International Centre for Genetic Engineering and Biotechnology THE ARTURO FALASCHI CONFERENCE SERIES ON MOLECULAR MEDICINE 2012
Frontiers in Cardiac and Vascular Regeneration
May 30 - June 2 Trieste, Italy Programme and Book of Abstracts
TOPICS: Vessel and heart formation during development - regulation of angiogenesis in adult tissues - stem and iPS cell differentiation towards vascular cells and cardiomyocytes - stimulation of cardiac and vessel regeneration in adults - bridging the gap between vascular and cardiac research.
Under the patronage of:
comune di trieste FACOLTA’ DI MEDICINA E CHIRURGIA
Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Welcome to the Trieste Conference on Frontiers in Cardiac and Vascular Regeneration, the first meeting of the ICGEB “Arturo Falaschi Conference Series on Molecular Medicine”.
This meeting inaugurates a new Conference Series, dedicated to the memory of Arturo Falaschi, founder and first Director-General of the ICGEB, who passed away on 1 June 2010. It is thanks to his very passion for research and his discerning managerial talent that ICGEB exists today. Beyond the mission of education that he established, lies the concept that you cannot perform good training if you do not undertake excellent research.
Twenty five years on, the ICGEB laboratories in Trieste, Cape Town and New Delhi, together with 40 Affiliated Centres worldwide, continue to provide intense training activities in the field of molecular research in medical and agricultural biotechnologies. Over 20 meetings and workshops are organised annually, counting over 1000 participants per year, and hosting international scientists of the highest standing in their fields. This conference alone brings together 30 such international speakers – world leaders in international experimental and clinical cardiology – for a rich programme juxtaposing problems related to cardiac cells with those concerning the formation of blood vessels.
I am equally proud to welcome the many scientists, students and medical practitioners amongst our participants, from across Italy, Europe, Asia, the Middle East, the USA and Canada, together with those from among the ICGEB Member States, which today number over 60.
I see this as a great opportunity for growth, for development, for innovation; a way to tackle the most serious, the most chronic of life threatening diseases, that causes more deaths, disability and economic strain than all other diseases.
The fact that this meeting takes place in Trieste is an additional source of satisfaction for me personally. It is my great pleasure to welcome you to this beautiful city and to wish you both professional and personal gratification over the next four days.
Mauro Giacca
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Table of Contents
General Information 5
Scientific Programme 6
Poster Session I 10
Poster Session II 12
Speakers’ Abstracts 14
Participants’ Abstracts 44
Abstract Author Index 96
List of Participants 102
About ICGEB 120
Sponsors and Acknowledgements 122
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General Information
Meeting Location FVG Region “Azienda Ospedaliero-Universitaria” Stazione Marittima Congress Centre for a total of 14 credits. These may be assigned to Molo Bersaglieri 3, 34100 Trieste, Italy registered Italian professionals, who are medical Tel. +39-040-304888 and biological staff of the local hospitals, who attend the entire meeting, and upon completion Hospitality arrangements of the relevant questionnaire (distributed directly Lunches and coffee breaks will be provided for at the Registration Desk). the duration of the meeting at the Stazione Marittima Congress Centre. A cocktail will be External services located close to the held on Friday 1st June, starting 7.30 PM at the Congress Centre Savoia Excelsior Starhotel, directly opposite the Bank in the vicinity of the Congress Centre Congress Centre. Thank you for wearing your UniCredit, Piazza della Borsa 9 badge at all times as this is required for admission. Tel: +39-040-6769311 Mon-Fri: 8.20-16.00 Internet Facilities Exchange Bureau Free internet access is available at the Congress Cambiavalute Giubbani, Piazza Ponterosso 3 Centre during the meeting. Tel: +39-040-368080 Mon: 10.00-12.00; Tue-Fri: 8.00 – 17.00 Poster Sessions Poster boards are situated in the centre Parking Facilities of the main hall of the Stazione Marittima Parking outside the Congress Centre Congress Centre. Posters should be displayed on “Park Si-Silos”, multi-storey (unattended) car park Wednesday, 30th May upon registration and by the railway and bus stations in Piazza Libertà 9. exhibited for the duration of the meeting. Specific Tel: +39-040-44924; open 24h/day at a cost of Poster Sessions will be held in the afternoon on Euro 16 per day. Thursday, 31st May and Friday, 1st June between Special rates for nearby parking facilities are 2.30 and 4.00 PM. A jury composed of selected offered by the local hotels. Please, check with your scientists will award a prize for the best poster for hotel reception. each of the two sessions. Taxi service Oral Presentations Tel: +39-040-307730 A technician is present in the conference room Pharmacies in the vicinity of the Congress Centre to provide any assistance that may be required. Farmacia al Lloyd, Via dell’Orologio 6 Speakers are requested to load and check their Tel: +39-040-300605 presentations by contacting the technician Mon-Fri: 8.30–13.00 and 16.00-19.30 present in the conference room, either at the time Farmacia Campi Elisi S.N.C., Via Combi 7 of Registration or during the coffee-breaks. Tel: +39-040 302800; Mon-Sun: 8.30-20.30 Oral presentations, selected from the abstracts Travel Agency submitted, have been allocated a 15 min time Cividin Viaggi, Via Imbriani 11 Tel: +39-040-3789111; slot in the programme (12 min presentation plus Mon-Fri: 9.00–13.00 and 15.30–19.15; Sat: 9.30-12.30 discussion). Presenters are kindly asked to strictly adhere to this timing. Tourist Information Agenzia Turismo FVG, Via dell’Orologio 1 Continuing Education in Medicine - ECM Tel: +39-040-3478312; E-mail: [email protected]; The conference has been accredited under the Mon–Fri: 9.00–19.00; Sat–Sun 9.00–13.00
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Programme
Wednesday, 30 May 2012 11.00 - 14.30 Registration
14.30 Welcome addresses by local Authorities Welcome by Francisco E. Baralle, Director-General, ICGEB 14.40 Mauro Giacca, ICGEB Trieste and Silvano Riva, A. Buzzati Traverso Foundation, Pavia Remembrance of Arturo Falaschi
Mechanisms of vascular development Charirman: Roger Hajjar, New York
Elisabetta Dejana, 15.00 Pathological development of brain microvasculature Milan Type-A plexins as versatile mediators of pro-angiogenic as 15.30 Gera Neufeld, Haifa well as of anti-angiogenic signal transduction Federico Bussolino, 16.00 Nervous vascular parallels: axon guidance and beyond Turin
Coffee Break
17.00 Eli Keshet, Jerusalem The vascular stem cell niche 17.30 Kari Alitalo, Helsinki Therapeutic potential of vascular endothelial growth factors VEGF overexpression in skeletal muscle induces vascular enlargement and intussusceptive angiogenesis through 18.00 Andrea Banfi, Basel reciprocal activation of Notch-1 in contiguous endothelial cells Neuropilin-1 expressing monocytes (NEMs), a novel Alessandro Carrer, 18.15 population of bone marrow-derived myeloid cells promoting Trieste vessel maturation
6 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy Thursday, 31 May 2012 Regulation of angiogenesis in normal and pathologic conditions Chairman: Elisabetta Dejana, Milan
Molecular regulation endothelial sprouting and angiogenic 9.00 Ralf Adams, Münster blood vessel growth Lena Claesson-Welsh, 9.30 How does VEGF regulate vascular permeability? Uppsala Peter Carmeliet, 10.00 Targeting endothelial metabolism: principles and strategies Leuven Sandro De Falco, Inhibition of pathological angiogenesis targeting vessels and 10.30 Naples inflammation
Coffee Break Gene therapy for cardiovascular disorders Chairman: Mauro Giacca, Trieste
SPECIAL TALK SPONSORED BY THE ADRIANO BUZZATI- Kenneth I. Berns, 11.30 TRAVERSO FOUNDATION Gainesville Evolution of AAV as a vector for gene therapy Roger Hajjar, 12.00 Gene therapy for the treatment of heart failure New York Andrew H. Baker, 12.30 Modifying vein grafts by gene therapy Glasgow
Lunch
14:40 - 16:00 Poster session I and coffee break
Gene therapy for cardiovascular disorders - continues Chairman: Andrew Baker, Glasgow Searching for secreted factors and microRNAs promoting 16.00 Mauro Giacca, Trieste myocardial protection and regeneration Christian Kupatt, AAV2.9 Thymosin beta 4 gene therapy: requirements for 16.30 Munich therapeutic neovascularization Controlled VEGF expression in a cardiac patch improves 17.00 Anna Marsano, Basel vascularization and cardiac function in a myocardial infarction model Tbx1 is required in brain endothelial cells to establish vascular 17.15 Sara Cioffi, Naples patterning
7 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy Friday, 1 June 2012 From cardiac development to cardiac regeneration Chairman: Stefan Engelhardt, Munich José Luis de la Pompa, 9.00 Notch signaling in cardiac development and disease Madrid Juan Carlos Izpisua Insights into the mechanisms underlying regeneration of the 9.30 Belmonte, San Diego vertebrate heart Jürgen Hescheler, Pluripotent stem cells: basic research for later clinical application 10.00 Cologne in the cardiovascular system 10.30 Piero Anversa, Boston Cardiac stem cell and human heart failure
Coffee Break Driving heart progenitor cell fate and regeneration in vivo via 11.30 Kenneth Chien, Boston chemically modified mRNA Maurizio Capogrossi, 12.00 Human cardiac stromal cell and heart regeneration Rome Functional screening identifies microRNAs inducing cardiac 12.30 Ana Eulalio, Trieste regeneration Roberto Gimmelli, Characterization of the c-kit receptor function in cardiac 12.45 Rome regeneration by transgenic mouse models Lunch 14:40 - 16:00 Poster session II and coffee break Cardiac regeneration Chairman: Mark Mercola, San Diego 16.00 Robert Passier, Leiden Human pluripotent stem cells and cardiac subtype specification Karl-Ludwig Laugwitz, 16.30 Human iPSC models of cardiac disease Munich Melusin and IQGAP1 are key players in ERK1/2 signaling and 17.00 Guido Tarone, Turin compensatory cardiac hypertrophy response to pressure overload Gianfranco Matrone, Role of Cyclin-Dependent Kinase-9 in cardiac injury-recovery in 17.30 Edinburgh the zebrafish embryo The G-protein coupled receptor APJ keeps the balance between Gabriella Minchiotti, 17.45 proliferation and cardiovascular differentiation of multipotent Naples mesodermal progenitors in embryonic stem cells Aftab Ahmad Chatta, Inhibition of CSC specific miRNAs results in differentiation of CSC 18.00 Lahore into cardiomyocytes Giuseppe Ristagno, Cardiac effect of modified embryonic mesoangioblasts expressing 18.15 Milan PlGF, MMP9 or both after myocardial infarction in the mouse
19.30 Cocktail and cultural event at the Savoia Excelsior Palace Starhotel
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Saturday, 2 June 2012
Cardiac regeneration continues Chairman: Thierry Pedrazzini, Lausanne
Mark Mercola, San Development of a pharmacological approach to myocardial 9.00 Diego regeneration Fabio Recchia, 9.30 Cardiac imaging in regenerative medicine Philadelphia and Pisa Ivonne Schulman, Cell therapy for chronic ischemic heart disease: from concept 10.00 Miami to clinic
Coffee Break
Cardiac regulatory RNAs Chairman: Jürgen Hescheler, Cologne
Thierry Pedrazzini, 11.00 Novel non-coding RNAs in cardiac regeneration Lausanne Stefan Engelhardt, 11.30 MicroRNA control of tissue fibrosis Munich Gianluigi Condorelli, 12.00 Translational control of myocardial function Rome and San Diego
12.30 Closing remarks and Meeting closure
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POSTER SESSION I Thursday, 31 May - 14.30-16.00
1. A proteomic evaluation of smooth muscle cells in thoracic aortic aneurisms Ceyda Acilan, Ph.D., Betul Baykal, Muge Serhatlı, MSc., Omer Kacar, MSc., Zelal Adiguzel, MSc., Altug Tuncer, M.D., Kemal Baysal, M.D., Ph.D., Ahmet T. Baykal, Ph.D.
2. Nkx2-3 homeodomain transcription factor as a master determinant for the vascular patterning of spleen and intestinal lymphoid tissues Péter Balogh, Zoltán Kellermayer, Árpád Lábadi, Tamás Czömpöly, Hans-Henning Arnold
3. VEGF over-expression in skeletal muscle induces vascular enlargement and intussusceptive angiogenesis through reciprocal activation of Notch-1 in contiguous endothelial cells Roberto Gianni-Barrera, Marianna Trani, Silvia Reginato, Ruslan Hlushchuk, Michael Heberer, Valentin Djonov and Andrea Banfi
4. Neuropilin-1 expressing monocytes (NEMs), a novel population of bone marrow-derived myeloid cells promoting vessel maturation Carrer A., Moimas S., Zacchigna S., Maione F., Mano M., Zentilin L., Kazemi, M., Bussolino F., Giraudo E., Giacca M.
