dazu der CIN, der I/R-I und der DNP, sowie pharmakologi- gehört der myogene Mechanismus, der tubulo- sche und prozedurale Möglichkeiten der Prävention glomeruläre Feedback (TGF), und ein dritter, erst bzw. Therapie dieser Erkrankungen.

285 ERDMANN SEELIGER – INTEGRATIVE KIDNEY RESEARCH

Research projects

1. Direct effects of Glucagon-like peptide 1 on renal hemodynamics and oxygenation – animal studies into potential prevention of diabetic nephropathy

Project leader Erdmann Seeliger Coworkers Bert Flemming, Mechthild Ladwig, Kathleen Cantow, Lilit Sargsyan, Andrea Gerhardt, Ariane Anger Funding DFG SE 998/2-1 External cooperations Michael Fähling (AG Molecular Physiology)

Patients who suffer from diabetes mellitus (DM) often the regulation of kidney hemodynamics and oxygena- develop renal impairment, i.e., diabetic nephropathy tion in rats. Previous studies of ours demonstrated that (DNP). The mechanisms that contribute to the devel- kidney-specific interactions among perfusion, tubular opment of DNP are not entirely clear, however, two resorption, and oxygenation play an outstanding role factors appear to play a pivotal role: oxygen defi- in the development of other forms of nephropathy. The ciency in the renal medulla, and deficiency of nitric present project aims at studying possible direct effects oxide (NO). Glucagon-like peptide 1 (GLP-1) facili- of GLP-1 on renal hemodynamics and oxygenation, tates the release of insulin, thereby reducing glucose both in healthy rats and in a rat model of DM. We concentration. Moreover, GLP-1 has effects that are want to assess potential glucose-independent reno- independent from glucose homeostasis. In the kidney, protective effects of GLP-1 in DNP, and clarify the roles GLP-1 receptors have been found that could medi- renal oxygen deficiency and NO deficiency play in this ate a reduction in tubular sodium resorption. We have context. established methods for integrative in vivo studies on

286 ERDMANN SEELIGER – INTEGRATIVE KIDNEY RESEARCH

2. Contrast-media induced nephropathy: role of physico-chemical properties of contrast media

Project leaders Bert Flemming, Erdmann Seeliger, Pontus B. Persson Coworkers Mechthild Ladwig, Thomas Wronski, Andrea Gerhardt Funding Bayer Schering Pharmaceuticals External cooperations Klaus Becker (Zürich University)

Interventional procedures such as cardiac catheteri- contrast media transiently increased renal blood flow, zation often require large amounts of contrast media and, even more, hindquarter blood flow. Iopromide (CM). Although available CM are well tolerated, and mannitol increased urine flow rate much more they potentially can cause kidney damage (contrast than iodixanol. Iodixanol led to a dramatic increase in medium induced nephropathy, CIN). Osmolality and urine viscosity and to marked transient decrease in viscosity are important physico-chemical properties GFR. Iopromide increased urine viscosity much less of CM, that have been suggested to play a role in and did not affect GFR. Only iodixanol and dextran (the CIN. Therefore, we instrumented anaesthetized rats to high viscous substances) decreased blood flux, eryth- assess either renal hemodynamics and oxygenation rocyte concentration and pO2 in the renal medulla. and hindquarter hemodynamics, or urine flow rate, Reduction in renal medullary blood flux by iodixanol urine viscosity, and glomerular filtration rate (GFR). A was most pronounced at renal perfusion pressure contrast medium with higher osmolality (iopromide) above 60 mmHg. Moreover, iodixanol prolonged the was compared to a contrast medium with higher vis- tubuloglomerular feedback response. In conclusion, cosity (iodixanol) at the same iodine concentrations iodixanol, by virtue of its high viscosity, reduced blood (320 mgI/ml). The effects of mannitol (same osmolality flux, erythrocyte concentration and pO2 of the renal as iopromide) and dextran (same viscosity as iodix- medulla and decreases GFR, by increasing tubular anol) were also studied. Kidney functions are pres- fluid viscosity, and, probably also by increasing plasma sure dependent, thus, pressure dependent effects viscosity. Iopromide, by virtue of its higher osmolality, on renal hemodynamics were investigated. Also the prevented excessive urine viscosity levels, medullary tubuloglomerular feedback response, a crucial con- hypoperfusion and hypoxia, and decline in GFR. troller of renal hemodynamics, was assessed. Both

287 ERDMANN SEELIGER – INTEGRATIVE KIDNEY RESEARCH

3. Renal safety of contrast media: mechanisms and prevention strategies

Project leaders Erdmann Seeliger, Pontus B. Persson Coworkers Bert Flemming, Mechthild Ladwig, Kathleen Cantow, Lilit Sargsyan, Andrea Gerhardt, Ariane Anger Funding Bayer Schering Pharmaceuticals

Current medical diagnosis and thereby therapeutic We found that the renal tubular concentration proc- success critically depends on imaging techniques ess concentrates high-viscous iso-osmolar CM to a using radiocontrast media. However, the application of great extent, thus, in non-hydrated rats, tubular fluid contrast media (CM) may cause severe complications viscosity dramatically increases and GFR declines. such as contrast media induced nephropathy (CIN). CM of higher osmolality (so called low-osmolar CM), Previous results of ours indicate that CM may cause on the other hand, bear the advantage of preventing renal medullary hypoperfusion and hypoxia, an effect excessive urine viscosity levels. In the present project, that is related to rheological properties of CM, in par- we set out to further underscore the significance of ticular to CM viscosity. High CM viscosity may cause renal fluid viscosity in CIN. The key question to be medullary hypoperfusion by two mechanisms. Along answered is if CM are retained in the kidney for longer the passage of blood through the vasa recta supplying periods of time, particulary iso-osmolar high-viscous the renal medulla, water escapes while CM cannot fol- CM. Furthermore, heavy ongoing debate on the pros low, which will increase blood viscosity, thereby hinder- and cons of fluids best suited in preventing CIN reflects ing perfusion. Likewise, water is reabsorbed along the the need to understand the basic mechanisms of vari- tubules, whereas CM is not, which increases tubular ous fluid expansion regimes. We set out to study dif- fluid viscosity, thereby hindering tubular fluid flow. The ferent regimen in the rat. Finally, we seek to elucidate latter will hinder glomerular filtration, in addition, it will the mechanism(s) behind the flush-phenomenon after prolong contact time of CM, thus, toxic effects, such giving CM. as vasoconstriction of vasa recta will be pronounced.

288 ERDMANN SEELIGER – INTEGRATIVE KIDNEY RESEARCH

4. Acute kidney injury, renal hemodynamics and potential benefit of nitrite

Project leaders Erdmann Seeliger, Bert Flemming, Pontus B. Persson Coworkers Mechthild Ladwig, Kathleen Cantow, Lilit Sargsyan, Andrea Gerhardt, Ariane Anger Funding Grant application for DFG FOR 1368/1 External cooperations D. Dragun, U. Hoff (Nephrology, Charité) Dr. rer. nat. W.-H. Schunck (MDC Berlin)

Renal outer medullary hypoxia plays a pivotal role constitute a reasonably specific measure to prevent in acute kidney injury (AKI). Reduced bioavailability AKI. We have established methods for integrative in of vasodilatory nitric oxide (NO) directly promotes vivo studies on regulation of kidney hemodynamics hypoperfusion and hypoxia; in addition, it disinhibits and oxygenation in rats, and applied these methods the synthesis of vasoconstrictive 20-hydroxyeicosa- to study contrast medium induced nephropathy (CIN) tetraenoic acid (20-HETE). According to a new para- and renal ischemia/reperfusion (I/R) injury. The project digm of hypoxic vasodilation, hemoglobin (Hb) is a that is part of an application for a DFG Forscher- regulated nitrite reductase. Hb reduces nitrite to NO as gruppe, aims at studying the role of nitrite-derived NO it deoxygenates, thus causing vasodilation in hypoxic for hemodynamics and oxygenation in rat models of regions. We hypothesize that, in the setting of AKI, low CIN and of (I/R) injury. In close cooperation with other dose nitrite may result in renal medullary vasodilation. participants of the Forschergruppe, we will also study As nitrite is reduced to NO in hypoxic areas only, it may the role of 20-HETE in I/R injury.

289 ERDMANN SEELIGER – INTEGRATIVE KIDNEY RESEARCH

Publications 2006 - 2010

Seeliger E, Ladwig M, Reinhardt HW (2006): Are large Seeliger E, Wronski T, Ladwig M, Dobrowolski L, Vogel amounts of sodium stored in an osmotically inactive form T, Godes M, Persson P, Flemming B (2009): The renin- during sodium retention? Balance studies in freely moving angiotensin system and the third mechanism of renal dogs. Am J Physiol Regul Integr Comp Physiol 290: blood flow autoregulation. Am J Physiol Renal Physiol R1429-R1435 296:F1334-F1345

Liss P, Persson PB, Hansell P, Lagerquist B (2006): Renal Seeliger E, Becker K, Ladwig M, Wronski T, Persson PB, failure in 57925 patients undergoing coronary procedures Flemming B (2010): Up to 50-fold increase in urine viscosity using iso-osmolar or low-osmolar contrast agents. Kidney by iso-osmolar contrast media in the rat. Radiology 256:406- Int 70: 1811-1817 414

Stauss HM, Persson PB (2006): Cardiovascular variability Seeliger E, Lunenburg T, Ladwig M, Reinhardt HW (2010): and the autonomic nervous system. J Hypertension 24: Role of the renin-angiotensin-aldosterone system for control 1902-1905 of arterial blood pressure following moderate deficit in total- body sodium – balance studies in freely moving dogs. Clin Seeliger E, Flemming B, Wronski T, Ladwig M, Arakelyan K, Exp Pharmacol Physiol 37:e43-e51 Godes M, Möckel M, Persson PB (2007): Viscosity of contrast media perturbs renal hemodynamics. J Am Soc Nephrol Hoff U, Lukitsch I, Chaykovska L, Ladwig M, Arnold C, Mant- 18:2912-2920 hati VL, Fuller FT, Schneider W, Gollasch M, Müller DN, Flem- ming B, Seeliger E, Luft FC, Falck JR, Dragun D, Schunck WH Bräutigam M, Persson PB (2006): Do iodinated contrast (2010): Inhibition of 20-HETE synthesis and action protects media interfere with renal tubular creatinine secretion? Radi from renal ischemia/reperfusion injury. Kidney Int in press ology 240: 615

Persson PB, Liss P, Hansell P (2007): Evaluation and com- Reviews & book chapters (2006-2010) parison between visipaque (Iodixanol) and hexabrix (Ioxaglate) in coronary angiography. J Am Coll Cardiol 49:1668-1671 Persson PB (2006): Pathophysiology of contrast-induced nephropathy. In: Contrast-Induced Nephropathy; eds. Patzak A, Steege A, Lai EY, Brinkmann O, Kupsch E, Spiel- Bartorelli AL, Marenzi G; Taylor & Francis Group, London, pp mann N, Gericke A, Skalweit A, Stegbauer J, Persson PB, 3-17 Seeliger E (2008): Angiotensin II response in afferent arterioles of mice lacking either the endothelial or neuronal isoform Persson PB, Tepel M (2006): Contrast medium-induced of nitric oxide synthase. Am J Physiol Regul Integr Comp nephropathy. Kindney Int Suppl. 100:S8-S10 Physiol 294:R429-437 Persson PB (2006): Dimeric contrast media: A true leap ahead? EJHPPractice 12: 73-74