5. Transcriptional analysis reveals new candidate modulators of IFN-alpha response in endothelial cells Francesco Ciccarese, Angela Grassi, Barbara Di Camillo, Gianna Toffolo and Stefano Indraccolo
6. Tbx1 is required in brain endothelial cells to establish vascular patterning Sara Cioffi, Marchesa Bilio, Stefania Martucciello, Elizabeth Illingworth
7. Nitric oxide modulation of endothelial progenitor cells: increased contribution to skeletal muscle remodeling? Valentina Conti, Emanuele Azzoni, Silvia Brunelli
8. Therapeutic potential of Askina Gel in regenerative medicine Delle Monache Simona, Giuliani Antonio, Evtoski Zoran, Sanità Patrizia, Amicucci Gianfranco and Angelucci Adriano
9. Engraftment responses of human bone marrow stromal cells are enhanced by Notch ligands in vitro Giulia Detela, Owen Bain, Prof. Anthony Mathur, Dr. Ivan Wall.
10. Soluble VEGFR-1 interaction with α5β1 integrin triggers an endothelial cell dynamic and angiogenic phenotype Angela Orecchia, Amel Mettouchi, Paolo Uva, Glenn Simon, Diego Arcelli, Simona Avitabile, Gianluca Ragone, Guerrino Meneguzzi, Karl Pfenninger, Giovanna Zambruno and Cristina Maria Failla
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11. Effect of sterilization by gamma-irradiation on biocompatibility of starch-based polymers and composites Abdel Wahab El Naggar, Magdy M Senna, Soheir Korraa and Sanaa Refaay
12. Controlled VEGF expression ensures safe angiogenesis and functional improvement in a model of myocardial infarction Melly L, Marsano A, Helmrich U, Heberer M, Eckstein F, Carrel T, Cook S, Giraud MN, Tevaearai H, Banfi A
13. VEGF and TNF up-regulate, NSAID down-regulate SOX18 protein level in HUVEC Milivojevic Milena, Petrovic Isidora, Nikcevic Gordana, Zaric Jelena, Ruegg Curzio, Stevanovic Milena
14. The role fo transcription factors ZBP-89, SP3, NF-Y and EGR1 in the regulation of the SOX18 promoter activity Petrovic Isidora, Kovacevic-Grujicic Natasa, Milivojevic Milena, Stevanovic Milena
15. Isolation and propagation of mesenchymal stem cells from human cord tissue and blood in Iraq Farooq I Mohammad, Majeed Irsheed Sabah, Ali Hayder, Shaimaa Yusif Abdualfatah, Ban Sabah and Baraa Abdualhadi
16. Cell-free cryopreserved arterial allografts from multiorgan donors: a new strategy to fabricate artificial blood vessels suited for peripheral vascular surgery F. Papadopulos, B. Weber, M. Buzzi, M. Gargiulo, G. Pasquinelli
17. Defining the role of Neuropilin 1 in the cardiac neural crest Alice Plein, Dr. Alex Fantin, Christiana Ruhrberg
18. Human umbilical cord blood progenitors coordinate a regenerative angiogenic microenvironment in ischemic tissues David M. Putman, Kevin Y. Liu, Heather C. Broughton, Gillian I. Bell, David A. Hess
19. Gene expression profile of the early chick hemangioblast Ana Teresa Tavares, Vera Teixeira, Antonio Duarte
20. Dose- and time-dependent angiogenesis by controlled delivery of matrix-bound growth factors Sacchi Veronica, Martino Mikael, Morton Tatjana, Hofmann Anna, Groppa Elena, Gianni-Barrera Roberto, Hubbell Jeffrey, Redl Heinz and Banfi Andrea
21. Ultrastructural characteristics of myogenic cells in failing myocardium of a patient on chemotherapy Ivan Zahradník, Jozef Bartunek, Marc Vanderheyden, Marta Novotová.
22. Post-translational modification by acetylation regulates VEGFR2 activity Annalisa Zecchin, Lucia Pattarini, Maria Ines Gutierrez, Miguel Mano, Mike P. Myers, S. Pantano and Mauro Giacca
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POSTER SESSION II Friday, 1 June - 14.30-16.00
23. ACE polymorphism and obesity: cardiovascular risk factors among Sudanese Fatima E. Abukunna, Nagwa M. Mohamed, Dina A. Hassan, Hussein A. M, Abdulhadi N. H
24. Potential treatment modality OF ischemic myocardial rat heart with mesenchymal stem cell transplantation Esin Akbay, Handan Sevim, Özer Aylin Gürpinar, Serdar Günaydin, Mehmet Ali Onur
25. Pharmacological attenuation of Cardiac Stem Cell senescence in vitro increases their reparative ability in vivo Elisa Avolio, Angela Caragnano, Carlo Vascotto, Giuseppe Gianfranceschi, Ugolino Livi, Rajesh Katare, Paolo Madeddu, Daniela Cesselli, Carlo Alberto Beltrami and Antonio Paolo Beltrami
26. Engraftment responses of human bone marrow stromal cells and unselected mononuclear cells are dependent on the physical extracellular matrix but not the chemotactic factor SDF-1 Owen Bain, Giulia Detela,Christopher Mason, Anthony Mathur, Ivan Wall
27. iPS technology as a tool to investigate atrial fibrillation Benzoni P., Bisleri G., De Luca A., Crescini E., Barbuti A., Baruscotti M., Muneretto C., Richaud Y., Raya A., and Dell’Era P.
28. Gap junctional coupling with cardiomyocytes is necessary but not sufficient for cardiomyogenic differentiation of cocultured human mesenchymal stem cells Arti A. Ramkisoensing, Daniël A. Pijnappels, Jim Swildens, Marie José Goumans, Willem E. Fibbe, Martin J. Schalij, Douwe E. Atsma, Antoine A.F. de Vries
29. Effects of SOX2 overexpression on relative cell cycle distribution and cell migration of NT2/D1 cell clones Danijela Drakulic, Aleksandar Krstic, Andrijana Klajn, Milena Stevanovic
30. Effects of SOX2 over-expression on pluripotency, proliferation and differentiation of embryonal carcinoma stem cells NT2/D1 Milena Stevanovic, Danijela Drakulic, Aleksandar Krstic and Andrijana Klajn
31. Open chromatin conformation at Notch1 target genes is essential to sustain neonatal cardiomyocyte proliferation Giulia Felician, Chiara Collesi, Marina Lusic, Matteo Dal Ferro, Lorena Zentilin and Mauro Giacca
32. The role of Ccbe1, a novel gene coding for an EGF-like domain protein, in the process of organogenesis of the heart João Furtado, Margaret Bento, and José A. Belo
33. Analysis of molecular profiling data to understand the whole-genome effects of HGF stimulus in the heart Stefano Gatti, Christian Leo, Simona Gallo, Valentina Sala, Enrico Bucci, Alice Poli, Daniela Cantarella, Enzo Medico, Tiziana Crepaldi
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34. Characterization of the c-Kit receptor function in cardiac regeneration by transgenic mouse models Gimmelli R., Di Siena S., Barbagallo F., Campolo F., Dolci S., Nori S., Isidori A., Lenzi A., Naro F., Pellegrini M.
35. AAV2.9 Thymosin beta 4 gene therapy: requirements fo therapeutic neovascularization R. Hinkel, T. Trenkwalder, F. Gesenhues, A. Pfosser, G. Stachel, F. Götz, C. Lebherz, I. Bock-Marquette, E. Hannappel, C. Kupatt
36. Expression profile of striated muscle signaling protein Ankrd2 in neonatal rat cardiomoyocytes and its putative role in heart function and development Snezana Kojic, Valentina Martinelli, Jovana Jasnic-Savovic, Ljiljana Rakicevic, Aleksandra Nestorovic, Dragica Radojkovic and Georgine Faulkner
37. Hyperpolarized 13C-pyruvate to study carbohydrate metabolism in beating heart by magnetic resonance imaging V. Lionetti, G. D. Aquaro, F. Frijia, L. Menchetti, F. Santarelli, V. Positano, S. L. Romano, G. Giovannetti, L. Landini, M. Lombardi, F.A. Recchia
38. Identification of novel genes regulating AAV transduction of cardiomyocytes and other post-mitotic cells by high-throughput, genome-wide siRNA screening Miguel Mano, Jasmina Lovric, Rudy Ippodrino, Lorena Zentilin and Mauro Giacca
39. Functional characterization of stem cell-derived cardiomoyocytes Irene C. Marcu, Pernilla Hoffmann, Marisa Jaconi, Nina D. Ullrich
40. Carbon nanotubes as a synthetic substrate for in vitro physiological heart tissue engineering Valentina Martinelli, Giada Cellot, Francesca Maria Toma, Carlin S. Long, John H. Caldwell, Lorena Zentilin, Mauro Giacca, Antonio Turco, Maurizio Prato, Laura Ballerini, and Luisa Mestroni
41. Role of Cyclin-Dependent Kinase 9 in cardiac injury-recovery in the zebrafish embryo G. Matrone, K.S. Wilson, J.J. Mullins, C.S. Tucker & M.A. Denvir
42. Ultrastructural characteristics of myogenic cells in hypertrophying myocardium of rodents Marta Novotová and Ivan Zahradník
43. Nuclear architecture dynamics during human myoblast in vitro differentiation analyzed by 2D and 3D FISH Natalia Rozwadowska, Tomasz Kolanowski, Piotr Pawlak, Marcin Siatkowski, Ewa Wiland, Marta Olszewska, Maciej Kurpisz
44. Identification of novel cardioprotective factors by in vivo gene selection using AAV vectors Giulia Ruozi, Serena Zacchigna, Matteo Dal Ferro, Francesca Bortolotti, Antonella Falcione, Lorena Zentilin and Mauro Giacca
45. Induction of cardiomyogenic markers in murine and human adult stem cells isolated from different sources upon electric stimulation Zamperone A., Pietronave S., Pavesi A., Oltolina F., Consolo F., Novelli E., Diena M., Fiore G.B., Soncini M., Radaelli A. and Prat M.
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Speakers’ Abstracts (in order of presentation)
Note: The Abstracts published in this meeting book should be treated as personal communications and be cited only with the consent of the author
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Pathological development of brain microvasculature Dejana E. IFOM-IEO-Campus and Milan University, Milan, Italy Brain microvasculature constitute a highly specialized and selective vascular barrier between blood and the central nervous system, called blood brain barrier (BBB). In these vessels endothelial cells present a highly developed system of tight junctions (TJs), absence of fenestration and low pinocytotic activity. Cells of the brain parenchyma, the astrocytes, contribute to the BBB-coverage with their foot processes, which constitute about the eighty percent of the basal aspect of the vessels. As a consequence, circulating solutes do not readily enter the brain parenchyma unless through specific endothelial “transporters”. Thus, BBB also limits the passage of anti-cancer drugs from the blood to the brain. Therefore, it would be therapeutically useful to develop systems to modulate BBB permeability. To this aim it is important to define the molecular mechanisms that regulate the establishment and maintenance of BBB properties. Data from our laboratory suggest a key role of the Wnt/β-catenin signaling pathways in the induction, regulation and maintenance of the BBB characteristics during embryonic and post-natal development. In endothelial cells, Wnt signaling induces barrier differentiation by increasing the stabilization and the transcriptional activity of β-catenin. On the contrary, inactivation of β-catenin causes significant downregulation of junctional proteins, and consequent BBB breakdown. Besides β-catenin, other three proteins, CCM1, CCM2 and CCM3, expressed by brain endothelial cells, are emerging as key modulators of the organization and function of the BBB. Indeed, mutations occurring in any of the genes encoding these proteins, leads to Cerebral Cavernous Malformation (CCM), a pathology characterized by brain vascular malformations. The endothelium in the lesions presents very few tight junctions and gaps are observed between endothelial cells. In addition, the vascular basal lamina is disorganized and the astrocytes do not take contact with the endothelial wall. The structural alterations of the BBB observed in CCM lesions are associated with severe clinical manifestations, such as cerebral haemorrhages and stroke. Additional data point to a possible link between the β-catenin and TGF beta pathways and the functions of CCM proteins in the regulation of BBB stability. These data open new therapeutic opportunities for this so far incurable disease
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Type-A plexins as versatile mediators of pro-angiogenic as well as of anti-angiogenic signal transduction Adi Sabag, Tanya Smolkin, Ofra Kessler and Gera Neufeld Cancer research and Vascular Biology Center, Faculty of Medicine, Technion, Israel Institute of Technology, Haifa, Israel. The seven members of the class-3 semaphorin subfamily are the only secreted factors of their class. In addition to their role as axon guidance factors have been found to function as inhibitors of angiogenesis and tumor progression. To transduce their inhibitory signals, most class-3 semaphorins, with the exception of sema3E, bind to one of the two neuropilin receptors. The neuropilins in turn cannot transduce semaphorin signals on their own and associate with one of the five plexin receptors capable of associating with neuropilins, and these serve as the signal transducing elements of the semaphorin receptor complex. We have recently observed that class-3 semaphorin receptor complexes contain more than one plexin. Thus, to transduce signals of sema3A in endothelial cells and glioblastoma cells three receptors, neuropilin-1, plexin-A1 and plexin-A4 are absolutely required and the lack of even one of them inhibits completely signal transduction. Likewise, we found that in these same cells signal transduction of sema3B requires the presence of plexin-A2 as well as plexin-A4 in addition to a neuropilin, and that none of the other plexins can substitutes for either of these plexins. These experiments demonstrate that receptor complexes for different semaphorins can contain shared components like the plexin-A4 receptor as well as unique components which determine specificity like plexin-A2. Furthermore both plexins and neuropilins can associate with other receptors that convey completely different signals and modulate their activities. For example, neuropilin-1 as well as plexin-A4 can associate with the VEGF receptor VEGFR-2 and potentiate VEGF165 induced signal transduction. Our experiments suggest that the characterization of receptor complexes for both pro- and anti-angiogenic factors is of crucial importance for the understanding of the mechanisms that fine-tune angiogenesis.
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Nervous vascular parallels: axon guidance and beyond Marco Arese, Enrico Giraudo, Guido Serini, Federico Bussolino Department of Oncological Sciences. University of Torino The vascular and nervous systems are organized with well defined and accurate networks, which represent the anatomical structure enabling their functions. In recent years, it has been clearly demonstrated that these two systems share in common several mechanisms and specificities. For instance, the networking properties of the nervous and vascular systems are governed by common cues that in the brain regulate axon connections and in the vasculature remodel the primitive plexus towards the vascular tree. As paradigmatic example of axon guidance molecule, we discuss the role of semaphorins in physiological and pathological angiogenesis. Finally, we discuss the presence in blood vessels of neurexin and neuroligin, two proteins that finely modulate synaptic activity in the brain. This observation is suggestive of an intriguing new class of molecular and functional parallels between neurons and vascular cells.
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The vascular stem cell niche Tamar Licht, Myriam Grunewald and Eli Keshet Dept. of Dev. Biol. & Cancer Res., The Hebrew University-Hadassah Med. Sch., Jerusalem 91120, Israel It is thought that the organ vasculature in a number of adult organs is an integral component of the respective stem cell niche. However, this proposition is mostly based on the intimate proximity of tissue stem cells to blood vessels and evidence that nearby vessels actually govern SC function is rudimentary. To address this issue, we use conditional VEGF-based vascular manipulations for expanding or, conversely, reducing the vasculature in selected adult organs. Neuronal stem cells (NSCs) residing in the hippocampus can promote adult neurogenesis associated with enhanced cognitive function. Previous studies by us and others have shown that hippocampus-specific induction VEGF augments adult neurogenesis and improves learning and memory. Via switching-off transgenic VEGF after it has induced durable vessels at the site where NSCs reside, we show that mere expansion of the vasculature is sufficient to maintain a 4-fold elevated neurogenic rate. Intriguingly, the sustained increase in neurogenesis rate does not come on the expanse of accelerated depletion of the exhaustible reservoir of hippocampal NSCs overriding the natural, rapid age-dependent decay of hippocampal neurogenesis. The hematopoietic stem cell (HSC) niche is thought to be composed of both osteoblasts and endothelial cells but the relative contribution of each is unknown. We show that switching-on VEGF in adult mice is sufficient for retaining mobilized BM HSCs in the liver and spleen and for generating a vessel-expanded niche conducive for full blown extra-medullary hematopoiesis associated with overall increase in HSC number. These studies thus provide a further support for a vascular stem cell niche.
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Therapeutic Potential of Vascular Endothelial Growth Factors Kari Alitalo and collaborators Molecular/Cancer Biology Program and Finnish Institute for Molecular Medicine My laboratory studies vascular growth factors to facilitate therapeutics development for cardiovascular diseases and cancer. – Because of the importance of the growth of new blood vessels, or angiogenesis, in tumor progression, the first anti-angiogenic agents have been approved for clinical use. However, most patients are either refractory or eventually acquire resistance to anti-angiogenic therapeutics. A combination of angiogenesis and lymphangiogenesis inhibitors based on based on solid knowledge of the major interacting angiogenesis signaling pathways could be used to significantly advance the efficacy of tumor therapy. – The idea of proangiogenic therapy is to grow new functional blood vessels and thus restore blood flow to ischemic tissue. Several attempts have been made to stimulate angiogenesis and arteriogenesis in tissue ischemia, with limited success. VEGF-B, a coronary vascular growth factor, has recently stimulated renewed interest in such therapy in cardiac ischemia. – The growth of lymphatic vessels, lymphangiogenesis, is actively involved in a number of pathological processes including tissue inflammation and tumor dissemination but is insufficient in patients suffering from lymphedema, a debilitating condition characterized by chronic tissue edema and impaired immunity. Lymphangiogenic growth factors, such as VEGF-C provide possibilities to treat lymphedema. – Thus, there is considerable potential for the development of therapeutics based the biological functions of vascular endothelial growth factors. The angiopoietin (Ang) - Tie signalling system has important functions in cardiovascular development and regulation of vessel stability. Ang1 is dispensable in quiescent vessels but modulates the vascular response after injury, while Ang2 functions as a context-dependent antagonist of Ang1. Inhibition of Ang2 attenuates tumor angiogenesis and improves the anti-angiogenic effect of VEGF inhibition. Recent results suggest that Ang1 and Ang2 inhibitors have applications also in cardiovascular medicine. Bry M et al., Vascular endothelial growth factor-B acts as a coronary growth factor in transgenic rats without inducing angiogenesis, vascular leak, or inflammation.Circulation 122:1725-33, 2010. Norrmén C et al., Biological basis of therapeutic lymphangiogenesis. Circulation 123: 1335-1351, 2011. Saharinen P, Alitalo K. The yin, the yang, and the angiopoietin-1. J Clin. Invest. 121: 2157- 2159, 2011.
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Molecular regulation endothelial sprouting and angiogenic blood vessel growth Ralf H. Adams1 and colleagues 1Max Planck Institute for Molecular Biomedicine, Department Tissue Morphogenesis, and University of Muenster, Faculty of Medicine, Muenster, Germany Angiogenesis is the main process mediating the expansion of the blood vessel network during development, tissue regeneration or in pathological conditions such as cancer. The formation of new endothelial sprouts, a key step in the angiogenic growth program, involves the selection of endothelial tip cells, which lack a lumen, are highly motile, extend numerous filopodia, and lead new sprouts. Angiogenic sprouting is induced by tissue–derived, pro–angiogenic signals such as vascular endothelial growth factor (VEGF), which activates and triggers signaling by cognate receptor tyrosine kinases in the endothelium. However, this response is strongly modulated by intrinsic signaling interactions between endothelial cells (ECs). For example, expression of the ligand Delta-like 4 (Dll4) in tip cells activates Notch receptors in adjacent (stalk) ECs and is thought to downregulate VEGF receptor expression in these cells. Thus, the tip cell phenotype is suppressed in stalk cells and a balance between sprouting and the necessary preservation of existing endothelial tubes is established. Our work is providing further insight into the regulation of sprouting angiogenesis. The Notch ligand Jagged1 is a potent pro-angiogenic regulator with the opposite role as Dll4. In contrast to current models, we found that Notch controls VEGFR2 only moderately whereas VEGFR3 is strongly regulated. Moreover, blocking of Notch enables angiogenic growth even in mutant animals lacking endothelial VEGFR2 expression. We also found that endothelial sprouting and proliferation extension depend on VEGF receptor endocytosis. Ephrin-B2, a ligand for Eph family receptor tyrosine kinases, is required for endothelial cell motility, VEGF receptor endocytosis and the activation of downstream signal transduction cascades. More recently, we have identified Disabled 2, a clathrin-associated sorting protein, and the cell polarity protein PAR-3 as novel interaction partners of ephrin-B2 and VEGF receptors. These results establish that regional VEGF receptor endocytosis, which is controlled by a complex containing Dab2, PAR-3 and ephrin-B2, play a key role in the spatial organization of angiogenic growth. Acknowledgement: This study has been supported by the Max Planck Society, the University of Muenster and the German Research Foundation.
20 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
How does VEGF regulate vascular permeability? Lena Claesson-Welsh Uppsala University Regulation of vascular endothelial growth factor (VEGF)-induced permeability is critical in physiological and pathological processes. Cardiovascular diseases as well as cancer growth and metastatic spread are progagated by excess vascular permeability. VEGF was originally identiifed as vascular permeability factor (VPF), but the molecular regulation downstream of VEGF has not been clarified We have identified a VEGF/VEGFR2-induced signaling chain that regulates vascular permeability. Autophosphorylation at Y951 in the kinase insert of VEGFR2 presents a binding site for the Src Homology 2-domain of T cell specific adaptor (TSAd), which in turn regulates VEGF-induced activation of the c-Src tyrosine kinase and vascular permeability. c-Src was activated in vivo and in vitro in a VEGF/TSAd-dependent manner, through increased phosphorylation at pY418 and reduced phosphorylation at pY527 in c-Src. Tsad silencing blocked VEGF-induced c-Src activation but did not affect other VEGF-induced pathways involving phospholipase Cgamma, extracellular regulated kinase and endothelial nitric oxide. VEGF-induced rearrangement of vascular endothelial (VE)-cadherin positive junctions in endothelial cells isolated from mouse lungs, or in mouse cremaster vessels, was dependent on TSAd expression and TSAd-mediated translocation of active c-Src to junctions. In accordance, TSAd formed a complex with vascular endothelial (VE)-cadherin, VEGFR2 and c-Src at endothelial junctions. Vessels in tsad-/- mice showed undisturbed flow and pressure but failed to respond to VEGF with extravasation of Evans blue, dextran and microspheres in the skin and the trachea. Moreover, macrophage inflammatory protein-2, but not VEGF, induced extravasation of inflammatory cells in the tsad-/- cremaster muscle. We conclude that TSAd is required for VEGF-induced c-Src-mediated regulation of endothelial cell junctions and for vascular permeability.
21 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Targeting endothelial metabolism: principles and strategies Peter Carmeliet Center for Transgene Technology & Gene Therapy, Flanders Interuniversity Institute for Biotechnology, University of Leuven Angiogenesis, the growth of new blood vessels, plays a crucial role in numerous diseases, including cancer. Anti-angiogenesis therapies have been developed to deprive the tumor of nutrients. Clinically approved anti-angiogenic drugs offered prolonged survival to numerous cancer patients. However, the success of anti-angiogenic VEGF-targeted therapy is limited in certain cases by intrinsic refractoriness and acquired resistance. New strategies are needed to block tumor angiogenesis via alternative mechanisms. We are therefore exploring whether targeting endothelial metabolism can be a possible alternative therapeutic strategy for anti-angiogenic therapy.
22 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Inhibition of pathological angiogenesis targeting vessels and inflammation. Laura Tudisco1, Ivana Apicella1, Menotti Ruvo2, Yoshio Hirano3, Jayakrishna Ambati3, Sandro De Falco1 1 Angiogenesis Lab, Institute of Genetics and Biophysics and 2 Institute of Biostructures and Bioimaging, CNR, Napoli Italy In the last years it has been definitively demonstrated that the pro-angiogenic members of vascular endothelial growth factor family, VEGF-A, VEGF-B and PlGF, which accomplish their action interacting with the two receptors VEGFR-1 (recognized by all three growth factors) and VEGFR-2 (specifically recognized by VEGF-A), play a central role in the modulation of pathological angiogenesis. These molecular players have become the preferential targets for anti-angiogenic therapy. Indeed, the first anti-angiogenic drugs approved for cancer and age-related macular degeneration treatments, have as target VEGF-A (Avastin and Lucentis) or the VEGF receptors (Sunitinib or Sorafenib, that are multi-target tyrosine kinase inhibitors). Currently a monoclonal antibody anti-PlGF is in phase 2 of clinical trials for tumor treatment. Recently, a great attention has been devoted on PlGF/VEGFR-1 axis because it is important not only for the stimulation of endothelial cells but mainly for the modulation of inflammatory response associated to the pathological angiogenesis, and for vessel stabilization. Indeed, it has been reported that overexpression of PlGF in tumor cells induced an impressive recruitment of inflammatory cells (F4/80 positive cells) and increased significantly the number of vessels surrounded by smooth muscle cells. It is important also consider that due to the strict biochemical and functional relationship between VEGFs and related receptors it appear evident how the ability to interfere with more than one of these factors may represent an advantage in term of therapeutic outcome. Indeed the upregulation of PlGF in tumors represents one of tumor escape strategy consequent to VEGF inhibition. We have recently reported the identification of a new synthetic tetrameric tripeptide named 4.23.5 that specifically binds VEGFR-1 preventing its activation by VEGF-A, VEGF-B and PlGF, at low micromolar range. Data on its anti-angiogenic properties in syngenic and xenograft cancer models as well as in choroid neovascularization model will be presented.