290 ERDMANN SEELIGER – INTEGRATIVE KIDNEY RESEARCH

General information Third party funding (2006-2010)

Project leader Project title Sponsor Period Seeliger E. Stellenwert von Nierennerven, Blut- DFG SE 998/1-3 2003-2006 druckoszillationen und Drucknatriurese bei der Regulation des Natriumbe- standes und des Blutdruckes – Bilanzun- tersuchungen an wachen Hunden Flemming B. Effects of SLV320 and furosemide on the Solvay Pharma-ceuticals GmbH 2003-2006 autoregulation of renal blood flow and renal tissue oxygen concentration Persson P.B. Einfluss maternaler Varianten des Gens DFG PE 388/20-1 2005-2008 der endothe-lialen NO-Synthase (eNOS) auf die Programmierung von Herz-Kreis- lauferkrankungen in der F1-Generation Seeliger E. Contrast medium induced nephropathy: Solvay Pharma-ceuticals GmbH 2005-2008 Persson P.B. Prevention of acute effects of iodixanol by SLV320 Flemming B. Contrast medium induced nephropathy: Schering AG 2005–2009 Persson P.B. importance of contrast medium viscosity Seeliger E. Contrast medium induced nephropathy: Bayer-Schering Pharmaceuticals 2007-2009 Persson P.B. Iopromide 370, Iodixanol, Iopromide 370 + osmodiuresis Flemming B. Contrast medium induced nephro-pathy: Bayer Healthcare 2007-2010 Persson P.B. modulation of acute effects of Iodixanol by the Adenosine A1-agonist, BR-4461 Seeliger E. Renal hemodynamic effects of the con- Bayer-Schering Pharmaceuticals 2008-2010 Patzak A. trast media, Iodixanol and Iopromide Persson P.B. Seeliger E. Direkte Wirkungen von Glukagon-like DFG SE 998/2-1 2009-2012 peptide 1 auf renale Hämodynamik und Oxygenierung – tierexperimentelle Unter- suchungen zur potentiellen Prävention der diabetischen Nephropathie Seeliger E. Renal safety of contrast media: mecha- Bayer-Schering Pharmaceuticals 2010-2011 Persson P.B. nisms and prevention strategies

291

MICHAEL FÄHLING – MOLECULAR PHYSIOLOGY

Head of the group

Dr. rer. nat. Michael Fähling

Prof. Dr. rer. nat. Bernd J. Thiele (until Oct. 2009) PD Dr. med. Benno Nafz (until April 2010)

Curriculum Vitae: Michael Fähling started his carrier 1996-2001 as a student at the Free University of Berlin, Faculty of Natural Sciences with the special subject Molecular Biology/ Genetics. He obtained his PhD degree in 2004 as a member of the Graduate Course GK 754 “Myocardial Hypertrophy” at the Charité - Universitätsmedizin Berlin/ German Heart Institute Berlin (DHZB). Following 3 years of post-doctoral experience he became group leader of the research group “Molecular Physiology” at the Institute of Vegetative Physiology. The main focus of his research is “Posttranscriptional control of gene expression – regulation at the mRNA layer” under stress conditions such as hypoxia, and during development. This involves regulative aspects that specifically modulate mRNA-stability, efficiency of mRNA translation or cellular mRNA-localization. Michael Fähling is a registered reviewer for several journals (e.g. Nucleic Acids Research, American Journal of Physiology, European Journal of Physiology) and Associate Editor of Frontiers in Molecular Neuroscience.

Members of the group Scientists Students Franjicevic, Monika Dipl. Biol. Benko, Edgar PhD student Skalweit, Angela Dr. med. Gardow, Stefanie-Josefin Medical student Perlewitz, Andrea PhD student Technicians Schmidt, Irina PhD student Neumann, Ulrike BTA (pro rata basis) Staudacher, Jonas Medical student Stöbe, Regine Dipl. Ing. (FH) Werner, Jeannette MTLA

Scientists and Technicans. From left to right first row: Students. From left to right: Andrea Perlewitz, Jonas Stau- Regine Stöbe, Ulrike Neumann, Jeannette Werner, back row: dacher, Stefanie-Josefin Gardow, Edgar Benko. Dr. Benno Nafz, Dr. Michael Fähling, Dr. Angela Skalweit, Prof. Dr. Bernd-Joachim Thiele

293 MICHAEL FÄHLING – MOLECULAR PHYSIOLOGY

Summary

The information for specific gene regulation is mainly Differentiation Control Elements – DICE) or AU-rich localized in the non-coding regions of the genome. elements (ARE). The latter motives (with the con- At the DNA level cis-elements that are essential for sensus: [AA]UAAAU[AA]) mainly convey alterations gene transcription can be found in the gene promoter in mRNA stability. The list of known trans-factors or in enhancer/ silencer regions upstream or down- is constantly growing. Currently more than 370 stream of the gene. Further regulatory elements are RNA-binding proteins have been identified (source: localized in the mRNA untranslated regions (UTRs). Human Protein Reference Database), as well as lots The message they carry is crucial to define mRNA- of miRNAs. Mechanisms of posttranscriptional gene half life time, mRNA localization and the efficiency of expression control seem to be crucial under stress translation. In our group we investigate these post- conditions and during development. Consequently, transcriptional - mRNA UTR based - mechanisms of several diseases are known which are a result of gene expression control in cellular models. loss of function (e.g. by mutations) of cis-elements Nearly all eucaryotic mRNAs contain a 5’- and or trans-factors. 3’-UTR. Specific mRNA UTR cis-elements are At present we focus in our studies on the posttran- responsible for the action of trans-factors like scriptional control of the Epithelial Na2+-Channel RNA-binding proteins (RNA-BPs) or microRNAs (ENaC) under the influence of hormones and on the (miRNAs). In an interspecies comparision mRNA transcription factor human Achaete-Scute Homo- 5’UTRs consist, on average, of ~200 nucleotides logue-1 (hASH1, ASCL1) during development and (nt). In contrast, mRNA 3’UTRs are more divergent, the hypoxia response. Furthermore, we investigate ranging from ~200 nt in plants and fungi to ~440 nt the influence of metabolic rate depression on mRNA in mammals. Human mRNA 3’UTRs, for instance, translation. have an average size of ~1.000 nt, thus, are similar in To address these questions we use a couple of length to human mRNA translated regions. The long- molecular standard methods as well as special est known 3’UTR is that of FMR2, containing 9.280 techniques such as Electrophoretic-Mobility Shift nt. Indicators of functionally (or regulatory) relevance Assays (EMSA), UV-Cross-linking, Affinity Chroma- in mRNA UTRs are repetetive sequences or con- tography, (Sucrose-) Polysomal Gradient Analysis, served regions. The best-defined cis-elements are UTR-dependent reporter gene assays or methods specific mRNA-secondary structures (e.g. Internal to estimate mRNA and protein half life times. Ribosome Entry Sites (IRES)), CU-rich elements (e.g.

294 MICHAEL FÄHLING – MOLECULAR PHYSIOLOGY

Zusammenfassung

Die Informationen für die Regulation der spezifi- Internal Ribosome Entry Site), CU-reiche Elemente schen Genexpression sind hauptsächlich in den (bspw. Differentiation Control Elements – DICE) nicht-kodierenden Regionen des Genoms lokalisiert. sowie ARE (AU-Rich Elements). Die Liste von poten- Ähnlich wie auf DNA-Ebene (Promotoren, Enhancer) tiell interagierenden trans-Faktoren wächst stetig verhält es sich auch auf der mRNA-Ebene, wo die und umfasst z.Z. über 370 RNA-Bindungsproteine mRNA untranslatierten Regionen (UTRs) wichtige (Quelle: Human Protein Reference Database) und regulative cis-Elemente enthalten, die die Eigen- eine Vielzahl von miRNAs. Die posttranskriptionelle schaften der jeweiligen mRNA bezüglich ihrer Sta- Expressionskontrolle scheint insbesondere bei der bilität, zellulären Lokalisation und Translatierbarkeit Anpassung von Zellen an veränderten Umweltbe- beeinflussen. Unsere Arbeitsgruppe befasst sich mit dingungen (Stress) sowie bei Wachstums- und Ent- der posttranskriptionellen Genexpressionskontrolle wicklungsprozessen von besonderer Bedeutung zu in zellbiologischen Untersuchungen. sein. Eine Veränderung der Bindungsmotive bzw. Nahezu alle eukaryotischen mRNAs enthalten eine der interagierenden trans-Faktoren durch Mutatio- 5’- und 3’-untranslatierte Region. Die in den mRNA 5’- nen oder differentielles Spleißen ist daher auch mit und 3’-UTRs lokalisierten cis-regulatorischen Infor- verschiedenen Krankheiten assoziiert. mationen werden hauptsächlich durch trans‑Fak- Der Fokus unserer Gruppe liegt auf der Untersu- toren wie RNA-Bindungsproteine (RNA-BPs), oder chung mRNA-basierter Expressionsveränderungen micro-RNAs (miRNAs) umgesetzt. In Vergleich zentraler Faktoren wie dem Epithelialen Na2+-Kanal einer Vielzahl untersuchter Spezies wurde ermittelt, (ENaC) unter hormoneller Stimulation oder dem dass mRNA 5’UTRs eine durchschnittliche Länge Transkriptionsfaktor human Achaete-Scute Homo- von ca. 200 nt aufweisen. Demgegenüber variieren logue-1 (hASH1, ASCL1) während der Entwicklung die Längen der mRNA 3’UTRs stärker (~200 nt bei und unter Hypoxie. Darüber hinaus untersuchen wir Pflanzen und Pilze, ~440 nt bei Säugern und speziell zentrale Prozesse wie den der mRNA Translation beim Menschen ~1 000 nt). Damit weisen humane unter Sauerstoff-Mangelbedingungen (Hypoxie). 3’UTRs eine vergleichbare durchschnittliche Länge Neben den molekularbiologischen Standardme- wie die der translatierten Bereiche auf. Die längste thoden kommen bei unseren Untersuchungen spe- bekannte 3’UTR umfasst 9 280 nt und wurde in der zielle Verfahren wie: Electrophoretic-Mobility Shift humanen FMR2 mRNA identifiziert. Indikatoren für Assays (EMSA), UV-Cross-linking, Affinitätschro- funktionell relevante Elemente sind hauptsächlich matographie, Polysomengradientenanalyse, UTR- repetitive Elemente und konservierte Bereiche in abhängige Reportergen Assays oder mRNA- bzw. den UTRs. Die bekanntesten RNA-Bindungsmotive Protein-Halbwertzeitbestimmungen zum Einsatz. sind spezifische Sekundärstrukturen (bspw. IRES –