23 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Evolution of AAV as a Vector for Gene Therapy Kenneth I. Berns University of Florida Genetics Institute, Gainesville, FL, USA Adeno-associated virus (AAV) is one of the most frequently used vectors for gene therapy. A successful vector must fulfill several criteria: it must not be toxic; it must be able to infect the target tissue; it must be able to be expressed in the nucleus; it must sccessfully contend with the host immune system; and, in most instances, expression should be long lasting. AAV vectors have been developed which meet all these criteria. A primary advantage is that AAV has not been implicated as the cause of any disease. Another potential advantage is that the only viral DNA that AAV vectors contain is the terminal repeat sequence of 145 bases at each end. The remainder of the vector genome contains the inserted transgene and associated regulatory sequences. The successful development of AAV as a vector has required detailed knowledge of the basic biology of AAV and its application. Among the issues which had to be resolved are differential targeting by different serotypes, differential interactions with the host immune system of different serotypes, and detailed knowledge of the 3 dimensional structure of the viral capsid and its various biological properties. Finally, knowledge of the factors affecting intracellular trafficking of the viral particle has proven to be of great importance. As a consequence of the basic knowledge which has been obtained, it has been possible to develop vectors which have been clinically efficacious in the treatment of one form of congenital blindness and hemophilia B.
24 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Gene Therapy for the Treatment of Heart Failure Roger J. Hajjar Cardiovascular Research Center, Mount Sinai School of Medicine, New York, NY, USA Congestive heart failure remains a progressive disease with a desperate need for innovative therapies to reverse the course of ventricular dysfunction. Recent advances in understanding the molecular basis of myocardial dysfunction, together with the evolution of increasingly efficient gene transfer technology have placed heart failure within reach of gene-based therapies. One of the key abnormalities in both human and experimental HF is a defect in sarcoplasmic reticulum (SR) function, which is responsible for abnormal intracellular Ca2+ handling. Deficient SR Ca2+ uptake during relaxation has been identified in failing hearts from both humans and animal models and has been associated with a decrease in the activity of the SR Ca2+-ATPase (SERCA2a). Over the last ten years we have undertaken a program of targeting important calcium cycling proteins in experimental models of heart by somatic gene transfer. This has led to the completion of a first-in-man phase 1 clinical trial of gene therapy for heart failure using adeno-associated vector (AAV) type 1 carrying SERCA2a. In this Phase I trial, there was evidence of clinically meaningful improvements in functional status and/or cardiac function which were observed in the majority of patients at various time points. The safety profile of AAV gene therapy along with the positive biological signals obtained from this phase 1 trial has led to the initiation and recent completion of a phase 2 trial of AAV1.SERCA2a in NYHA class III/IV patients. In the phase 2 trial, gene transfer of SERCA2a was found to be safe and associated with benefit in clinical outcomes, symptoms, functional status, NT-proBNP and cardiac structure.Furthermore, the recent successful and safe completion of the CUPID trial along with the start of more recent phase 1 trials usher a new era for gene therapy for the treatment of heart failure.
25 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Modifying Vein Grafts by Gene Therapy Andrew H. Baker Institute of Cardiovascular and Medical Sciences University of Glasgow Patency rates for coronary artery bypass grafting (CABG) procedures using autologous saphenous vein (SV) remain poor. The vast majority of failures are due to neointima formation and superimposed atherosclerosis. There remains a requirement to develop a novel therapy through which to improve patency rates. Since many of the molecular and cellular mechanisms that lead to neointima formation have been identified, a number of strategies have emerged. These include modulation of smooth muscle cell migration, proliferation and/or apoptosis, acceleration of endothelial regeneration and improvement in endothelial function. Vein grafting is highly suited for human gene therapy since it allows ex vivo manipulation of the vein prior to grafting into the coronary circulation of the patient. This has clear safety advantages over in vivo gene therapeutic applications but, due to the short clinical window through which gene transfer can be administered, efficient vector systems are required. Adenoviral vectors have proven efficient for gene delivery in this context although expression of transgenes using first-generation vectors is transient in nature and associated with inflammation. However, this may be advantageous since a number of candidate therapeutic transgenes have been targeted to either block vascular smooth muscle cell migration and/or proliferation or to promote apoptosis. Our research has shown that overexpression of certain genes, including TIMP-3, p53 and NogoB has a beneficial effect by reducing neointima formation. This represents a valid therapeutic strategy to prevent vein graft failure in patients. We have also developed novel adenovirus vectors with improved capacity to transduce vascular cells.
26 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Searching for secreted factors and microRNAs promoting myocardial protection and regeneration Mauro Giacca Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy The identification of novel genes and pathways controlling prenatal cardiomyocyte proliferation or regulating cardiomyocyte survival during the adult life holds paramount interest in view of developing new therapeutic approaches for patients with ischemic cardiomyopathy and heart failure. We are undertaking two complementary approaches in this respect. The first one exploits the capacity of viral vectors based on the Adeno-Associated Virus (AAV) to transduce myocardial cells in vivo with high efficiency and to promote the expression of their transgenes for prolonged periods of time. The exquisite tropism of these vectors for post-mitotic tissues such as cardiomyocytes and skeletal muscle fibers is explained by the lack of expression of DNA damage response proteins in these cells, in particular the members of the Mre11-Rad50-Nbs1 complex, which in replicating cells restrict vector transduction. Using AAV vectors, we have undertaken an exhaustive approach to select factors exerting beneficial effect upon myocardial damage by the construction of a library of AAV vectors delivering the entire mouse secretome (1600+ mouse secreted proteins). Preliminary screening of a subset of 100 vectors from this library has led to the identification of a few novel factors exerting a powerful cardioprotective action in vitro and in vivo. Among these factors is the neuropeptide hormone ghrelin, which we found to markedly ameliorate myocardial function upon AAV delivery after infarction in vivo. A second approach entails high throughput screening of microRNAs promoting primary neonatal cardiomyocyte proliferation in vitro. Out of a library consisting of ~1000 human microRNAs, we identified 40 human microRNAs with outstanding activity in promoting expansion of cardiomyocytes in cell culture, massive cardiac hyperplasty in the neonatal heart and remarkable improvement of cardiac function after infarction in adult animals upon cardiac delivery using an AAV9 vector. The identified factors and microRNAs hold great promise for the development of innovative therapeutic strategies for myocardial infarction and heart failure.
This work was supported by Advanced Grant 250124 “FunSel” from the European Research Council (ERC)
27 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
AAV2.9 Thymosin beta 4 gene therapy: requirements for therapeutic neovascularization R. Hinkel1, T. Trenkwalder1, F. Gesenhues1, A. Pfosser1, G. Stachel1, F. Götz1, C. Lebherz1, I. Bock-Marquette2, E. Hannappel3, C. Kupatt1 1Internal Medicine I, Klinikum Großhadern - LMU, München; Germany 2UT Southwestern Medical Center, Dallas, USA; 3Institut für Biochemie, Universität Nürnberg-Erlangen, Erlangen, Germany Thymosin beta4 (TB4), a vasoactive peptide, has been shown to promote angiogenesis in the developing heart. For therapeutic use, however, conductance vessel growth is viewed as inevitable, which is not provided by all vessel growth factors at the same rate. For this process, microvessel maturation and smooth muscle cell proliferation of collaterals appear necessary. We investigated the potential of TB4 to induce therapeutic neovascularization. Methods: In vivo, rabbits (n=5) underwent femoral artery excision on day0 and 5x1012 AAV2/9 expressing Tb4, Ang-2 or Lac-Z were injected intramuscular. Application of Tb4 was performed over the whole limb or in the upper and lower limb separately. Injection of AAV2/9-Ang-2 was performed selectively into the lower limb. In an additional group L-NAME, an unselective NO inhibitor was applied. Quantification of collaterals was measured via angiography on day7 and day35 (%d7). Regional perfusion was assessed by application of fluorescent microspheres (%d7). HPLC-Anaylsis of Tb4 protein levels and Immunohistochemistry for PECAM-1 positive cells (capillaries/muscle fiber = c/mf) and vessel maturation (NG-2 positive pericytes/capillary = p/c) were quantified. Results: Overexpression of Tb4 in chronic ischemia increased capillary density (1.96±0.1 vs. control 1.37±0.1c/mf), collateral growth (175±15% vs. 95±6 %d7 in controls) and blood perfusion (187±5%d7 vs. control 110±6%d7). Furthermore, pericyte coverage was stimulated in the Tb4-treated group (0.69±0.1p/c) compared to control (0.46±0.1 p/c). Transduction of Tb4 only into the lower limb was sufficient to induce collateral growth (177±4%d7) and blood perfusion in the upper limb (207±8%d7), whereas selective injection into the region of collateral formation in the upper limb resulted only in a slight improvement of arteriogenesis (150±3%d7) and blooflow (142±6%d7). Furthermore, nitric oxide mediated increases in flow and shear stress were necessary for collateral growth, since the unselective NO-inhibitor L-NAME prevented arteriogenesis (118±8%) and perfusion gain (123±13%) after LL-TB4 transduction. Conclusion: TB4 induces arteriogenesis of conductance vessels in the upper limb, even when applied remotely in the lower limb. The mechanisms include microcirculatory maturation, i.e.pericyte coverage, and backward signaling via shear stress and nitric oxide formation.
28 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Notch signaling in cardiac development and disease José Luis de la Pompa CNIC, Madrid Spain Notch is an evolutionarily conserved signaling pathway that regulates cell-fate specification, differentiation, and patterning. Mutations of Notch ligands and receptors are implicated in numerous congenital and acquired human diseases. Notch acts locally, specifying individual fates among a group of equivalent neighboring cells or directing a field of cells towards a given developmental fate. Notch activity is crucial in organs with complex architecture, such as the heart, that requires the coordinated development of multiple parts. In this talk I will discuss our recent advances in the field of Notch signaling in cardiac development and its impact in disease. I will describe how Notch signaling patterns the embryonic endocardium, enabling region-specific differentiation and critical interactions of the endocardium (or its derived mesenchyme) with other cardiac tissues (cardiac neural crest, myocardium), so that specialized structures (cardiac valves and chambers) are generated. I will also discuss extensively the implication of Notch in cardiac pathologies, including aortic valve disease, and cardiomyopathies.
29 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Insights into the mechanisms underlying regeneration of the vertebrate heart Chris Jopling1, Guillermo Sune1, Eduard Sleep1, Marina Raya1, Merce Marti1, Carme Fabregat1, Angel Raya1, Juan Carlos Izpisua Belmonte1,2 1 Center of Regenerative Medicine in Barcelona, Barcelona, Spain 2 Salk Institute for Biological Studies, La Jolla, USA Although mammalian hearts show almost no ability to regenerate, there is a growing initiative to determine whether existing cardiomyocytes or progenitor cells can be coaxed into eliciting a regenerative response. In contrast to mammals, several non-mammalian vertebrate species are able to regenerate their hearts, including the zebrafish (zf) which can fully regenerate its heart after amputation. To address the source of newly formed cardiomyocytes during zf heart regeneration, we first established a genetic strategy to trace the lineage of cardiomyocytes in the adult fish, based on the Cre/lox system widely used in the mouse. Utilizing this system, we will show that regenerated heart muscle cells are derived from the proliferation of differentiated cardiomyocytes. Furthermore, we will show that proliferating cardiomyocytes undergo limited dedifferentiation characterized by the disassembly of their sarcomeric structure, detachment from one another and the expression of cell-cycle progression regulators. Specifically, we will show that the gene product of polo-like kinase 1 is an essential component of cardiomyocyte proliferation during heart regeneration. Our data provide the first direct evidence for the source of proliferating cardiomyocytes during zf heart regeneration and indicate that stem or progenitor cells are not significantly involved in this process. Hypoxia plays an important role in many biological/pathological processes. In particular, hypoxia is associated with cardiac ischemia which, while initially inducing a protective response, will ultimately lead to the death of cardiomyocytes and loss of tissue, severely impacting cardiac functionality. We surmised that ventricular amputation would lead to hypoxia induction in the myocardium of zf and that this may play a role in regulating the regeneration of the missing cardiac tissue. Using a combination of O2 perturbation, conditional transgenics and in vitro cell culture, we will show how hypoxia induces cardiomyocytes to dedifferentiate and proliferate in zf. These results indicate that hypoxia plays a positive role during heart regeneration, which should be taken into account in future strategies aimed at inducing heart regeneration in humans.
30 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Pluripotent stem cells: basic research for later clinical application in the cardiovascular system Juergen Hescheler Institute for Neurophysiology, University of Cologne It is our aim to provide a fundamental basis to the development of new medical treatments. This presentation will give an overview on our recent research work on human embryonic in comparison with induced pluripotent stem cells. Starting from basic investigations on the physiological properties of cardiomyocytes developed from pluripotent stem cells we have established in vitro and in vivo transplantation models enabling us to systematically investigate and optimize the physiological integration and regeneration of the diseased tissue. Our main focus is the cardiac infarction model. Induced pluripotent stem cell-derived cardiomyocytes (iPSCM) are regarded as the most promising cell type for cardiac cell replacement therapy. iPS cells are functionally highly similar to embryonic stem (ES) cells, but in addition have the advantage of being ethically uncontroversial and obtainable from readily accessible autologous sources. Moreover, iPSCMs also provide an interesting new tool to study the pathophysiology of monogenetic diseases of the cardiovascular system. A functional integration of iPSCMs is crucial for efficiency and safety, but has not been demonstrated, yet. Thus, we investigated the electrical integration of transplanted CMs into host tissue. Genetically modified murine iPSCM, expressing eGFP and a puromycin resistance under control of the alpha-MHC promoter, were purified by antibiotic selection. Purified iPSCM were injected into adult mouse hearts. At different times after transplantation recipients were sacrificed and viable ventricular tissue slices were prepared. Slices were focally stimulated by a u iPSCMs iPSCMs nipolar electrode placed in host tissue. Recordings of action potentials were performed by glass microelectrodes in transplanted iPSCM, which could be identified by their green fluorescence, and in host cardiomyocytes within the tissue slices. Translation from the laboratory into the clinic is one of the key problems of stem cell research. Although proof of principle for the therapeutic use of iPS cells in cardiac diseases has been shown both at the laboratory scale and in animal models, the methods used today for generation, cultivation, differentiation and selection are not yet suitable for the clinic.
31 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Cardiac stem cell and human heart failure Piero Anversa Departments of Anaesthesia and Medicine, Center for Regenerative Medicine Brigham & Women's Hospital, Harvard Medical School, Boston, MA The identification of cardiac progenitor cells in small and large mammals raises the possibility that the human heart contains a population of stem cells capable of generating cardiomyocytes and coronary vessels. We established the conditions for the isolation and expansion of c-kit-positive hCSCs from small fragments of myocardium discarded at surgery. Additionally, we tested whether these cells have the ability to form functionally competent human myocardium after infarction in immunodeficient and immunosuppressed animals independently of cell fusion. We have documented that c-kit-positive human cardiac cells possess the fundamental properties of stem cells: they are self-renewing, clonogenic and multipotent. hCSCs differentiate predominantly into cardiomyocytes and to a lesser extent into smooth muscle cells and endothelial cells. When locally injected in the infarcted myocardium of immunodeficient mice and immunosuppressed rats, hCSCs generate a chimeric heart, which contains human myocardium composed of myocytes, coronary resistance arterioles and capillary profiles. Importantly, the differentiated human cardiac cells possess only one set of human sex chromosomes excluding cell fusion. Although the human myocardium shows an immature phenotype, it contracts regionally and contributes to the improvement in the hemodynamic performance of the infarcted left ventricle. These experimental findings constituted the basis for pre-clinical studies in large animal models and for the recent phase 1 trial SCIPIO (Stem Cell Infusion in Patients with Ischemic cardiOmyopathy). One million autologous hCSCs were administered by intracoronary infusion ~4 months after multi-by-pass surgery in patients with chronic ischemic cardiomyopathy. No hCSC-related adverse effects were reported. The initial results of the SCIPIO trial are very encouraging; intracoronary infusion of autologous CSCs improved left ventricular systolic function and reduced infarct size in patients with severe heart failure after myocardial infarction.
32 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Driving heart progenitor cell fate and regeneration in vivo via chemically modified mRNA Kenneth R. Chien Department of Stem Cell and Regenerative Biology, 7 Divinity Avenue, Fairchild Lab, Harvard University, Cambridge, MA 02138, USA and MGH Cardiovascular Research Center A family of multipotent heart progenitors is responsible for the diversification and expansion of cardiac myocyte, smooth muscle, and endothelial cell lineages during cardiogenesis1-3. These progenitors persist after birth4-10 and may represent an opportunity for cell-based regenerative therapeutics. However, an effort to unlocking their potential has met with limited success11-14. The localized, transient and efficient delivery to the heart of paracrine factors which control the expansion and differentiation of resident heart progenitors might represent a viable alternative therapeutic strategy, akin to the known clinical utility of cytokines to selectively augment specific blood cell lineages. Herein, we establish chemically modified mRNA (modRNA) as a platform for localized, transient and highly efficient expression of paracrine factors in the murine heart. We show that VEGF-A modRNA administered in the setting of myocardial infarction (MI) markedly expanded the pool of Wilm’s tumor 1 (Wt1)- expressing epicardial heart progenitors. Moreover, VEGF-A acted as a fate switch, driving rare pre-existing epicardial progenitors away from a previously described interstitial fibroblast-like state7 and towards a cardiac and vascular fate following MI. As a result, VEGF-A modRNA markedly increased epicardial progenitors and their vascular cell lineages, increase capillary density, reduced cardiac fibrosis, and improved cardiac function. These results show that VEGF-A modRNA represents a novel heart progenitor cell fate switch following injury and provide a new cell-free therapeutic paradigm to achieve in vivo recruitment and subsequent differentiation of endogenous heart progenitors for cardiovascular regeneration.
33 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Human cardiac stromal cell and heart regeneration Maurizio Capogrossi Istituto Dermopatico dell’Imamcolata-IRCCS, Via dei Monti di Creta 104, 00167 Rome Stromal cells can be isolated from a variety of adult tissues and organs, the most extensively characterized being those of bone marrow origin [bone marrow mesenchymal stromal cells (BMStC)]. Recently, stromal cells have been also isolated from the cardiac tissue (CStC). The objective of the present work was to compare adult human CStC to syngeneic BMStC in order to establish their ability to differentiate into cardiovascular cells in vitro and repair the infarcted rat heart. Although CStC and BMStC exhibited a similar immunophenotype, their gene, microRNA, and protein expression profiles were remarkably different. Biologically, CStC, compared with BMStC, were less competent in acquiring the adipogenic and osteogenic phenotype but more efficiently expressed cardiovascular markers. When injected into the heart, in rat a model of chronic myocardial infarction, CStC persisted longer within the tissue, migrated into the scar, and differentiated into adult cardiomyocytes better than BMStC. In conclusion, although CStC and BMStC share a common phenotype, CStC present cardiovascular-associated features and may represent an important cell source for more efficient cardiac repair.
34 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Human pluripotent stem cells and cardiac subtype specification Robert Passier LUMC, Dept of Anatomy & Embryology, 2300 RC Leiden, the Netherlands Pluripotent human embryonic stem cells (hESC) have the potential to differentiate to any cell type of the human body. This characteristic has sparked researchers to study the use of hESC for regenerative medicine, drug screenings and embryonic development. We have recently optimized differentiation of hESC to cardiomyocytes, including growth factor directed differentiations as monolayers or as three-dimensional aggregates (embryoid bodies or EBs). Previously, we have demonstrated that hESC-derived cardiomyocytes (hESC-CM) faithfully recapitulate the early molecular events during embryonic development. Recently, we have generated a cardiac reporter line by introducing Green Fluorescent Protein (GFP), in the genomic locus of the early cardiac transcription factor NKX2-5, which enables us to visualize the derivation of NKX2-5+ cardiomyocytes during in vitro differentiation and purify these cells by Fluorescent Activated Cell Sorting (FACS). The combination of different transcription factor-coupled fluorescent reporters in this so-called “rainbow” hESC cell line, covering sequential stages of the cardiac lineage, will allow us to identify and characterize pathways for specific subtypes of the cardiac lineage at early and later stages during differentiation. Furthermore, a better understanding of these developmentally related processes will be further important for progress in fields of tissue engineering, disease modelling, drug toxicity and discovery, which most likely will lead to improved tailor-made therapies and better and safer medicines on the market.
35 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Human iPSC models of cardiac disease Laugwitz K-L Klinikum rechts der Isar und Deutsches Herzzentrum, Technische Universität München Much of what is known about the molecular pathways that lead to human cardiovascular disorders has come from studying animal models, particularly genetically modified mice. In some cases it is possible to translate genetic discoveries from humans to mice (e.g. non-sense mutations), but in most circumstances there are no direct correlates for human genetic variants such as single nucleotide polymorphisms (SNPs) or copy number variants. Therefore, it is imperative to replicate relevant features of human cardiovascular physiology in the context of the human genome. Recent advances in stem cell biology now raise the possibility of generating human models of cardiovascular physiology and disease. Generating patient-specific cells and tissues has recently emerged with the demonstration that exogenous expression of four proteins in human skin fibroblasts (e.g. c-MYC, KLF4, OCT4, and SOX2) is sufficient to induce pluripotency in the cells. Although so-called induced pluripotent stem cells (iPSCs) are not perfectly equivalent to human ES cells, they retain important properties of ES cells such as the capacity for long-term propagation and the ability to differentiate into all human somatic cell types. Factor-based reprogramming enables us of the long-standing ambitions of stem cell biology: the ability to generate pluripotent cells from specific patients and figuratively, move a patient`s disease into the Petri dish. This will be discussed for human monogenetic cardiovascular diseases, e.g. LQT syndromes, catecholaminergic polymorphic ventricular tachycardia (CPVT) and arrhythmogenic right ventricular dysplasia (ARVC). Recent advances describing the derivation of human iPSCs from peripheral, frozen blood brings the stem cell field an important step closer to bio-banked blood samples and eventual clinical use.
Moretti A, Bellin M, Welling A, Jung CB, Lam JT, Bott-Flügel L, Dorn T, Gödel A, Höhnke C, Hofmann F, Seyfarth M, Sinnecker D, Schömig A & Laugwitz K-L (2010). Patient-specific induced pluripotent stem cell models for long-QT syndrome. N Engl J Med., Epub Jul 21. Jung B, Moretti A, Mederos y Schnitzler M, Iop L, Storch U, Pfeiffer S, Bellin M, Dorn T, Goedel A, Dirschinger R, Seyfarth M, Lam JT, Sinnecker D, Gudermann T, Lipp P, Laugwitz KL. (2011). Dantrolene rescues arrhythmogenic RYR2 defect in a patient-specific stem cell model of catecholaminergic polymorphic ventricular tachycardia. EMBO Mol. Med., Epub Dec 15.
36 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Melusin and IQGAP1 are key players in ERK1/2 Signaling and compensatory cardiac hypertrophy response to pressure overload Mauro Sbroggiò, Alessandro Bertero, Daniela Carnevale, Giuseppe Lembo, Mara Brancaccio and Guido Tarone Molecular Biotechnology Center University of Torino, and Department of Angiocardioneurology, I.R.C.C.S. “Neuromed”, Pozzilli, ITALY Melusin is a muscle-specific protein involved in triggering compensatory cardiac hypertrophy in response to pressure overload via activation of AKT and ERK1/2 MAPKs signaling pathways. To define the mechanism of melusin action, we searched for molecular partners involved in the melusin-dependent signal transduction. We demonstrated that melusin forms a supramolecular complex with c-Raf, MEK1/2 and ERK1/2 and that melusin-bound MAPKs are activated by pressure overload. Both the cytosolic tyrosine kinase FAK (Focal Adhesion Kinase) and the scaffold protein IQGAP1 are part of the melusin supramolecular complex and are required for ERK1/2 activation in response to pressure overload. To asses the in vivo role of this molecular complex we investigate the heart remodeling in mice lacking IQGAP1 expression. IQGAP1-null mice have unaltered basal heart function and develop normal hypertrophy in response to acute aortic banding. When subjected to chronic pressure overload, however, they develop thinning of left ventricular walls, chamber dilation, and a decrease in contractility, in an accelerated fashion compared to wild type mice. This unfavourable cardiac remodeling is characterized by blunted reactivation of the fetal gene program, impaired cardiomyocyte hypertrophy and increased cardiomyocyte apoptosis. IQGAP1-null mice show strongly impaired phosphorylation of MEK1/2-ERK1/2 and AKT following 4 days of pressure overload, but normal activation these kinases after 10 min, indicating that IQGAP1 is required for a specific, temporally regulated wave of both ERK1/2 and AKT signaling in response to pressure overload. These findings point to melusin and IQGAP1 as a key players in organizing a specific ERK1/2 signalosome in cardiomyocytes and in regulating adaptive long-term left ventricle remodeling in response to pressure overload.
37 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Development of a pharmacological approach to myocardial regeneration Erik Willems, Wenqing Cai, Marion Lanier, Karl Okolotowicz, Marcia Dawson, John Cashman, and Mark Mercola Sanford-Burnham Medical Research Institute, La Jolla, CA 92037 USA; Human Biomolecular Research Institute, San Diego, CA 92121 USA Since current therapies for heart failure do not specifically create new myocytes, there has been tremendous interest in defining natural and synthetic molecules to regenerate myocardium from endogenous stem or progenitor cells. Towards this end, we have developed automated assays using progenitors from mouse and human embryonic stem cells expected to resemble functionally adult cardiac stem cells. Using these assays in high throughput screens of chemical and microRNA libraries, we have discovered chemical compounds and miRs that promote cardiomyocyte differentiation through the production of committed cardiac progenitors and differentiation of cardiomyocytes. An example of such a compound is a novel inhibitor of TGFbeta signaling. This compound selectively clears the type II TGFbeta receptor (TGFBR2) from the cell surface and targets it for proteasomal degradation in a ubiquitination-independent manner. Unlike previous inhibitors of TGFbeta signaling that target the receptor kinase activity, this compound is selective for TGFbeta over the closely related Activin receptor mediated signaling. Probing cardiogenesis, the compound aided in revealing a bimodal role for Nodal and TGFbeta signaling. During development, Nodal and TGFbeta both induce cardiogenic mesoderm, but subsequently Nodal expression declines and TGFbeta becomes uniquely responsible for inhibiting cardiomyocyte differentiation while simultaneously promoting vascular endothelial and smooth muscle differentiation. Mechanistically, Nodal/TGFbeta signaling represses cardiomyogenic differentiation, functioning by blocking an epigenetic switch involving Baf60c, Gata4 and Tbx5 that open chromatin of cardiac enhancers enhancing accessibility to transcriptional machinery. Since TGFbeta is involved in fibrosis and inflammation after myocardial injury, our results indicate that it might also antagonize creation of new myocytes and, therefore, suggest that transient inhibition of might facilitate regeneration.