295 MICHAEL FÄHLING – MOLECULAR PHYSIOLOGY

Research projects

1. Posttranscriptional control of epithelial sodium channel (ENaC) expression

Project leader Bernd-Joachim Thiele, Benno Nafz Coworkers Angela Skalweit, Michael Fähling, Andrea Perlewitz, Jeannette Werner, Ulrike Neumann Funding German Research Foundation (DFG), Research Unit #667 „Tubular Mechanisms of renal Volume Regulation“ TP8

The sodium channel ENaC consists of three subunits: efficiency, playing an important role in the synthesis α, β, and γ and is found in the apical membrane of salt- of ENaC subunits. absorbing epithelia of organs such as the kidney, colon In this project, we investigate the influence of hormones, or lung, where it constitutes the rate-limiting step in e.g. aldosterone and vasopressin, at the level of post- Na+ and water absorption. In the kidney, vasopressin transcriptional control of ENaC subunit expression. We and aldosterone play key roles in the minute adjust- identified a wide range of RNA binding proteins (RBP) ments of sodium re-absorption in the distal nephron. as trans-factors and mRNA sequence motifs involved Both hormones act via ENaC. The number of func- in translational control of gamma-ENaC synthesis. For tional ENaC channels at the cell surface depends on instance, the γ-ENaC mRNA 3’UTR contains an AU- basic molecular processes like transcription of the rich element (ARE), which was shown by RNA-affinity ENaC genes, synthesis of protein subunits, storage chromatography to interact with AU-rich element bind- in vesicles, vesicle trafficking and channel assembly ing proteins (ARE-BP) like HuR, AUF1 and TTP. Some in the membrane, and finally on removal of channels RBPs associate with γ-ENaC mRNA in polysomes in from the surface by endocytosis and degradation by a hormone-dependent manner and are crucial for the the ubiquitine/proteasome system. Basically, hormo- up-regulation of γ-ENaC synthesis. Furthermore, we nal and environmental regulation of ENaC expression could demonstrate that aldosterone and vasopressin, is exerted through transcriptional control. In addition, for instance, affect the synthesis of the α- and γ-, but there is evidence of mRNA related posttranscriptional not the β-ENaC subunit, utilizing RBPs as effectors of processes, which affect mRNA stability or translational translational control.

296 MICHAEL FÄHLING – MOLECULAR PHYSIOLOGY

Figure 1: Sucrose polysome gradient analysis: Aldosterone and dDAVP increase translational efficiency of alpha- and gamma-ENaC mRNA. Cytosolic extracts of aldosterone- or dDAVP-stimulated mpkCCD- cells (S10) were separated by ultracentrifugation over 10-50% sucrose gradients and divided into 12 fractions. Total RNA was prepared and alpha-, beta- and gamma-ENaC mRNAs were visualized by RT-PCR. Polysome-bound mRNAs (which are translationally active) sediment in fractions 2-8, monosomes and free mRNPs in fractions 9-12.

(from: Perlewitz,A., B.Nafz, A.Skalweit, M.Fähling, P.B.Persson, and B.J.Thiele. “Aldosterone and vasopressin affect alpha- and gamma- ENaC mRNA translation.” Nucleic Acids Research (2010). accepted.)

2. Posttranscriptional control of human achaete-scute homologue-1 (hASH1) synthesis

Project leader Michael Fähling, Holger Scholz Coworkers Edgar Benko, Angelika Richter, Regine Stöbe, Funding German Research Foundation (DFG); FA 845/2-1 External cooperations Prof. Dr. Dr. Joachim Klose, Institute of Human Genetics, Charité, CVK) Junior-Prof. Dr. Jochen C. Meier, Max-Delbrück-Centrum for Molecular Medicine (MDC)

The human achaete-scute homologue-1 (hASH1; pression of Mash1/ hASH1 is correlated with a broad ASCL1; Mash1) belongs to the bHLH (basic helix-loop- variety of tumors, including lung cancer, neuroendo- helix) family of transcription factors. Mash1/ hASH1 is crine tumors, foregut and midgut carcinoid tumors, expressed in both the central and the peripheral nerv- pancreatic adenocarcinomas, islet cell tumors, gas- ous system during development and promotes early trinomas, as well as parathyroid adenomas. Although neuronal differentiation as well as the specification the importance of Mash1/ hASH1 in neurogenesis and of neuronal subtype identities. Furthermore, misex- cancer has been well documented, the mechanism

297 MICHAEL FÄHLING – MOLECULAR PHYSIOLOGY of its regulation and downstream targets are largely ciency of hASH1 mRNA by FMRP may represent an unknown. The aim of this project is the identification important regulatory switch in neuronal differentiation. of trans-factors which modulate hASH1 gene expres- Currently we investigate the role of oxygen in the alter- sion at the level of posttranscriptional control. For ation of hASH1 gene expression. Hypoxic suppression instance, we identified hASH1 as a novel downstream of hASH1 synthesis may participate in the hypoxia- target of FMRP (Fragile X-Mental Retardation Protein). induced dedifferentiation of tumor cells, especially FMRP was found to gate the translation of a large set of neuroblastoma cells. Although the initial cellular of mRNAs in dendrites that are involved in synaptic response to low oxygen concentrations is the sup- plasticity. Dysfunction of the Fragile X-Mental Retar- pression of energy consuming processes, including dation-1 (FMR1) gene transcription is associated with mRNA translation, our data suggest that inhibition of neuronal disorders, such as the Fragile X-Syndrome hASH1 in hypoxia is actively controlled by cis-regula- (FXS) and the Fragile X-Associated Tremor/Ataxia tory elements, located in the hASH1 mRNA 3’UTR. Syndrome (FXTAS). Thus, improved translational effi-

Figure 2: Sucrose polysome gradient analysis: Neuroblastoma-derived Kelly cells were incubated either under control or hypoxic conditions (21% vs. 0.5% O2) for 6 h. Cytosolic extracts were separated by sucrose gradient centrifugation as described in Figure 1. A: Ribosomal profile recorded by absorbance at 254 nm. B, C: mRNA levels for hASH1 (B) and beta-Actin (C) were quantified by real-time PCR and visualized by RT-PCR. The data indicate no dramatic alteration, neither in global translational efficiency (A) nor translation rate of beta-Actin mRNA (C). In contrast, the hASH1 mRNA (B) shifts from polysomes to the translationally inactive non-polysomal fractions, indicating an inhibition of hASH1 mRNA translation under hypoxic conditions.

298 MICHAEL FÄHLING – MOLECULAR PHYSIOLOGY

3. Rate of protein synthesis under hypometabolic conditions

Project leader Michael Fähling Coworkers Stefanie-Josefin Gardow, Jonas Staudacher, Regine Stöbe, Funding Charité - Universitätsmedizin Berlin External cooperations Dr. Andreas Steege, Universitätsklinikum Regensburg

Under normoxic conditions, oxidative phosphoryla- for 25-30% under resting conditions. Consequently, tion constitutes the major source of the cellular energy hypoxia causes metabolic rate depression and overall intermediate adenosine triphosphate (ATP). High-rate protein synthesis drops to ~10% compared to control energy production is a prerequisite for cells and tis- level. Interestingly, it is well known that hundreds of sues to achieve a high metabolic rate. Increased oxy- hypoxia-sensitive genes are increasingly expressed, gen consumption or disturbance in oxygen supply especially under prolonged hypoxic conditions. Thus, result in decreased oxygen levels. Thus, hypoxia is a effective mRNA translation following oxygen depriva- consequence of inadequate oxygen availability. As a tion seems to be possible. consequence, multiple metabolic processes must be The aim of this project is to find out how specific coordinated to achieve a net suppression that bal- mRNAs can avoid the global suppression of mRNA ances the rates of ATP production and ATP consuming translation. Our hypothesis is that mRNAs of hypoxia- processes at a new lower net rate of ATP turnover. sensitive genes are recruited to membrane-bound The process of protein synthesis belongs to the most polysomes, a process mediated by trans-factors inter- energy-consuming processes in the cell, accounting acting with cis-elements in the mRNA UTRs.

299 MICHAEL FÄHLING – MOLECULAR PHYSIOLOGY

Figure 3: Simplified model of cellular adaptation in moderate hypoxia and its influence on mRNA translation. Upon rapid suppression of global protein synthesis by PERK mediated inhibition of translational initiation, cells respond to hypoxia by activation of glycolysis. Restored ATP levels ensure the expression of survival factors, which may be released such as signal molecules (e.g. endothelin-1 (ET1), vascular endothelial growth factor (VEGF), or adenosine). Furthermore, newly synthesized factors can be involved in glucose uptake, glycolysis, prevention of acidosis, or may represent factors affecting mRNA specific translation. Under prolonged hypoxic conditions glucose may be consumed to a large extent, which causes a second drop in ATP levels. A combined action of AMPK and mTOR, as well as unknown factors cause a global translational arrest accompanied by sustained specific mRNA translation. (from: Fähling,M. “Surviving hypoxia by modulation of mRNA translation rate.” Journal of Cellular and Molecular Medicine (2009). 13(9A):2770-2779.)

300 MICHAEL FÄHLING – MOLECULAR PHYSIOLOGY

Publications 2006 - 2010 Ufer C, Wang CC, Fähling M, Schiebel H, Thiele BJ, Billett EE, Kuhn HH, Borchert A (2008): Translational regulation of Schildroth J, Rettig-Zimmermann J, Kalk P, Steege A, Fähling glutathione peroxidase 4 expression through guanine-rich M, Sendeski M, Paliege A, Lai EY, Bachmann S, Persson PB, sequence binding factor 1 is essential for embryonic brain Hocher B, Patzak A (2010): Endothelin type A and B recep- development. Genes Dev 22(13):1838-50 tors in the control of afferent and efferent arterioles in mice. Nephrol Dial Transplant accepted Steege A, Fähling M, Paliege A, Bondke A, Kaps C, Kirschner KM, Martinka P, Patzak A, Scholz H, Persson PB, Thiele BJ, Perlewitz A, Nafz B, Skalweit A, Fähling M, Persson PB, Mrowka R (2008): Wilms’ tumor protein WT1(-KTS) modu- Thiele BJ (2010): Aldosterone and vasopressin affect alpha- lates renin gene transcription. Kidney Int 74(4):458-66 and gamma-ENaC mRNA translation. Nucleic Acids Res accepted Mrowka R, Blüthgen N, Fähling M (2008): Seed based systematic discovery of specific transcription factor target genes. Kielbasa SA, Blüthgen N, Fähling M, Mrowka R (2010): FEBS J 275(12):3178-92 Targetfinder.org: A resource for systematic discovery of transcription factor target genes. Nucleic Acids Res 38 Martinka P, Lai EY, Fähling M, Jankowski V, Jankowski J, Suppl:W233-8 Schubert R, Gaestel M, Persson AE, Persson PB, Patzak A (2008): Adenosine increases calcium sensitivity via receptor- Westphal K, Stangl V, Fähling M, Dreger H, Weller A, Bau- independent activation of p38 MAPK/MK2 pathway in iso- mann G, Stangl K, Meiners S (2009): Human-specific Induc- lated mesenteric arteries. Acta Physiol (Oxf.) 193(1):37-46 tion of Glutathione Peroxidase-3 by Proteasome Inhibition in Cardiovascular Cells. Free Radic Biol Med 47(11):1652-60 Patzak A, Steege A, Lai EY, Brinkmann JO, Kupsch E, Spiel- mann N, Gericke A, Skalweit A, Stegbauer J, Persson PB, Eichler SA, Förstera B, Smolinsky B, Jüttner R, Lehmann TN, Seeliger E (2008): Angiotensin II response in afferent arteri- Fähling M, Schwarz G, Legendre P, Meier JC (2009): Splice- oles of mice lacking either the endothelial or neuronal isoform specific roles of glycine receptor a3 in the hippocampus. Eur of nitric oxide synthase. Am J Physiol Regul Integr Comp J Neurosci 30(6):1077-91 Physiol 294(2):R429-37