38 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Cardiac imaging in regenerative medicine Fabio A. Recchia Institute of Life Sciences, Scuola Superiore Sant’Anna, Pisa, Italy; Department of Physiology, Temple University School of Medicine, Philadelphia, USA Cardiac stem cell therapy typically targets selected regions of the heart, therefore classical functional evaluations of global function, such as ejection fraction or dP/dtmax, may largely underestimate its efficacy. This is particularly important during the first few months following cell delivery, when the cardioreparative effects may not rapidly translate into an overall improvement of contractile function. Unfortunately, most of the published studies on stem cell therapy, both in experimental models and in humans, have assessed the functional outcome based on ejection fraction. While this approach can be justified by practical aspects that render ejection fraction a standard and widely used index, its limitation might explain the inconsistent findings relative to the therapeutic efficacy of stem cell delivery. Modern technology offers better tools for non-invasive measurements of cardiac function and metabolism at regional level. In our laboratory, we have induced small size myocardial infarctions in pigs, which did not affect ejection fraction both in untreated and stem cell- treated hearts. However, standard and “tagged” cardiac magnetic resonance imaging (MRI), combined with positron emission tomography (PET), revealed marked differences in infarct size, contractile function, blood perfusion and glucose uptake between treated and untreated hearts. The effectiveness of stem cell delivery, documented by our study, would have been completely ignored without the “magnifying lens” of MRI and PET imaging, which was focused on pre-defined segments of the ventricular wall in order to obtain rigorous comparisons of equivalent regions. A more recently developed technology, named 13C hyperpolarization, will likely evolve into a new precious tool for regional assessment of ventricular metabolism with MRI, thus replacing PET and the associated use of radioisotopes. Our Center is currently testing 13C-labeled myocardial energy substrates such as pyruvate and acetate. They are first subjected to a strong magnetic field and then injected intravenously during MRI performed with a clinical standard scanner. This imaging allows a global and segmental evaluation of substrate uptake and generation of the catabolites lactic acid and CO2 and even of Krebs cycle intermediate metabolites. Finally, at least in pre-clinical studies, stem cells can be transduced with genes encoding for proteins that render them detectable with MRI. In infarcted rats, we have successfully tested ferritin as a reporter protein detectable with a clinical MRI scanner, showing that stem cells can be tracked in vivo also after their differentiation into mature lineages. Based on these premises, it is conceivable that, in a near future, MRI, widely available at many research and clinical centers, might become the gold standard for simultaneous assessment of cardiac morphological, functional and metabolic changes occurring after stem cell delivery, with the additional possibility to track cell homing and differentiation in pre-clinical studies.
39 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Cell therapy for chronic ischemic heart disease: from concept to clinic Ivonne H. Schulman, MD University of Miami Miller School of Medicine, Miami, USA. Heart failure is an increasingly frequent cardiovascular disorder that affects 15-20 million people worldwide. After myocardial infarction, the heart has insufficient regenerative capacity and undergoes progressive remodeling events that lead to left ventricular dysfunction and ultimately heart failure. Although pharmacological and interventional advances have reduced the morbidity and mortality of heart failure, there is an ongoing need for novel therapeutic strategies that prevent or reverse progressive ventricular remodeling. The development of cell-based therapy as a strategy to repair or regenerate injured cardiac tissue offers extraordinary promise for a powerful anti-remodeling therapy. Clinical trials are currently underway in our laboratory testing autologous and allogeneic bone marrow-derived mesenchymal stem cells (MSCs) for myocardial regeneration and reverse remodeling in heart failure patients with ischemic cardiomyopathy. In addition, the effectiveness of various methods of cell delivery and accuracy of diverse imaging modalities to assess therapeutic efficacy is under investigation. Optimization of stem cell therapy has the potential to provide long-term improvements for those suffering from heart failure. MSCs contribute to cardiac repair after myocardial injury via mechanisms that include trilineage differentiation, anti-inflammatory effects, immunomodulation, neovascularization, and modulation of the cardiac stem cell niche. These findings have in turn guided rationally designed translational clinical investigations. Collectively, there is a growing understanding of the parameters that underlie successful cell-based approaches for improving heart structure and function in ischemic cardiomyopathy.
40 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Novel non-coding RNAs in cardiac regeneration Samir Ounzain1, Mark Ibberson2, Stefania Crippa1, Mohammed Nemir1, Alexandre Sarre3, Gaelle Boisset4, Keith Harshman5, Ioannis Xenarios2, Dario Diviani6, Daniel Schorderet4, Thierry Pedrazzini1 1Experimental Cardiology Unit, Department of Medicine, University of Lausanne Medical School, Lausanne; Switzerland; 2Swiss Institute of Bioinformatics, Lausanne, Switzerland; 3Cardiovascular Assessment Facility, University of Lausanne, Lausanne, Switzerland; 4Institute for Research in Ophthalmology, Sion, Switzerland; 5Genomic Technologies Facility, University of Lausanne, Lausanne, Switzerland; 6Department of Pharmacology and Toxicology, University of Lausanne, Lausanne, Switzerland Recently, the notion of promoting cardiac regeneration has engendered considerable research interest. Unlike the mammalian heart, which has a poor regenerative capacity, certain fish retain a robust capacity for regeneration into adult life. This includes the zebrafish, which can fully regenerate its heart after amputation of up to 20% of the ventricle. This capacity is a consequence of the differential utilization of regenerative genetic circuits, many of which are absent in the adult mammalian heart. These circuits are enacted by the integrated execution of specific transcriptional programs, which themselves are modulated by coding and noncoding regulatory factors, including small noncoding microRNAs (miRNAs). Recent studies have revealed central roles for miRNAs as core regulators of gene expression during cardiac development and disease, with the integration of miRNAs into the regulatory circuitry of the heart providing regulatory interactions to control cardiac gene expression. To systematically characterize these regulatory circuits we have generated global gene and miRNA expression profiles in the poorly regenerating mouse (ligation of the left descending coronary artery) and regenerating zebrafish (20% ventricular apex resection) models of cardiac injury and regeneration. We have developed and employed a novel integrated bioinformatic approach to identify differentially regulated miRNA dependant genetic programs in the mouse and zebrafish injury models. Many well characterized miRNA networks implicated in cardiac fibrosis (miR-133, -29, -30, -21, -208, -499) and hypertrophy (miR-1, -133, -208) were differentially modulated in the two species, highlighting the fundamentally different response of the mouse and zebrafish to cardiac injury at the miRNA level (miRome). Additionally, this global miRome characterization has allowed us to identify novel miRNA regulatory circuits potentially involved in critical biological pathways. These pathways have been implicated in the regenerative response including ECM deposition/ fibrosis, cell cycle control, cytokinesis and sarcomeric assembly/disassembly. Through the direct manipulation of these novel candidate miRNA circuits in the mouse, we hope to reactivate the regenerative potential of the adult mammalian heart in response to cardiac injury.
41 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
MicroRNA control of tissue fibrosis Stefan Engelhardt Institut für Pharmakologie und Toxikologie, Technische Universitaet Muenchen (TUM) Munich Heart Alliance, Munich, Germany Muscle responds to a wide variety of stressors by hypertrophic growth of myocytes and interstitial fibrosis. In various muscular disorders, the hypertrophic response may temporarily serve a compensatory role but becomes detrimental when prohypertrophic stimulation persists. As such, cardiac hypertrophy has been identified as a risk factor for impaired cardiac function, future heart failure and life threatening arrhythmias. In contrast, muscle fibrosis has long been regarded as a secondary phenomenon thought to primarily result from myocyte pathology and cell death. Our recent work and that of others on small RNAs expressed in muscle suggests that fibroblasts may play an early and important role in muscular disease. MiRNAs interact specifically with mRNAs by repressing their translation or inducing their degradation. They are thus, by their impact on gene expression, key factors in the development and maintenance of tissue, both in health and disease states. The majority of work in the cardiovascular field has focussed on miRNAs whose cellular levels change under disease-causing conditions, based on the assumption that disease relevance and deregulation are tightly linked. In this context, we have identified miR-21 as one of the strongest upregulated microRNAs in myocardial disease and to be serve a key role in tissue fibrosis. We found that miR-21 regulates ERK-MAPkinase signalling in cardiac fibroblasts, which impacts on cardiomyocyte hypertrophy and cardiac function. Experimental therapeutic studies in animal models demonstrated inhibition of miR-21 as an effective therapeutic strategy against cardiac, pulmonary, kindney and skeletal muscle fibrosis. This talk will summarize our current state of knowledge on the role of miRNAs in tissue fibrosis and discuss prospects for RNA-based therapeutic approaches to target tissue fibrosis.
42 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Translational control of myocardial function Deng Hong Zhang1, Riccardo Contu1, Juliana Smetana1, Gianluigi Condorelli1-2 1University of California San Diego, La Jolla; 2National Research Council of Italy and Istituto Clinico Humanitas IRCCS, Milan mRNA translation is fundamentally involved in the regulation of cardiac hypertrophy. One of the critical molecules controlling mRNA translation and thus cell growth and in the response to energy state changes is a gene product called Mechanistic target of rapamycin (MTOR), a serine-threonine kinase. mTOR can be found in multiprotein complexes containing, among other partners, either raptor, forming TORC1, or raptor, forming TORC2. TORC1 and TORC2 selectively phosphorylate substrates with specific activities. Myocardial mTOR activity changes during hypertrophy and heart failure (HF). However, whether MTOR exerts a positive or a negative effect on myocardial function remains to be fully elucidated. We previously demonstrated that ablation of Mtor in the adult mouse myocardium results in a fatal, dilated cardiomyopathy that is characterized by apoptosis, autophagy, altered mitochondrial structure, and accumulation of eukaryotic translation initiation factor 4E–binding protein 1 (4E-BP1). 4E-BP1 is an MTOR-containing multiprotein complex-1 (MTORC1) substrate that inhibits translation initiation. When subjected to pressure overload, Mtor-ablated mice demonstrated an impaired hypertrophic response and accelerated HF progression. When the gene encoding 4E-BP1 was ablated together with Mtor, marked improvements were observed in apoptosis, heart function, and survival. We found that deletion of 4E-BP1 determined an increase of 4E-BP2, another member of the 4E-BP family. Deleting 4E-BP2 in the context of mTOR/4E-BP1 double knockout, further ameliorated cardiac funciton and survival, strenghthening the concept that 4E-BP is a critical regulator of the effects of mTOR in the heart. Our results demonstrated a critical role for the MTORC1 signaling network in the myocardial response to stress. In particular, they highlight the role of 4E-BP in regulating cardiomyocyte viability and in HF. Because the effects of reduced MTOR activity were mediated through increased 4E-BP1 inhibitory activity, blunting this mechanism may represent a novel therapeutic strategy for improving cardiac function in clinical HF.
43 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Participants’ Abstracts (in alphabetical order according to presenter)
44 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
ACE polymorphism and obesity a cardiovascular risk factors among Sudanese Fatima E. Abukunna, Nagwa M. Mohamed, Dina A. Hassan, Hussein A. M, Abdulhadi N. H Central Laboratory-Ministry of Sciences& Technology- Khartoum- Sudan In this study, the association of angiotensin converting enzyme (ACE) insertion (I)/deletion (D) polymorphism and obesity a cardiovascular disease factors in Sudanese adults was investigated.The subjects were divided according to the BMI, into two groups (Obese& over weight/ Normal weight). Body composition, lipid profile and C reactive protein were compared among groups. Obesity related dyslipidemia seems to affect overweight/obese males more than females. While obesity related hypertension is a phenomenon among females probably due to release of CRP.
* The finding of ACE polymorphism will be included later.
45 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
A Proteomic Evaluation of Smooth Muscle Cells in Thoracic Aortic Aneurysms Ceyda Acilan1, Ph.D., Betul Baykal1,2, Muge Serhatlı1, MSc., Omer Kacar1, MSc., Zelal Adiguzel1, MSc., Altug Tuncer3, M.D., Kemal Baysal1,4, M.D., Ph.D., Ahmet T. Baykal1*, Ph.D. 1TUBITAK, MRC, Genetic Engineering & Biotechnology Institute, 2International Centre for Genetic Engineering and Biotechnology, 3Kartal Kosuyolu Advanced Training and Research Hospital, 4Dokuz Eylul University, Medical Faculty, Biochemistry Department Aortic aneurysms (AA), a common disease affecting up to ~9% of individuals above the age 65, are characterized as localized degeneration of the aorta leading to weakening of the wall and widening of the vessel. While the exact mechanisms are yet to be determined, current studies indicate that the degradation of extracellular matrix proteins and apoptosis of vascular smooth muscle cells (SMCs) within the aorta may result in extendibility, dilatation and rupture of the vessel. Among the cells that form the aortic wall, SMCs are implicated as key components involved in disease development as numerous molecular changes have been reported to occur in these cells. Most current studies involve either investigation of proteins comprising a group or pathway in SMCs or global analyses in the whole aortic tissue. In order to determine which proteins are important in the development of thoracic aortic aneurysms, we performed comparative proteomic analyses using isolated, cultured SMCs from thoracic aortic aneurysms (TAA) versus controls. We hypothesized that cultured SMCs represent tissue characteristics and investigation of molecular changes in this sub-fraction of aorta should be able to better reveal the underlying reasons for this disease. Label-free LC-MS/MS analysis of cell extracts resulted in the identification of 256 proteins from which 26 significant differentially regulated proteins were identified and characterized. Both previously described and new proteins were identified that were involved in oxidative stress, extracellular matrix formation, protein folding, energy metabolism, and 14-3-3 pathway. Here, we attempted to build a multi protein model to shed light on the cellular mechanism of TAA. Keywords: Label-free proteomics, thoracic aortic aneurysm, smooth muscle cell, SerpinH1/ HSP47, oxidative stress, protein expression
46 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Inhibition of CSC specific miRNAs result in differentiation of CSC into cardiomyocytes 1Aftab Ahmad, 2Edilamar M. de Oliveira, 3Dafina Ibrani, 4Yao Liang Tang, 5M.Ian Phillips 1 School of Biological Sciences, University of the Punjab, Lahore. Pakistan 2 University of Sao Paulo, Sao Paulo, Brazil. 3University of California. Los Angeles, CA, USA 4,5 KGI, Claremont. CA. USA Cardiomyocytes can be derived from hESC and human cardiomyocyte progenitor cells by treatment with activin A (Acv), bone morphogenic protein (BMP) or transforming growth factor, beta receptor III (TGF-βIII). We hypothesized that inhibition of specific miRNA in mouse cardiac stem cells (CSC) and CSC with GATA-4 (CSCG) could induce differentiation in cardiomyocytes by activation of their target genes. We studied 569 unique miRNAs probes in mouse heart cells (MHtC), CSC and CSCG and identified high expression of miR762, 21 and 31 in CSC and CSCG compared to MHtC. CSC and CSCG were cultured in matrigel and anti-mRNAs were transfected with oligofectamine reagent twice (3 and 15 days) after plating cells. We inhibited miR-762; 762+21; 762+31 and 762+21+31, which present as target genes activin A, BMP and TGF-β receptors. The target genes as TGF-βIII, activin A, BMP as well as Smad-4 and Dicer were analyzed by RT-PCR. Beating cells were recorded on Confocal microscopy. As a positive control the cells were treated with activin A and BMP-4 cytokines and also compared to MHtC exrpession. The anti-sense was tested at 1, 5, 10, 50 and 100nM concentration. After 72hs of the treatment the target genes were analyzed by RT-PCR. In CSC and CSCG the TGF-βIII, activin AR, BMPR and Smad4 expression increased up 50nM and beginning inhibition with 100nM concentration. The maximum miRNA expression inhibition was observed 6hs after the treatment, and 24hs the miRNA expression was similar of the control cells, however the target genes expression was increased. As expected cytokines treatment increased the target genes and signaling pathway increasing Smad-4. In CSC the miR-762, 762+21 and 762+21+31 anti-sense increased the target genes TGFβIII, AcvR, but not BMPR. Furthermore, increase the transcription factor Smad-4 and MLC-2a. α-MHC expression increased with 762+21 anti-sense association. In CSCG the 762+31 and 762+21+31 inhibition induced the genes, including TGFβIII, AcvR, but not BMPR. Also, increase Smad4 and MLC-2a and α-MHC. The results show that CSC and CSCG differentiate in cardiomyocytes by activation of TGFβIII and AcvR signaling pathway, but not BMPR.
47 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Potential treatment modality for ischemic myocardial rat heart with mesenchymal stem cell transplantation Esin Akbay a, Handan Sevim a , Özer Aylin Gürpinar a, Serdar Günaydin b,Mehmet Ali Onur a a Hacettepe University, Faculty of Science, Department of Biology, Beytepe,06800, Ankara, Turkey b Kirikkale University, Faculty of Medicine, Department of Thoracic and Cardiovascular Surgery, Kirikkale, Turkey Stem cells are investigated for their potential use in regenerative medicine (1). Several human organs are not capable of functional regeneration following a tissue defect and react with scar formation (2). For example, after ischemic cardiomyopathy and myocardial infarction (MI), the damaged myocardium is replaced by fibrotic noncontractile cells. These diseases are typified by the irreversible loss of cardiac muscle cells, which are essential for maintaining cardiac integrity and function. This ischemic heart disease accounts for 50% of all cardiovascular deaths. (3). For stem cell transplantation, undifferentiated precursor cells are applied to defective tissue for therapeutic regeneration (2). In this study, mesenchymal stem cells collecting from rat bone marrow were transplanted into the ischemic rat myocardium and healing of damaged tissue was investigated by using electrocardiographic and histological methods. In our study, the experiments were performed in 20 male Wistar rats with an initial body weight of ~ 300 gram. Bone marrow was collected from tibias and femurs of five rats. After the ether inhalation, both tibias and femurs were removed and proximal ends were cut. Samples were collected in a centrifuge tube with normal cell culture medium (DMEM/F12) containing 1% penicillin/streptomicin. Bone marrow were homogenized by pipetting and centrifuged at 800 rpm for 5 minutes. The marrow pellet were resuspended in special medium and incubated in a humidified atmosphere of 95% air and 5% CO2 at 37 ºC. After three days of incubation, unattached cells were removed by changing medium. The attached cells were incubated in same medium under standard culture conditions. When the cells become confluent, they were trypsinized. Mesenchymal stem cells were passaged three times. Mesenchymal stem cells were characterized for CD13 and CD29 by using immunoflourescence staining. To evaluate the effects of trypsinization, cells were characterized by using same antibodies after 1st, 2nd and 3rd passages. Ischemic rat heart model was performed with a modified method of Pfeffer et al. (4). The rats were anesthetized by ketalar injection (0.2 mg/kg). After thoracotomy was performed, the left coronary artery was heat-cauterized between the pulmonary artery outlow tract. Cell transplantation was performed within 5 minute after induction MI. The thorax and the skin then immediately closed and rats were left to heal.
48 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Pharmacological attenuation of Cardiac Stem Cell senescence in vitro increases their reparative ability in vivo Elisa Avolio, Angela Caragnano, Carlo Vascotto, Giuseppe Gianfranceschi, Ugolino Livi, Rajesh Katare, Paolo Madeddu, Daniela Cesselli, Carlo Alberto Beltrami and Antonio Paolo Beltrami Department of Medical and Biological Sciences, University of Udine Recently, we demonstrated that both age and pathology exert detrimental effects on human Cardiac Stem Cells (CSC) by attenuating their telomerase activity, reducing telomeric length, determining telomere erosion and the occurrence of telomere induced dysfunction foci, eventually leading to an impairment of CSC function in vitro. Aims: to investigate whether CSC senescence is associated with a reduction in their reparative ability in vivo, to identify the molecular determinants possibly responsible for CSC senescence, to screen drugs able to interfere with CSC senescence and to verify if CSC drug treatment in vitro is effective in restoring the reparative potential of senescent CSC in vivo. Methods and Results: Western blot analysis of CSC obtained from either controls (C) or failing hearts (F) was carried out to identify pathways possibly associated with CSC senescence. Drug screening assays were performed on growing cultures of F-CSCs, exposing them to Rapamycin, DETA/NO or Resveratrol for three days. At the end of the treatment, cells were analyzed to quantify cellular senescence, cell proliferation, and cell death. The reparative capacity of CSC was evaluated in a mouse model of acute myocardial infarction (AMI). Cardiac function and histology were studied 2 weeks post-AMI. F-CSC are characterized by a significant activation of TORC1 complex and reduced in vivo reparative potential. A short term pharmacological treatment of F-CSCs resulted in a significant: a. reduction of p16+ cells, and increase of apoptosis in rapamycin treated cells, b. reduction of p21+ and γH2A.X+Ki67- cells, together with an increase in CSC proliferation in resveratrol treated cells, and c. decrease of γH2A.X+Ki67- cells in DETA/NO treated cells. In vitro treatment of CSC obtained from pathologic hearts with 10nM Rapamycin and 0.5µM Resveratrol prior to their in vivo administration to infarcted mice restored their reparative ability in vivo.
49 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Engraftment responses of human bone marrow stromal cells and unselected mononuclear cells are dependent on the physical extracellular matrix but not the chemotactic factor SDF1 Owen Bain, Giulia Detela,Christopher Mason, Anthony Mathur, Ivan Wall University College London, Barts and the London NHS Trust Recently, a growing number of clinical trials have assessed bone marrow-derived cells as candidate therapies for ischemic injury to the myocardium. However, only mild improvements in cardiac function are reported. Furthermore, cell engraftment and survival are very low as many cells either undergo anoikis or migrate away. Several pre-clinical studies have reported that preconditioning bone marrow-derived stromal cells (MSCs) with factors such as SDF1 increases their engraftment in ischemic myocardial tissue, resulting in improved cardiac output. Therefore, we hypothesised that pre-stimulating MSCs with SDF1 would enhance cellular responses associated with cell engraftment and provide clinically relevant data about how to improve engraftment of cells in the heart. We tested the effect of SDF1 on early attachment and subsequent migration of human MSCs on fibronectin versus BSA-blocked tissue culture plastic. Additionally, we sought to address whether conditions that support angiogenesis induce formation of vessel-like structures by MSCs in vitro. We also addressed the effect of SDF1 on mediating these same cellular functions in whole bone marrow mononuclear cells (BMCs) isolated from patients with ischemic heart disease. To mimic the ischemic environment that prevails in injured myocardial tissue, assays were conducted under hypoxic conditions (2% oxygen tension), with normoxia (20%) used as a control. We found that early attachment to fibronectin was significantly greater than BSA control, both in normoxia and hypoxia (p<0.01). However, in hypoxia, attachment was greatly diminished at 5 min (p<0.01) and to a lesser extent at 20 min compared to normoxia, indicating delayed activation of adhesion receptors. However, SDF1 did not rescue MSCs from the effects of hypoxia or enhance the responsiveness to fibronectin. Preconditioning the cells with SDF1 did not enhance migration of MSCs towards SDF1 in transwell migration assays. Hypoxia seemed to marginally enhance MSC capacity to form vessel-like structures, although none of the data was statistically significant. In the case of BMCs from trial patients, a similar pattern was seen for attachment whereby fibronectin enhanced cell attachment and hypoxia diminished the early (5 min) response. In conclusion, hypoxia substantially diminished attachment of MSCs and patient-derived BMCs. SDF1 did not lend any positive effect to the functional response of MSCs in low oxygen tension and/or on fibronectin matrix.
50 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Nkx2-3 homeodomain transcription factor as a master determinant for the vascular patterning of spleen and intestinal lymphoid tissues Péter Balogh, Zoltán Kellermayer, Árpád Lábadi, Tamás Czömpöly, Hans-Henning Arnold University of Pécs, Hungary, and Technical University of Braunschweig, Germany In addition to supplying lymphoid tissues with nutrients, specialized local vasculature is required for the efficient entry of leukocytes to peripheral lymphoid organs. Little is known either in human or mouse about those developmental factors that direct the patterning of endothelial cells to display the tissue-specific addressin signature in these vessels, distinguishing peripheral lymph nodes, mucosal lymphoid tissues and spleen. We present our findings on the role of Nkx2-3 homeodomain-containing transcription factor in determining the vascular specification of the spleen and gut-associated lymphoid tissues including Peyer's patches and mesenteric lymph nodes using immunohistological, functional and gene expression approaches. In the absence of Nkx2-3 the spleen undergoes a near-complete reversal for peripheral lymph node-like vascular patterning, including the formation of lymphoid sacs expressing LYVE-1 lymphatic endothelium-associated marker and also the development of ectopic high endothelial venules with PNAd expression, in a lymphotoxin beta receptor dependent pathway. In intestinal lymphoid tissues a MAdCAM-1→PNAd shift occurs postnatally in high endothelial venules, thus licensing these vessels to perform an altered lymphocyte homing mechanism. We propose that the role of Nkx2-3 is to preserve the unique vascular patterning of the spleen and gut-associated lymphoid tissues via determining their addressin specification.