Lai EY, Fähling M, Ma Z, Källskog Ö, Persson PB, Patzak A, Patzak A, Lai EY, Fähling M, Sendeski M, Martinka P, Persson Persson AE, Hultström M (2009): Norepinephrine increases PB, Persson AE (2007): Adenosine enhances long-term the calcium sensitivity of mouse afferent arteriole, thereby contractile response to angiotensin II in afferent arterioles. Am enhancing angiotensin II-mediated vasoconstriction. Kidney J Physiol Regul Integr Comp Physiol 293(6):R2232-42 Int 76(9):953-9 Mrowka R, Steege A, Kaps C, Herzel H, Thiele BJ, Persson Fähling M, Mrowka R, Steege A, Kirschner KM, Benko E, PB, Blüthgen N (2007): Dissecting the action of an evolution- Förstera B, Persson PB, Thiele BJ, Meier JC, Scholz H ary conserved non-coding region on renin promoter activity. (2009): Translational regulation of the human Achaete-Scute Nucleic Acids Res 35(15):5120-9 Homologue-1 (hASH1) by Fragile X Mental Retardation Protein (FMRP). J Biol Chem 284(7):4255-66 Schmidt I, Fähling M, Nafz B, Skalweit A, Thiele BJ (2007): Induction of translationally controlled tumour protein (TCTP) Lüdemann L, Nafz B, Elsner F, Grosse-Siestrup C, Meissler by transcriptional and posttranscriptional mechanisms. FEBS M, Kaufels N, Rehbein H, Persson PB, Michaely HJ, Lengs- J 274(20):5416-24 feld P, Voth M, Gutberlet M (2009): Absolute quantification of regional renal blood flow in swine by dynamic contrast- Lai EY, Martinka P, Fähling M, Mrowka R, Steege A, Gericke enhanced magnetic resonance imaging using a blood pool A, Sendeski M, Persson PB, Persson AE, Patzak A (2006): contrast agent. Invest Radiol 44(3):125-34 Adenosine Restores Angiotensin II-Induced Contractions by Receptor-Independent Enhancement of Calcium Sensitivity in Renal Arterioles. Circulation Res 99:1117-1124

301 MICHAEL FÄHLING – MOLECULAR PHYSIOLOGY

Fähling M, Mrowka R, Steege A, Nebrich G, Perlewitz A, Theilig F, Debiec H, Nafz B, Ronco P, Nüsing R, Seyberth HW, Persson PB, Thiele BJ (2006): Translational Control of Col- Pavenstädt H, Bouby N, Bachmann S (2006): Renal cortical lagen Prolyl 4-Hydroxylase alpha (I) Gene Expression under regulation of COX-1 and functionally related products in early Hypoxia. J Biol Chem 281(36):26089-10 renovascular hypertension (rat). Am J Physiol Renal Physiol 291(5):F987-94 Andree H, Thiele H, Fähling M, Schmidt I, Thiele BJ (2006): Expression of the human TPT1 gene coding for translation- Reviews & book chapters (2006-2010) ally controlled tumor protein (TCTP) is regulated by CREB transcription factors. Gene 380(2):95-103 Fähling M (2009): Surviving hypoxia by modulation of mRNA translation rate. J Cell Mol Med 13(9A):2770-2779 Fähling M, Mrowka R, Steege A, Martinka P, Persson PB, Thiele BJ (2006): Heterogeneous Nuclear Ribonucleoprotein- Fähling M (2009): Cellular oxygen sensing, signalling and how A2/B1 Modulate Collagen Prolyl 4-Hydroxylase alpha (I) to survive translational arrest in hypoxia. Acta Physiol (Oxf.) mRNA Stability. J Biol Chem 281(14):9279-86 195(2):205-230

General information Third party funding (2006-2010)

Project leader Project title Sponsor Period Thiele BJ. Tubuläre Mechanismen der renalen Deutsche Forschungsgemein- 2005-2010 Nafz B. Volumenregulation schaft (DFG); FoGr 667, TP8, TH 459/5-1 Nafz B. Functional genomics of cardiovascular NGFN 2, 2004-2007 damage in hypertension BMBF KBCV 1.2, 01GS0416 Fähling M. Posttranskriptionelle Kontrolle der Deutsche Forschungsgemein- 2008-2011 Scholz H. Genexpression des bHLH-Transkrip- schaft (DFG) tionsfaktors Mash1 FA 845/2-1

302 SCIENTIFIC COORDINATION

As a Scientific Coordinator of the CCR Dr. rer. nat. Karin Effertz is responsible for the webpage (www.ccr. charite.de), the bi-annual report of the CCR and for general public relations. Moreover she gives strategic and administrative support for the preparation of cooperative grant proposals and for the realisation of joint research projects at the CCR.

Frau Dr. rer. nat. Karin Effertz kümmert sich als Wissenschaftliche Koordinatorin um die Belange der Öffentlich- keitsarbeit, wie die CCR Webseite (www.ccr.charite.de), den CCR Jahresbericht, und die allgemeine Außen- darstellung. Darüber hinaus unterstützt sie strategisch, inhaltlich und administrativ die Erstellung von Verbund- anträgen und die Durchführung von Verbundprojekten am CCR.

303 VASCULAR TRAINING

GefäSStraining: Von der Stange zum molekularen MaSSanzug Nicht jede Art von Sport ist die richtige Antwort auf eine Gefäßerkrankung

Von Ivo Buschmann

„Treiben Sie mehr Sport!“ So richtig dieser ärztliche Rat Pulsatilität bestehen bleibt bzw. erhöht vorliegt, umso auch für Patienten mit Gefäßkrankheiten ist, so kom- besser kann sich ein kollateraler Kreislauf ausbilden. plex und facettenreich stellt er sich im klinischen Alltag Und das ist auch der Grund, warum eine regelmäßige dar. Welchen Sport soll ein Patient mit peripherer arte- Belastung dem Herzen und den Beinen so gut tut rieller Verschlusskrankheit (PAVK) machen? Radfah- und zum Beispiel Hobbysportler statistisch gesehen ren oder lieber laufen? Wie lange muss ein Herzpatient länger leben. Zwar treten auch bei ihnen atheroskle- am Tag laufen? Eher morgens oder besser abends? rotische Veränderungen der Arterien auf, durch deren Was macht der Patient der nicht richtig trainieren biologische Regeneration über eigene biologische kann? Durch die molekulare Grundlagenforschung Bypässe sind sie jedoch im Falle von Gefäßverengun der letzten Jahrzehnte kommt nun langsam Licht in gen geschützt. dieses Dunkel und erstmals zeichnet sich am Horizont eine zunehmend verbesserte Behandlungsform von Hohe Pulsatilität gibt das Signal zur Bildung Gefäßkrankheiten ab: vom unspezifischen Gefäßsport von Kollateralen zum patientenindividuellen, personalisierten Gefäß- Das wichtigste Wachstumssignal für solche Kollate- training. Dabei stehen insbesondere 3 Säulen der ralen ist ein hoher pulsatiler Blutfluss. Demnach kön- Therapie im Vordergrund: nen sich biologische Bypässe (z. B. im Unterschenkel) - die Nutzung der patienteneigenen Regeneration von besser vergrößern, wenn dieses Blutflusssignal auch biologischen Bypässen. in der Peripherie bei ihnen ankommt. Ist also eine - die Kombination von gefäßtherapeutischer Interven- große Beckenarterie verschlossen, ist es sinnvoll, eine tion (Ballondilatation und Stent) mit Gefäßtraining und solche Einengung– entweder mithilfe eines Ballonka - die Personalisierung der Gefäßtherapie (Maßanzug) theters sowie gegebenenfalls auch einer Gefäßstütze statt unspezifischem Training (von der Stange). (Stent) – zunächst wieder zu öffnen, bevor der Patient mit einem strukturierten Gefäßtraining beginnen kann. Regelmäßige Belastung tut den Beinen gut Durch die Erfindung eines „Gefäßtachometers“ an der Biologische Bypässe sind kleine Kollateralkreisläufe, die Charité und im Gefäßzentrum Berlin lassen sich diese sich zu vollwertigen Arterien umbilden können. Fließt kollateralspezifischen Beschleunigungswerte im Blut das Blut in einem Umgehungskreislauf schneller und strom nun mithilfe von Ultraschallverfahren messen und damit mit hoher Pulsatilität, erweitert sich als biologi- visualisieren [1]. Möglicherweise ergeben sich dadurch sche Antwort der Querschnitt dieser Arterie und kom- auch neue Einsichten zu den interessanten Ergebnissen pensiert somit das Blutflussdefizit auf der verschlos der MIMIC1-Studie [2], in der eine optimale Medikation senen Hauptstrecke. Je länger und effizienter diese 1 Mild to Intermittent Claudication

304 VASCULAR TRAINING plus Gefäßtraining gegen eine optimale Medikation plus Gefäßtraining plus eine Angioplastie der Becken- und der Oberschenkeletage verglichen wurden. Wurde auch eine Angioplastie durchgeführt, verbesserten sich sowohl der Knöchel-Arm-Index („ankle brachial index“; ABI) als auch die Gehstrecke signifi- kant. Ferner wissen wir, dass Therapieverfahren additiv wirken können.

Der Schlüssel sind individuell angepasste Therapieschemata Ein unspezifisches Training ist nicht immer die richtige Antwort auf eine Gefäßerkrankung. Beispielsweise ist ein Training mit dem Fahrradergometer bekann termaßen bei Weitem nicht so effizient wie ein struktu- trum Berlin in Kooperation mit der Charité mehrere riertes Gehtraining. Interessanterweise verbessert aber Studien, die diesen neuen individualisierten Gefäßan- ein Arm-ErgometerTraining die periphere arterielle Ver- satz verfolgen. Spannende Zeiten für alle Blutfluss- schlusskrankheit. Dass hierbei die pulsatile Beschleu- Physiologen also: „Go with the flow!“ nigung des Blutstromes eine viel wichtigere Rolle spielt als bisher vermutet [1], eröffnet neue Behandlungspfade Literatur für stenosierende Gefäßkrankheiten. 1 Buschmann I, Pries A, Styp-Rekowska B et al. Pul- Der bereits erwähnte Gefäßtachometer wird es in satile shear and Gja5 modulate arterial identity and Zukunft möglich machen, das Training der Patienten remodeling events during flow-driven arteriogene- (wie bei einem Tachometer im Auto) zu dosieren und sis. Development 2010; 137: 2187–2196 damit zu spezifizieren. Erstmals ergibt sich hiermit 2 Greenhalgh RM, Belch JJ, Brown LC et al. The adju- auch die Möglichkeit, passive Trainingsverfahren wie vant benefit of angioplasty in patients with mild to die externe Gegenpulsation spezifisch anzuwenden: moderate intermittent claudication (MIMIC) mana- Wurde bisher mit zum Teil noch sehr hohen Drü ged by supervised exercise, smoking cessation cken, die bei einigen Patienten zu deutlichen Neben- advice and best medical therapy: results from two wirkungen führten („enhanced counterpulsation“; randomised trials for stenotic femoropopliteal and EECP), blind behandelt, lässt sich jetzt das scho- aortoiliac arterial disease. Eur J Vasc Endovasc Surg nende Verfahren der Herzhose [3] in Kombination mit 2008; 36: 680–688 einer Gefäßtachometer-Darstellung kombinieren und 3 Buschmann EE, Utz W, Pagonas N et al. Improve- spezifizieren. ment of fractional flow reserve and collateral flow by Damit ist es möglich, statt einer Standardtherapie treatment with external counterpulsation (Art.Net.-2 von 6–7 Wochen ein individuell angepasstes The Trial). Eur J Clin Invest 2009; 39: 866–87 5 rapieschema zu starten. Derzeit laufen am Gefäßzen-

305 Research Foci at the CCR

Drug Development

Principle Investigators Heiko Funke-Kaiser, Ulrike Steckelings, Franz Theuring, Thomas Unger, Berthold Hocher Partners Prof. Jürgen Scholze , Charité Campus Mitte, Berlin, Klinik für Innere Medizin/ Kardiologie am Deutschen Herzzentrum Berlin

Drug development is a complex, time- and cost- process to narrow down the hundreds and thousands intensive process involving different disciplines from of hits generated by the HTS/ secondary screening, medicine and natural sciences. Classically, drug devel- but also comprises synthetic chemistry (medicinal opment can be divided into two sequential modules chemistry (MC)) steps. Leads - chemical structures called drug discovery and drug development with a potency in the 1-10 microM range, selectivity (subsequent figure). Discovery comprises all steps and appropriate ADMET parameters - represent the from the therapeutic concept, i.e. from the biological milestone of the hit-to-lead programme. target, to the molecule, whereas development encom- The lead-to-candidate programme, also named passes the operating processes from the molecule to lead optimisation phase, aims to further increase the registered drug. Discovery often starts with target affinity/ potency, specificity/ selectivity and ADMET identification, e.g. the cloning and (patho)physiologi- properties by using e.g. in vivo ADMET analyses. The cal characterisation of a novel gene. A crucial step in in vivo proof-of-concept (PoC) is a crucial milestone early drug discovery is assay development. This can within the lead-to-candidate programme to analyse be accomplished through the generation of stable if a certain compound is effective with respect to a cell lines and a luciferase-based read-out to meas- certain indication in an animal model. So-called devel ure receptor activity. A high-throughput screening opmental candidates have passed the PoC and are (HTS; primary screening) to explore the chemical space further optimised by MC to attain e.g. a potency in the for pharmacological activities represents the next step 1-10 nM range, which is a usual prerequisite for clini which can be performed in cooperation with contract cal candidates for the first trials in humans. research organisations (CROs). So-called confirmed The clinical phase of drug development comprises compounds resulting from the HTS are validated by for defined phases: Phase I studies are performed a secondary screening to yield hits. The hit-to-lead on a small group of healthy volunteers with the aim to programme addresses aspects such as structure- check for drug safety, tolerability, and pharmacokinetic activity-relationships (SAR)/ definition of a pharma- properties. Phase II studies are performed on groups cophore, determination of affinity (KD) and potency of patients with a defined set of indications to test for (IC50), specificity/ selectivity (panel-screens), phar- clinical efficacy, and for dose finding analysis. Often macokinetic (i.e., absorption, distribution, metabolism, such studies will cover several distinct clinical disor- excretion (ADME)) and toxicological (T) parameters. ders to identify the possible therapeutic indications for The hit-to-lead programme can be viewed as a filtering the new compound. Phase III trials are the definitive

306 Research Foci at the CCR double-blind randomised trials, commonly performed CCR´s research teams cover a broad spectrum within as multicentre trials on 1000-3000 patients, aimed at the drug discovery and development process as illus- comparing the new drug with commonly used alterna- trated in the next table: tives. At the end of a “successful” phase III, the drug will be submitted to the relevant regulatory authority target identification Funke-Kaiser/ for licensing. Phase IV studies comprise the obliga- and validation Unger, Kintscher tory postmarketing surveillance designed to detect assay development to Funke-Kaiser/ any long-term adverse effects. More importantly, hit-to-lead-programme Unger, Kintscher these studies are important to identify new potential lead-to-candidate- Steckelings/ programme, e.g. Unger, Hocher. Theuring indications for the drug in a clinical setting in many preclinical in vivo proof- thousands of patients. of-concept/ Clinical studies are a major cornerstone of the drug in vivo evaluation of dif- development agenda and the translational research ferent indications approach at the CCR. In close collaborations with Phase II Hocher clinical departments, in particular the Outpatient Phase III Theuring Department at the Charité Campus Mitte under Phase IV Kintscher/ Unger/ Scholze the guidance of Prof. Jürgen Scholze, and the Department of Internal Medicine/ Cardiology at To give an example, Prof. Dr Thomas Unger and Dr. the German Heart Institute, multiple clinical trials are Ulrike Steckelings are in strategic partnership with the currently conducted in different clinical phases and car- company Vicore Pharma to contribute to the preclinical diovascular indications. In addition, clinical investigators and clinical development of AT2 Receptor Agonists. with a focus on cardiovascular research from various Moreover two drug developing companies were departments at the Charité have their laboratories at founded by members of the CCR as spin offs of the the CCR, and provide an optimal platform for mutual Charité. CCR Pharma was founded by PD Dr. Heiko research interactions. Funke-Kaiser, Prof. Dr. Thomas Unger, Frank Zollmann and Dr. Jan Schefe and is developing prorenin/ ren- I. Drug discovery nin receptor (RER) antagonists as potential future car- target target assay secondary HTS identification validation development screening diovascular drugs, a project funded by the “Go Bio” program of the BMBF. II. Drug discovery & preclinical drug development http://www.bmbf.de/de/6868.php, hit-to-lead-programme lead-to-candidate programme http://www.ccr-pharma.de developmental clinical hits leads candidates candidate The company of Prof. Dr. Franz Theuring is targeting neurodegenerative diseases. http://www.taurx.com III. Clinical drug development For more detailed information on the various projects

phase I phase II phase III approval phase IV please refer to the chapters of the individual research groups.

307 Research Foci at the CCR

Gender in Cardiovascular Research

Principle Investigators Vera Regitz-Zagrosek (Research Focus-, GK-, RG-coordinator), Duska Dragun (GK-, RG-Member), Ulrich Kintscher (GK-, RG-member), Andreas Patzak, Pontus Persson (GK) Thomas Unger/ Ulrike Steckelings/ Heiko Funke-Kaiser (GK-members) Partners national: Gender in Medicine (GIM) Charité, Members of the Graduate Course GK 754 and of the Research Group FOR 1054 international: European curriculum gender in medicine (EUGIM) members (Karin Schenck-Gustafsson, Center on Gender in Medicine at the Karolinska Institute Stockholm, Ineke Klinge, University of Maastricht, Netherlands; Maria Koop, Semmelweiss University Budapest, Hungary; Margarete Hochleitner, Womens Health Center of Insbruck Medical University, Austria; Flavia Franconi, Universita di Sassari, Italy, Toine Lagro Janssen, University of Njimegen, Netherlands); Eugeneheart Gender Task members (Fred Jaisser, Paris, H Jarry, Goettingen, Leon de Windt, Maastricht, Guido Tarone, Turin, Karin Sipido, Leuven), International Society of Gender in Medicine Board members

Gender differences in widespread diseases like car- ing Program GK 754: “Sex and gender specific diovascular disease, stroke, immunological disease mechanisms in myocardial hypertrophy This GK etc. are well known, but the underlying molecular and was particularly successful. It has been funded from cellular mechanisms are still poorly understood. The 2001 – 2011 and about 55 MD, veterinarian and PhD hypothesis of steroid hormones as molecular regu- students will obtain their degrees in this graduate lators of gender specific pathological phenotypes course, most of them already finished with summa is tempting, but in many cases still to be proven. and magna cum laude. Sex differences in myo- Research groups at the CCR, which are interested in cardial hypertrophy A Research Group (DFG, this topic, work together with groups from Charité and FOR 1054) - Forschergruppe – Sex differences in MDC in the Research Focus “Gender in Cardiovascu- myocardial hypertrophy is funded from September lar Disease”. The common aim of the group is to inves- 2008- 2011 with the contribution of the CCR-groups of tigate sex and gender differences in cardiovascular Dragun, Kintscher and Regitz-Zagrosek (coordinator). and cardiometabolic and -renal diseases and to define Animal models are an appropriate tool for analyzing the role of sex hormones. Eleven groups including 4 molecular and cellular mechanisms of cardiovascular from CCR, one from DHZB (Deutsches Herzzentrum and cardiorenal diseases and show often sex specific Berlin), other from Charité and MDC are cooperating differences. Three such models with known gender since 2001 with similar goals in the Research Train- differences are established at the CCR: a mouse

308 Research Foci at the CCR model for pressure induced hypertrophy (transverse Furthermore gender aspects in cardiovascular dis- aortic banding), a mouse model for salt induced eases are investigated in international cooperations hypertrophy (DOCA salt) and a model for physiologi- with the INSERM Institute Toulouse, France and the cal hypertrophy after voluntary exercise.. Forced exer- John Hopkins School of Public Health, Baltimore US. cise has been established as another physiological At the European level gender aspects are analyzed hypertrophy model. In these models, sex differences in Task 4 of the EUGENE project (consortium of 21 and estrogen/androgen mediated signal transduc- partners from research institutes and SMEs from 10 tion are investigated. Moreover, in hypothesis-driven different countries). approaches, the interaction of sex differences and sex An additional goal is the implementation of Gender hormones on cardiomyocyte signalling will be ana- aspects in the medical education all over Europe with lyzed and, in a more systemic approach, influence of the Erasmus Project EUGIM, developing a Bologna sex hormones on gene expression and protein pattern compatible module „Gender Medicine“ with 7 Euro- will be examined. pean partner universities. Karima Schwab and Nicolas Vignon-Zellweger from At the national level Gender aspects will be imple- the Group of F. Theuring work on effects of estrogens mented systematically in the novel medical education and phytoestrogenes on protein expression pattern in at the Charité Universitätsmedizin Berlin („Modellstu- systemic approaches. diengang Medizin“), which will start in October 2010.

309 Research Foci at the CCR

Metabolism

Principle Investigators Ulrich Kintscher, Michael Schupp, Berthold Hocher, Stefan Anker Partners Joachim Spranger (Endocrinology, Charité), Jürgen Scholze (Outpatient Clinic, Charité), Philipp Stawowy (Cardiology, DHZB), Simone Spuler (ECRC, Charité), M. Boschmann (ECRC, Charité)

Deregulation of body weight control occurs in two old age lead to pathophysiologically relevant car- opposite directions: progressive gain of weight which diovascular alterations, namely chronic heart failure, leads to obesity or progressive loss of weight resulting which is clinically relevant for prognosis. Disturbance in cachexia. Between these two extremes an inter- of the endothelial function and the regulation of periph- facial area exists with common molecular mediators eral perfusion result in ischemia and hypoxia of the of pathogenesis. Based on these joint pathologies, peripheral tissue as well as the induction of reactive the present research focus metabolism concentrates oxygen species and inflammation. We have some on molecular mechanisms involved in body weight evidence that all forms of cachexia, independently of regulation in a bidirectional manner. Moreover studies their underlying chronic disease, show neurohumoral of genetic factors and epigenetic DNA modifications activation, which is commonly associated with chronic shed light on their contribution to metabolic control. heart failure. Furthermore, cachectic patients almost with no exception are short of breath, fatigue easily Cachexia (Anker) and very often show edema, all of which are hallmarks An underlying hypothesis of our work is that all forms of chronic heart failure. We have shown that several of cachexia as seen in COPD, cancer, infections and therapies of chronic heart failure display anti-cachectic properties, e.g. ACE inhibitors and beta-blockers. Hence, we are testing whether these cardiovascular therapies are effective in other therapeutical areas than cardiac cachexia. Currently, several projects are focused on cardiovascular drugs and novel compounds on survival, heart function and body composition in a rat model of cancer cachexia.

Obesity (Kintscher) Obesity is a major risk factor for cardiovascular disease (CVD). Our work is focussing on mechanisms involved in obesity-mediated CVD. Hereby we are con- centrating on tissue cross-talk between metabolic tissues such as adipose tissue, skeletal muscle and liver

310 Research Foci at the CCR and CV-organs such as the heart and the vasculature. unfavourable effects on the offspring into this cur- Regulation of tissue crosstalk by endocrine media- rently rapidly growing research field. We were further- tors such as nuclear hormone receptors is one of the more able to demonstrate for the first time that insulin main topics. We follow a strict translational research resistance may also be present in otherwise healthy approach from cell culture to animal to proof-of-con- human newborns already at birth. Our animal stud- cept studies in humans. ies demonstrated that also over-nutrition may exert harmful effects on the offspring in a gender depend- Genetic Mechanisms and Impact of Epigenetic ent manner. Current clinical and preclinical studies Modifications (Hocher) are designed to detect molecular pathways of fetal Fetal programming of cardiovascular disease programming including epigenetic DNA modifications. There is meanwhile clear evidence indicating that early The Research Focus Metabolism is in close collabora- life events play an important role in the pathogene- tion with a series of other clinical and basic research sis of cardiovascular diseases in later life. We have groups. The schematic presentation below shows introduced the concept of maternal genes exerting currently funded collaboration projects at the Charité.

311 Research Foci at the CCR

Vascular Stress

Principle Investigators Axel Pries, Thomas Unger, Kai Kappert, Ivo Buschmann, Harm Peters, Elena Kaschina, Ulrike Steckelings, Heiko Funke-Kaiser, Duska Dragun, Ullrich Kintscher, Andreas Zakrzewicz, Vera Regitz-Zagrosek Partner Ferdinand le Noble (MDC Berlin-Buch), Gilbert Schönfelder (Charité), Michael Bader (MDC, Berlin-Buch), Norbert Hübner (MDC, Berlin-Buch), Heinz-Peter Schultheiss (Charité)

For a major group of vascular diseases (arterioscle- lasts) and the extracellular matrix. Within the process rosis, myocardial infarction, stroke, hypertension, of vascular remodelling number and form of all com- tumours) and clinically relevant adaptations (wound ponents of the vascular wall are continuously varied healing, development, aging, exercise) vascular reac- resulting in a structural reorganisation of the vascular tions to hemodynamic, inflammatory, oxidative or met- wall. It responds to physiologic stimuli to guarantee abolic stimuli play an important role. In this context all its stability under normal conditions. In the case of functional and pathologic scenarios are seen as vas- cardiovascular disease pathological stimuli cause cular stressors, as long as they increase vascular load. maladaptation of the vascular wall. Frontiers between functionally adequate response and Schematic representation of the CCR focus maladaptation are fluent and are determined by the “Vascular Stress”. Tissue remodelling of the type of stimuli, genetic predisposition and other pro- vascular wall is based on the capability of the tective or harmful factors. artery to alter in size and shape, and occurs in response to numerous stress factors. This CCR focus addresses aspects of pathological inward remodelling in terms of atherosclerosis, restenosis and transplant vasculopathy, as well as outward remodelling regarding arteriogenesis and aneurysm formation. Integrating both clinicians and basic scientists we are focussing at unravelling the biological and molecular basis of these physiological and pathological remodelling processes, which should pave the way for developing therapeutic and preventive strategies for the benefit of patients suffering or not yet suffering from vascular disease. The vascular wall is a flexible and integrating organ The involved principle investigators at the CCR are consisting of cellular components (endothelial cells, listed along the sphere. smooth muscle cells, cells of the adventitia and fibrob-

312 Research Foci at the CCR

an interdisciplinary team of researchers (internists, cardiologists, nephrologists, cell biologists, biochem- ists, physiologists and pharmacologists) is studying vascular changes in different organs (heart, brain, kidney, aorta and extremities). Diseases like stroke, myocardial infarction, cardiac hypertrophy, aortic aneurisms, cardiac, retinal or nephrotic changes due to adipositas or diabetes, chonic kidney disease, glomerulosclerosis, transplant vasculopathy but also beneficial adaptations like arteriogenesis after stenosis are explored. On a molecular level, contributions of the renin-angiotensin-aldosteron-system. NO- cGMP-, TGF beta-, endothelin- estrogen- and PPAR Within the Research Focus „Vascular Stress“ at ( Peroxisom-Proliferator-Activated Receptor)- and HIF the CCR clinicians and basic scientists are working (hypoxia-inducible factors)-mediated signal transduc- together to understand physiological and pathophysi- tion pathways are investigated. ological remodelling of the vascular wall. One of the major claims of the research focus “Vas- A number of current research projects address topics cular Stress” is the advancement of internal and in the field of „Vascular Stress”. Some of the house external co-operations in order to increase the quality intern co-operations were initiated within the Euro- of scientific output. This is why the research focus pean early stage research training programm “Car- concentrates on projects which need the expertise of diovasc”. Within the CCR “Vascular Stress” focus two or more groups.

313 DFG-RESEARCH GROUP MYOCARDIAL HYPERTROPHY

Research Group Myocardial Hypertrophy (DFG, FOR 1054)

Coordinator Vera Regitz-Zagrosek (CCR, GIM) Members Duska Dragun (CCR), Ulrich Kintscher (CCR), Michael Bader (MDC), Otmar Huber (CBF), Joachim Klose (CVK), Wolf-Hagen Schunck (MDC), Dominik Müller (MDC), Shokoufeh Mahmoodzadeh (CCR)

The main aim of the research group “Myocardial lipid turnover is investigated. Several collaborations Hypertrophy” (FOR 1054) is the analysis of gender with clinical projects exist; in this area we mainly focus differences in the adaptation of the heart to mechani- on gender differences in myocardial reaction to over- cal stress. Women develop a more advantageous and load due to aortic valve disease. physiological adaptation of the myocardium to stress compared to men. This includes the synergistic activation of numerous cell-based protective pathways at the molecular and cellular level, which are most often linked to the activation of estrogen receptors or estrogens themselves. This influences mitochondria and the myocardial energy metabolism and limits apoptosis and fibrosis. Testosterone, on the other hand, appears to favour fibrosis and a more pronounced and unfavourable myocardial proliferation. Our research group performs a series of mechanical analysis using cell cultures and animal models. This includes models of physiological and pathological myocardial stress, e.g. strength training and hypertension. The role of estrogen and androgen receptors is selectively analyzed. Moreover, the effect of sex hormones on the

314 Graduate Courses

Graduate Courses

1. MARIE CURIE early stage research training programme CARDIOVASC

Project Coordinator Patricia Ruiz Noppinger Project Manager Dian Michel Steering Committee Eva Becher, Ivo Buschmann, Karin Effertz, Harm Peters, Catarina Curato, EST fellow, George Kararigas, EST fellow Funding European Commission, FP6, Human Resources and Mobility Activity, MEST-CT-2005-020268

CARDIOVASC is the acronym for cellular and molec- human disease educated by specialists of the different ular mechanisms of cardiovascular diseases repre- disciplines with the possibility of interacting across the senting a Marie Curie Early Stage Research Training traditional boundaries. Programme for PhD students that started in January The training programme is based upon three columns 2006. The aim of CARDIOVASC is to provide training • Personalized training guaranteed by a direct supervi- for young researchers in the specialized and highly sor and a mentor, as well as the steering committee competitive area of cardiovascular diseases (CVD) • Instruments for structured training, such as semi- under the tutorial of highly experienced scientists at nars, lectures, workshops, meetings and e-coop- the Center for Cardiovascular Research (CCR). eration Research projects apply systematic and hypothesis- • Regular and interactive quality control of the fellows driven approaches in molecular, cellular and physi and the supervisors ological biology focusing on heart, kidney and the vascular wall. The scientific programme of CAR- DIOVASC focuses on the following questions: (i) Gender-specific aspects of cardiac function and dysfunction (ii) Molecular mechanisms of endothelial dysfunction in cardiovascular disease, and (iii) New strategies in the prevention and treatment of myocardial infarction. A structured training programme with special emphasis on the integration of basic and clinical research is provided to bundle and integrate these questions. Our objective is to train a new generation of scientists and Fellows and supervisors of the Marie Curie EST programme clinicians with the capacity of working at the interface CARDIOVASC at the Harnack-Haus, Berlin-Dahlem at the Day of genetics, developmental biology, physiology and of-Science Meeting.

315 Graduate Courses

2. Graduate course 754-III: Gender specific mechanisms in myocardial hypertrophy

Project leader Vera Regitz-Zagrosek Coworkers Duska Dragun, Heiko Funke-Kaiser, Roland Hetzer (DHZB) Ulrich Kintscher, Joachim Klose, C.Knossalla (DHZB), Jun Li, Shokufeh Mahmoodzadeh, Ingo Morano (MDC Berlin), Andreas Patzak, Martin Paul, Pontus Persson, Patricia Ruiz Noppinger, Gilbert Schönfelder, Carola Schubert, Ulrike Steckelings, Thomas Unger Funding German Research Foundation

GK754 III analyses sex specific mechanisms in myo- renin-angiotensin system, the cardial hypertrophy and heart failure. It is based endothelin system, lipid and glu- on the established projects and collaborations in cose metabolism, expression of GK754 II and I. The promotion period was extended contractile proteins and calcium to 2010 because of a very good evaluation in 2006. handling, neo-aniogenesis, and An impaired cardiac function heart failure (HF) is one aging processes. The scientific of the most common health problems in our present aim of this program (GK) is to analyse the mechanisms society. It develops often on the basis of myocardial of sex differences in the pathophysiology of myocardial hypertrophy and manifests differently in women and hypertrophy. This focus is the base for a very close men. HF appears in women at a higher age compared cooperation of all working groups and finds outstand- to men and shows specific features in its epidemiol- ing partners in the new focus of gender research in ogy, clinical manifestation, pathophysiological mecha- Berlin and in our international cooperation partners. All nisms, genetic bases and molecular phenotypes in the scientific approaches are well organized in an interdis- myocardium. At the comparable levels of mechanical ciplinary manner. Aims and systematic teaching con- stress and neurohumoral stimulation women develop tents are related to biology, genomics, endocrinology, less cardiac hypertrophy and less systolic dysfunction cardiology, nephrology, pharmacology, physiology compared to men. However, severe cardiac hypertro- and veterinary medicine. The involved medical stu- phy has more adverse consequences for women. The dents learn principles and methods of basic research, molecular basis of gender differences may be related while the scientists get across the basics of medical to the effect of sex hormones on the myocardium or to science. The table shows all projects with project lead- X-chromosomal gene products. Experimental results ers and involved students from 2006 to 2010. point to an impact of sexual hormones on NO-synthesis, on the gluco- and mineralocorticoidsystem, the

316 Graduate Courses

TP Project leader Project title PhD students

Models of hypertrophy

1 Patzak / Influence of sex on mechanisms in the development of cardiac M. Sendeski, P. Persson hypertrophy and fibrosis in NO-deficient mice O. Zavaritskaya 2 C. Schubert / Sex differences in aortic stenosis; effects of sex hormones and D. Fliegner, U. Kintscher modulation of estrogen receptors C. Westphal 3 Dragun Sex specific differences in the development of left ventricular A. Karatas, hypertrophy in hypertensive kidney disease in mice D. Gürgen

Hormonal effects and hormone receptors; sex dependent effects in the myocardium

4a V. Regitz-Zagrosek Effects of estrogen and testosterone on myocardial hypertrophy M. Schanz, and matrix synthesis H. Hampl, A. Penkalla 4b S.Mahmoodzadeh Organ specific transcriptional and translational regulation of E. Dworatzek, estrogen receptor ER α und ER β J. Leber 5 I. Morano Analysis of sex specific changes in cardiac function by tran- L. Schulz, scriptional regulation of hALC-1 J. Lossie 6 G. Schönfelder / Sex specific differences in the regulation of VEGF receptor 1 N. Wendt, M. Paul (FLT1) J. Langen, W. Weiss, M. Martinelli 7a T. Unger / AT2-receptor and ATBP in cardiac hypertrophy: role of sex J. Reinemund, H. Funke-Kaiser / differences K. Ströder U. Steckelings 7b T. Unger / Role of estrogen in modulation of myocardial collagen M. Brinckmann, J. Li degradation S. Slavic 8 U. Kintscher Influence of adipose tissue on myocardial hypertrophy: rel- M. Clemenz, evance of Peroxisome Proliferator-Activated Receptors and C. Böhm estrogen receptors

Clinics Dependence of age and sex on gene and protein expression profile

9 P. Ruiz Age and sex specific profiles of gene expression in myocardial J. Isensee, hypertrophy and relevant mechanisms A. Queiros 10 J. Klose Proteins of cardiac muscles in dependence on sex and age M. Diedrich, S. Forler 11 R. Hetzer / Aortic stenosis H. Pham, C. Knosalla C. Eschen, G. Petrov

317 Graduate Courses

3. International PHD Programme on arterial hypertension and vascular biology

The PhD Programme on Arterial Hypertension and of human disease processes (e.g. stroke, myocardial Vascular Biology has been established in coopera- infarction, hypertension, vasculopathies).The candi- tion with the Università degli Studi di Padova (Italy) dates fulfil the requirements for admittance to the the- and the Medical University of Gdansk (Poland) for sis programme (Dr. rer. Medic) at the Medical Faculty graduates wishing to attend a three-year doctoral of the Charite-Universtitätsmedizin Berlin (or adequate course, and started in the academic year 2005/06. in Italy: laurea/laurea specialistica-magistrale). Each Research emphasises are placed on collaborative and PhD-student is admitted to the course at only one of interdisciplinary research that ranges from molecular the partner universities (the Home University) but will biology and cell culture studies on the pathophysiol- attend the other universities involved in the coopera- ogy of hypertension and hypertensive end-organ dis- tion agreement for at least 9 months. ease, obesity and diabetes to defined animal models

318 Graduate Courses

4. Initiative „Young CCR“

Coordinators Daniela Fliegner, Pawel Namsolleck

Since 2003 we offered a weekly seminar for PhD fel- selves. The agenda of the seminar comprises not only lows, which was part of the structured research train- the discussion of scientific projects and the exchange ing programmes “Gender-specific mechanisms in of knowledge, experience and methods, but also the myocardial hypertrophy” (graduate course 754, DFG) training of communication skills. Right in the begin- and “CARDIOVASC” (Marie Curie Early Stage Training ning, it is planned to give an introduction to presenta- program, EU). As this seminar was very successful tion techniques. In the course of the seminar, feedback and well accepted, we wanted to keep the tradition on the quality and the content of each presentation beyond the end of the program funding periods, albeit will be elaborated by the audience. In addition, invited in a slightly modified way. As an initiative of the junior speakers will give introductions to statistical analysis, post-doctoral fellows Daniela Fliegner (Prof. Regitz- literature databases and soft skills. Zagrosek´s lab) and Pawel Namsollek (Prof. Unger`s The seminar is especially addressed to diploma lab) the main objective here is to have a platform for students, Master students and Ph.D. fellows of the scientific exchange of young researchers. To lower CCR. The registration is optional, but once registered the barrier for free communication among them, the attendance is obligatory. This seminar should provide management and moderation of the seminar will be a platform for networking and the initiation of future performed by D. Fliegner and P. Namsolleck them- collaborations.

319 RAS Symposium

Spotlight on the Renin Angiotensin System (RAS) – the period from 1950 to 2010 Symposium at the Tagungszentrum Katholische Akademie in Berlin, 29th October 2010

The spotlight symposium about tant regulator of cardiovascular function. The RAS the RAS honoured one of the out- has been successfully targeted by various pharma- standing investigators in the field of cological approaches to reduce cardiovascular risk the RAS during the last decades, such as ACE-inhibitors, AT1 receptor blockers, and Professor Thomas Unger. On the direct renin inhibitors. Recently, this work has been occasion of Professor Unger´s 60th expanded and new pharmacological RAS interven- birthday, we have invited national tions are now being tested inpreclinical development and international experts to discuss including AT2 receptor agonists and rennin receptor past, recent, and future develop- blockers. In summary, the RAS has been extensively ments in the RAS and hypertension studied in preclinical and clinical settings, and more research. The topics cover a wide range of RAS phar- importantly, drugs acting on the RAS have markedly macology from preclinical studies to clinical trials. The improved the outcome of millions of patients suffer- renin-angiotensin-system (RAS) is one of the major ing from cardiovascular disease such as myocardial peptide hormone systems in our body and an impor- infarction or heart failure.

Programme

Chair: Axel Pries, Berlin 12.00 – 12.30 The renin-angiotensin-system – what didn´t work? Detlev Ganten, Berlin Martin Paul, Maastricht, The Netherlands

9.00 – 09.30 Introduction 12.30 – 14.00 Lunch Break

Ulrich Kintscher, Berlin Chair: Monika Stoll, Münster Thomas Unger, Berlin Jürgen Scholze, Berlin

9.30 – 10.00 Exploiting the potential of the RAS in therapeutic 14.00 – 14.30 Renin – A new therapeutic target for approaches Bernward Schölkens, Frankfurt cardiac hypertrophy Massimo Volpe, Rome, Italy 10.00 – 10.30 Arterial hypertension and RAS-blockade Björn Dahlöf, Göteborg, Sweden 14.30 – 15.00 Renal mechanisms of hypertension Rainer Rettig, Greifswald 10.30 – 11.00 Coffee Break 15.00 – 15.30 Hypertension therapy in 2010 Chair: Reinhold Kreutz, Berlin Peter Sever, London, UK Gilbert Schönfelder, Berlin 15.30 – 16.00 New avenues from Berlin targeting the RAS 11.00 – 11.30 Blockade of the RAS in Cardiology Heiko Funke-Kaiser, Berlin Georg Nickenig, Bonn Ulrike Steckelings, Berlin

11.30 – 12.00 Angiotensin receptors – an experimental view 16.00 End of symposium Lutz Hein, Freiburg

320 FCVB Congress

FCVB Congress in Berlin, from 16th to 19th July

From July 16th to 19th the first meeting for basic and abroad. The programme included translational research ‚Frontiers in Cardio-Vascular keynote speeches and sympo- Biology’ (FCVB 2010 – conference president Axel R sia by outstanding experts from Pries) took place at the Institute of Anatomy of the Europe and beyond, excellent Charité and the CCR. abstracts of researchers from the FCVB has been brought whole world in the cardiovascular into being by the ‚Euro- field as well as very stimulating pean Society for Cardiol- contributions by young colleagues. ogy’ (ESC) und its‚ Coun- The congress was aimed to be the starting step for the cil for Basic Cardiovas- development of the most important congress in the field cular Research’ (CBCS) of basic cardiovascular research in Europe. According which is constituted by Working Groups of the ESC to the members of the CBCS, this goal was met and and scientific Societies in Europe in the cardiovascular the next FCVB will be held in 2012 at Imperial College, field (EVBO, ISHR, ESM, EAS, ECCR, AECVP). The London, UK under the chairmanship of Sian Harding. ESC is one of the greatest medical specialist societies A lot of researchers from the CCR participated (e.g. in Europe and organizes annually the world biggest Kintscher, Le Noble, Mammoozadeh, Regitz-Zagro- congress in the field of cardiovascular research and has zek, Unger) contributed and helped to strengthen the founded the ‚Council for Basic Cardiovascular Science’ cardiovascular ‘community’ in Berlin. A special ‘break- including relevant external specialist societies with the fast’ symposium organized at the CCR by Ulrich Kint- aim to support basic and translational research. scher got a lot of positive comments.

The FCVB was designed as a central forum for exchange with a special emphasis on young researchers. The congress came out with a great success including an unexpected high participation (700 registrations, 550 free scientific abstracts) by scientists of Europe and

321 BI Lectures

BI Lectures

Since 2004, a lecture serial – called BI-Lectures– takes place at the CCR with speakers representing international experts in the field of cardiovascular medicine. BI-Lectures are supported by Boehringer Ingelheim Pharma GmbH & Co. KG.

Programme 2010

19.05.2010 Sebastian Frische 06.10.2010 Rukshana Shroff Regulation of renal acid excretion: how does the Which cellular mechanisms produce progressive kidney know what to do? vascular damage in chronic kidney Anatomisk Institut, Åarhus Universitet, disease? Århus, Denmark Institute of Child Health, Great Ormond Street Hospital, London, UK 09.06.2010 Stephan Herzig Transcriptional checkpoints in metabolism 13.10.2010 Kurt A. Jäger and Molecularmetabolic disorders Modern vascular therapy - Intervention or no inter- German Cancer Research Center (DKFZ), Meta- vention? Novel insights into training physiology bolic Control, Heidelberg, Germany Universitätsspital Basel, Universität Basel, Basel, Switzerland 19.07.2010 Dennis Bruemmer (Monday) Telomeres and telomerase in vascular biology 20.10.2010 Thomas Thum Cardiovascular Research Center, University of Development of microRNA-based therapeutic Kentucky, Lexington, USA strategies in cardiovascular disease Institute for Molecular and Translational Therapeutic 15.09.2010 Florian Lang Strategies, MHH, Hannover, Germany Physiology and pathophysiology of SGK1 Physiology, University of Tübingen, Tübingen, 03.11.2010 Maria Grazia Modena Germany Metabolic syndrome and gender Dipartimento ad Attività Integrata di Emergenza, 22.09.2010 Daniel Henrion Urgenza, Università degli Studi di Flow induced arterial remodeling Modena e Reggio Emilia, Modena, Italy Laboratoire de Biologie Neurovasculaire Intégrée, UMR CNRS 6214 - INSERM 771 10.11.2010 Michael Ristow Faculté de Médecine, Université d’Angers, Angers, Promoting metabolic health and life expectancy by France increasing oxidative stress Institute of Nutrition, Friedrich-Schiller-University 29.09.2010 Detlef Böckenhauer Jena, Jena, Germany Go EAST: Walking the frontier of neuro- and nephrology 24.11.2010 Denise Hilfiker-Kleiner Pediatric Nephrology, Great Ormond Street STAT-3 in cardiovascular pathology Hospital, London, UK Molecular Cardiology, Dept. of Cardiology and Angiology, MHH, Hannover, Germany

322 Perspectives

Drug Development in Industry and Academia: the Emergence of TMAT

Garret A. FitzGerald MD, Institute for Translational Medicine and Therapeutics, University of Pennsylvania, Philadelphia, Pa.

Background than is the cautious perestroika evident at the margins of It is now widely appreciated that the current model the boundaries of intellectual property (IP (2). of drug discovery and development is unsustainable. Perhaps the most striking hint of the future is in the “not While the number of new drugs approved by the FDA for profit” sector where initiatives to find new treatment has remained quite constant since 1950, the estimates classes for previously neglected diseases is burgeon- of the cost of having a drug approved have increased in ing. Here, driven by altruism, the perception that there a log linear fashion (1). While some argue that the “low is little money to be made (perhaps not so true as one hanging fruit” has been snatched, that this is a cyclical witnesses the rising middle classes of the semi tropi- phenomenon or that the segmentation of previous clas- cal BRICs) and charismatic leadership, central funds sifications of disease account for this failure, it is worth have been accumulated. These are used to incent par- pondering two realities. The first is that the revolution in ticipants from both academia and industry along the approaches to target discovery has been remarkably chain from target identification to large scale trials to successful; we have more rationally selected targets, collapse their IP, adhere to timelines and hand off their often with attendant chemistry, than ever. The second results to the next group along the chain. is that the ballooning cost of bringing a drug to market Although conversion of this model to the for profit sec- is mostly accounted for by the ballooning cost of failure tor will not be instant, a truly adventurous approach to of the current model which is built into these estimates. venture capital, revisions of our outmoded and unreal- One might conclude that what we face is (i) a failure in istic concepts of IP, revived capacity to participate in drug development rather than discovery and (ii) that the academic sector and perhaps above all, a sense the current cost estimates largely do not apply ab initio of crisis within the industry, might afford the necessary to a novel approach to drug development and should ingredients(3). not deter such innovation. So, how are we to move to a modular approach to drug discovery and development (4), harnessing the The Emerging Paradigm of Drug Development heterogeneous talents necessary across sectors and We are presently witnessing the progressive disintegra- geographical boundaries? tion of the vertically integrated pharmaceutical industry. Roughly half of the companies that produced drugs The Deficit of Human Capital that were approved since 1950 are no longer with us (1) Thirty years ago, the leading exemplars of the dis- and the industry’s response to production failure of its cipline of clinical pharmacology housed within one research enterprise has been an ever more aggressive organizational structure experts in cellular biology, strategy of merger. The temporary little fix of firing people model systems, mechanistic studies of physiology, is no more likely to rescue the productivity of the industry disease and drug action in humans, pharmacokinet-

323 Perspectives ics and modeling, chemistry, statistics and information the discipline in the heart of medicine and indicates the handling, toxicology, poisoning and clinical trials (5). focus on the development of novel therapeutics (5). Initia- These heterogeneous talents were blended to foster tives on both sides of the Atlantic, by the Wellcome Trust both training and the execution of what we now call and the NIH, are congruent with this term. interdisciplinary science, specifically “T1” translational The development of TMAT will require the development research. Graduates of such programs populated of new interdisciplinary training structures. We favor a chairs of medicine and pharmacology, rose to leader- Masters as a general introductory degree with subse- ship positions in industry and currently lead the exem- quent areas of focus within the spectrum of TMAT. The plar UK organizations in comparative effectiveness. flexibility of a Masters to combine with lead degrees Gradually, particularly as Departments of Medicine in medicine, science, dentistry, veterinary medicine shifted to a cost center model, they lost interest in this etc makes it attractive. However, training support and discipline so that it atrophied to an essentially industry grant cycles will need to lengthen to support the par- based activity, often pursuing rather unimaginative and ticular challenges of this type of science. routine studies of drug availability and disposition. We have paid a price. Firstly, the most critical and expen- Revision of Intellectual Property sive mistakes in drug development occur in phase II – There was a time when people patented transistors and we either failure to see an impact on surrogates of efficacy, have reached that level of absurdity in drug development. ignoring surrogates of risk or errors in dose selection Access to compound libraries, transparency and access for phase III. Secondly, physicians in the US, just like to “failed” compounds for their potential repurposing, their patients, get most of their information from direct expansion of the precompetitive space to foster systems to consumer advertising. Thirdly, the regulatory bodies pharmacology and physiology – in cells, model systems have minimal intramural capacity to address the mecha- and humans - all offer the promise of releasing value pres- nistic plausibility of apparent signals of risk detected by ently locked unproductively in industry. Similarly, academic epidemiological methods and finally expertise in basic systems, patenting everything in the slim hope of finding a or human pharmacology is unapparent in approaches Gatorade, need to get real. The unmeasured cost of these to comparative effectiveness in the US. IP stockades is the inertia that they bring to much potential We must rebrand and update a translational discipline collaborative interaction across the sectors (3). that integrates expertise in pharmacology with contem- A more contentious proposal relates to the foundation of porary science. The term “Clinical Pharmacology” has IP, configured as it is on composition of matter. Most of lost its luster and in contemporary perception only covers this matter never makes it to become an approved drug some of what we need (6). To attract the best and bright- and, if it does, many people of diverse skill sets have est we need a new brand, backed by funders, academics to work effectively together. It is easy, within a vertically and industry, one that transcends geography. Potential integrated company, to restrict IP reward to composi- trainees must perceive the field to be “hot”, where the tion of matter as the fiscal benefits that derive from this excitement, the money and the jobs will be. We sug- can be distributed internally in a way that recognizes the gested the term “Translational Medicine and Therapeutics efforts of all the actors. However, it is a different game in (TMAT)” as it captures the fashion for translation, places academia. How do we motivate the cardinal efforts of

324 Perspectives experts in TMAT if the fiscal reward is restricted to the expire, while the value of drugs in development in the chemist? Perhaps we should consider new models of industry’s collective pipeline that could be launched IP as well as just expanding the domains where it does during that period are worth just $30bn. That is the not apply. A postponed and more evenly distributed challenge to the present model of drug discovery and system of reward might be necessary within the emerg- development. ing context of trans-national and trans-sectoral modular It seems likely that a potentially abrupt change beck- drug discovery and development. ons, such as we have previously observed in the air- line, print media, computing and music industries. It The Informational Infrastructure is unlikely that a spot of glasnost and a touch of per- What are the foundations on which this new model estroika will rescue current mores. might be built? Here the challenge is to establish structures that permit the secure and compliant sharing References: of highly heterogeneous data, including clinical data, 1. Munos B. Lessons from 60 years of pharmaceu- across not just geographies but also sectors. In some tical innovation. Nat Rev Drug Discov 2009; 8: ways, this is the greatest challenge of all. Partial solu- 959–968. tions are emerging however and here, the global 2. FitzGerald G.A. Perestroika in Pharma; Evolution Pharma industry can contribute greatly to establish- or Revolution in Drug Development? Mt. Sinai ing best practice. Med. J. 2010; 77: 327-332. 3. FitzGerald GA. Drugs, industry and academia. Adventurous Venture Science 2008; 320: 1563. Finally, what provides the incentive? The Venture Capi- 4. Skarke C, FitzGerald GA. Training translators for tal (VC) industry has always been rather conventional smart drug discovery. Sci Transl Med 2010; 2: in this space and the recent recession has only served 26 cm12. to heighten its conservative instincts, much to the det- 5. FitzGerald GA. Opinion: anticipating change in riment of the Biotech industry in particular. Will there drug development: the emerging era of transla- be a VC investment analogous to the pot of money tional medicine and therapeutics. Nat Rev Drug contributed to by Governments, industries and indi- Discov 2005; 4: 815–818. viduals to incent novel behavior in the altruistic sector? 6. FitzGerald GA. Clinical pharmacology or trans- Traditionally, the experts in drug development that sit lational medicine and therapeutics: reinvent or in the Venture industry are alumni of the unsustainable rebrand and expand? Clin Pharmacol Ther 2007; current model. When will the VC industry break free 81: 19–20. of convention and provoke a disruptive approach to drug development? Acknowledgements: Dr. FitzGerald is the McNeill Professor of Translational Conclusion Medicine and Therapeutics. Roughly $100bn (€79bn, £67bn) in sales from medi- Email: [email protected] cines will be lost in the next five years as IP protections

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