51 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
VEGF over-expression in skeletal muscle induces vascular enlargement and intussusceptive angiogenesis through reciprocal activation of Notch-1 in contiguous endothelial cells. Roberto Gianni-Barrera (1), Marianna Trani (1), Silvia Reginato (1), Ruslan Hlushchuk (2), Michael Heberer (1), Valentin Djonov (2) and Andrea Banfi (1) (1) Cell and Gene Therapy, Departments of Biomedicine and of Surgery, Basel University Hospital, Switzerland (2) Institute of Anatomy, University of Bern, Switzerland VEGF can induce either normal or aberrant angiogenesis depending strictly on its dose in the microenvironment around each producing cell in vivo. During sprouting of new vessels, an alternate pattern of Dll4 expression and Notch1 activation in contiguous endothelial cells leads to the formation of migrating tip and proliferating stalk cells. Here we sought to determine the mechanisms by which physiologic and aberrant vessels are induced by over-expression of different VEGF doses in adult skeletal muscle. Clonal populations of retrovirally transduced myoblasts were implanted in limb muscles of SCID mice to homogeneously express specific low and high VEGF levels (60 and 120 ng/million cells/day), previously shown to induce physiologic and therapeutic angiogenesis, or aberrant angioma growth, respectively. Three independent and complementary methods (confocal microscopy, vascular casting and 3D-reconstruction of serial semi-thin sections) showed that, at both VEGF doses, angiogenesis took place without sprouting. Rather, an initial circumferential enlargement of pre-existing vessels took place by 4 days through VEGF-induced endothelial proliferation, followed by intussusception, or vascular splitting, by 7 days. Remarkably, an alternate pattern of Dll4 expression and Notch1 activation was never detected. During initial enlargement by both VEGF doses, long stretches of contiguous endothelial cells both expressed Dll4 and at the same time had activated Notch-1 intracellular domain in the nuclei. After 7 days, intussusceptive remodeling generated either normal capillaries or aberrant angiomas, depending on VEGF dose, but neither structure showed Dll4 expression or Notch1 activation anymore. Notch inhibition, by either systemic DAPT treatment or co-expression of a soluble Dll4 isoform, disrupted the initial vascular enlargements and led to disorganized aggregates of endothelial cells. In summary, these data suggest that: 1) both normal and aberrant angiogenesis induced by over-expression of different VEGF doses arise through a process of enlargement, followed by intussusceptive remodeling, rather than sprouting; 2) the enlargement stage depends on the simultaneous and reciprocal activation of Notch1 in contiguous endothelial cells, leading to an all-stalk phenotype, rather than an alternate pattern of tip and stalk cells; 3) the subsequent intussuceptive remodeling into either normal or aberrant vessels is not regulated by differential Notch1 activation.
52 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy iPS technology as a tool to investigate atrial fibrillation Benzoni P.1, Bisleri G.2, De Luca A.1, Crescini E.1, Barbuti A.3, Baruscotti M.3, Muneretto C.2, Richaud Y.4, Raya A.4, and Dell'Era P.1 1Dept. Biomedical Sciences and Biotechnology and 2Division of Cardiac Surgery, University of Brescia, Brescia 3Dept. Biomolecular Sciences and Biotechnology, University of Milan, Milan 4Control of Stem Cell Potency Group, IBEC, Barcelona. Atrial fibrillation (AF) is the most common arrhythmic disorder in adults, with a prevalence ranging around 0.5-2% in the general population. Atrial fibrillation is characterized by rapid and irregular activation of the atria, loss of coordinated atrial contraction thus resulting in reduced ventricular filling and blood stasis in the atria, and finally leading to heart failure and thromboembolic stroke. Several therapeutic strategies are currently available, either pharmacological or ablative, albeit the rate of success in terms of sinus rhythm restoration may consistently vary among patients. AF has traditionally been described as a multifactorial sporadic disease; however some hints about AF hereditability have recently been issued from epidemiological studies.Therefore in vitro AF models to identify the genetic basis, to examine cell differentiation, to characterize interactions of cells belonging to cardiovascular lineage, are urgently needed in order to improve the comprehension of the pathophysiological basis of AF. We began to characterize a familial group with continuous AF who were scheduled to undergo surgical ablation following failed pharmacological treatment. Screening for mutation of the most common genes associated to AF (KCNQ1,KCNH2, KCNE1, KCNE2, and SCN5A) did not show any modification. We are now in process of sequencing some other recently identified putative candidate (MiRP1, CAV3, and HCN4), but a more extensive analysis needs to be carried out. In order to provide a valuable experimental platform to model AF disease, primary cultures of dermal fibroblasts were established from all individuals. At low passage number, cells were induced to pluripotency by retroviral infection with the classical set of factors (OCT4, KLF4, SOX2 and c-MYC). Then, we isolated 2-4 independent lines per individual of AF-specific induced pluripotent stem cells (iPSC). We have begun to characterize the pluripotency state of our colonies, by colony morphology and growth dynamics, alkaline phosphatase staining, expression of pluripotency associated transcription factors (OCT4, SOX2, NANOG, REX1), surface markers (SSEA3, SSEA4, TRA1-60, TRA1-81), and silencing of retroviral transgenes. Following the assessment of pluripotency, we aim to differentiate AF-derived iPSC into cells of the cardiovascular lineage, such as cardiomyocytes and endothelial cells. We believe that the model we are developing will help us in clarifying the molecular basis of AF.
53 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Neuropilin-1 expressing monocytes (NEMs), a novel population of bone marrow-derived myeloid cells promoting vessel maturation Carrer A.1, Moimas S.1, Zacchigna S.1, Maione F.2, Mano M.1, Zentilin L.1, Kazemi, M.1, Bussolino F.2, Giraudo E.2, Giacca M.1 1International Centre for Genetic Engineering and Biotechnology, Trieste, Italy 2Department of Oncological Sciences, University of Torino School of Medicine, Candiolo, Italy In the attempt to satisfy the ever increasing demand for oxygen and nutrients, malignant cells incessantly produce angiogenic factors and co-opt a variegated assortment of accessory cells to sustain accelerated angiogenesis. The net result of this process is the generation of a leaky, chaotic and poorly functional vascular network, which paradoxically favors further cellular infiltration and tumor growth in a vicious cycle that fosters tumor progression. We have previously described a novel population of Semaphorin3A (Sema3A)-responsive monocytes, characterized by the unique ability to sustain vessel maturation. These cells express Neuropilin-1 (Nrp1), a receptor crucially involved in their mobilization. Hence, we wondered whether this peculiar cell population also infiltrates the tumor microenvironment and which is its contribution to tumor angiogenesis. Infiltrating cells were isolated from Sema3A-overexpressing muscles and injected into syngeneic tumor xenografts eventually causing a significant inhibition of tumor growth. Extensive characterization of Sema3A-recruited cells led to the definition of these cells as CD11b+/Nrp-1+/Gr1- (nicknamed Neuropilin-1 Expressing Monocytes – NEMs – for short). When directly purified from the bone marrow, NEMs migrated in response to Nrp1 ligands in a dose dependent manner, did not affect tumor or endothelial cell proliferation in vitro, but strongly promoted vascular smooth muscle cells migration. Accordingly, injection of these cells into growing tumors induced vascular normalization as determined by the relative increase in α-SMA+ and NG2+ mural cell coverage and by the reduction of tumor leakage. Notably, no vessel pruning was noticed as vessel density was not affected. Tumor-associated hypoxia was also decreased, as unveiled by pimonidazole staining. Most importantly, cell administration reduced tumor growth in mice inoculated either with B16. F10 melanoma or 4T1 breast carcinoma cells and inhibited 4T1 metastatization to the lung. Similarly, injection of purified CD11b+/Nrp-1+/Gr1- cells into the mesenteric artery of 12 weeks-old RipTag mice, strongly impeded tumor progression by inducing vessel normalization. RNA-Seq analysis of purified NEMs revealed that they express abundant levels of molecules known to be involved in vessel maturation, among which PDGFb, TGF-β, Thrombospondin-1 and Oncostatin-A. Ingenuity Software analysis significantly associated such expression profile with arteriogenic capability. qPCR confirmed this signature, which clearly distinguishes these cells from other tumor infiltrating cells, such as TAM, TEM and Gr1+-MDSC. To assess the relevance of this novel cell population in human malignancy, we analyzed the expression of Sema3A in human colo-rectal carcinoma. We observed that high levels of Sema3A expression correlated with less aggressive tumor growth, longer patient survival, improved mural cell coverage and, most strikingly, tumor infiltration with CD11b+/Nrp1+ mononuclear cells. This observation is consistent with the conclusion that Sema3A might lead to tumor vessel normalization through the recruitment of the specific NEM population also in humans.
54 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Transcriptional analysis reveals new candidate modulators of IFN-α response in endothelial cells Francesco Ciccarese 1,2, Angela Grassi 2,3, Barbara Di Camillo 4, Gianna Toffolo 4 and Stefano Indraccolo2 1 DiSCOG, Università degli Studi di Padova, Italy 2 Istituto Oncologico Veneto I.R.C.C.S., Padova, Italy 3 Institute of Biomedical Engineering, National Research Council, Padova, Italy 4 DEI, Università degli Studi di Padova, Italy Interferon (IFN)-α is a cytokine endowed with potent pleiotropic biological activities, achieved through the induction of hundreds of interferon-stimulated genes (ISGs). Among its actions, inhibition of tumor angiogenesis is one of the most interesting. Although some mediators of IFN-α effects have been identified among ISGs, it is still unknown whether they may account for individual differences in the response to this cytokine. Aim of this study was to investigate the possibility that some IFN-α-stimulated genes might influence the entire signal transduction pathway, in order to find out new modulators of IFN-α actions. Primary endothelial cells (HUVECs) were pulsed in vitro with human recombinant IFN-α under different perturbation conditions (siRNA inactivation of target genes) and the transcriptional response of a panel of 96 transcripts was analysed. The monitored genes were screened by custom TaqMan Low-Density Arrays. Stealth siRNA were used for RNAi-mediated knockdown of candidate IFN-α modulators: STAT1, IRF1, IRF7, GBP1, OAS2, IFIH1 and USP18. Some of the identified significant modulations are currently being evaluated at the functional level. STAT1 and USP18 silencing produced the most evident effect among the perturbations performed. Following the silencing of STAT1, several genes were significantly down-regulated and only few genes up-regulated, including IFNα-1 and IFNα-R1. USP18 silencing, on the other hand, caused a massive up-regulation of genes, including some involved in cell-to-cell adhesion (e.g. DSP). The possibility that USP18 could influence vascular biology is currently being evaluated through analysis of apoptosis, proliferation and migration ability of endothelial cells transfected with USP18 targeting siRNA. Identification of new modulators of IFN-α response by a systems biology approach may contribute to uncover novel aspects of its anti-angiogenic activity. Our results may be important in defining the mechanisms through which inflammatory cytokines could influence the pathophysiological process of neoangiogenesis in tumors and the embryonic process of vasculogenesis.
55 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Tbx1 is required in brain endothelial cells to establish vascular patterning Sara Cioffi1, Marchesa Bilio1, Stefania Martucciello2, Elizabeth Illingworth1 1 CNR/institute of genetics and biophysics, Naples, Italy; 2 University of Salerno, Salerno, Italy Purpose: TBX1 mutation causes most of the physical features of 22q11.2 deletion syndrome (22q11.2DS), in humans and mice, but it is not known whether it contributes to the behavioral disorders and psychiatric diseases that characterize this syndrome. It has been hypothesized that some neuroanatomical defects in 22q11.2DS have a vascular basis. The purpose of this study was to determine in mice whether brain vascular development is affected by Tbx1 mutation. Methods: We evaluated gene and protein expression by quantitative RTPCR and western blotting respectively on histological sections of whole brains or on brain endothelial cells (ECs) isolated from Tbx1 mutants. Brain vessel density was measured by the reconstruction of confocal images from histological sections where brain ECs were identified by Glut1 immunostaining. Vessel permeability and perfusion were evaluated by the infusion of appropriate tracer molecules. For experiments performed on cultured ECs, Tbx1 expression was knocked down by RNA-interference. Results: We show that in embryonic and adult mouse brain, Tbx1 is expressed in ECs and cell-fate mapping indicates that most brain vessels derive from Tbx1-expressing cells. Loss of Tbx1 causes brain vascular abnormalities consisting mainly of a widespread increase in vessel numbers, increased angiogenic sprouting and branching and vessel disorganization. This phenotypic picture is reproducible in 3D matrigel cultures of ECs after Tbx1 knockdown by siRNA, where there is increased microtubule branching. Thus, the in vivo vascular phenotype is likely to be cell-autonomous. Tbx1 mutant brain vessels have normal permeability, but in EC-specific Tbx1 null embryos some brain vessels are poorly perfused. At the molecular level, we show that in ECs TBX1 regulates Delta-like ligand 4 (DLL4), a transmembrane ligand for the Notch family of receptors that plays a key role in angiogenesis. Like Tbx1, Dll4 is anti-angiogenic, and Dll4+/- mice have a striking hypervascularization phenotype that is similar to that of Tbx1 null embryos. Conclusions: We show that Tbx1 is required in brain endothelial cells to establish vascular patterning. Our data suggest that a newly identified molecular pathway, Tbx1-Dll4/Notch1, may regulate brain angiogenesis in mice. Our study should encourage clinicians to look for brain microvasculature defects in 22q11.2DS patients and, if found, seek correlations with 22q11.2DS-related neuroanatomical defects.
56 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Nitric Oxide Modulation of Endothelial Progenitor Cells: Increased Contribution to Skeletal Muscle Remodeling? Valentina Conti 1, Emanuele Azzoni 2, Silvia Brunelli 1,2 1 Department of Experimental Medicine, University of Milano-Bicocca, Monza (MB), Italy 2 Division of Regenerative Medicine, Stem Cells and Gene Therapy, DIBIT, San Raffaele Scientific Institute, Milan, Italy Skeletal muscle (skm) is a stable tissue with slow cell turnover, but it has a remarkable regenerative capacity after injury. Skm healing is mediated by satellite cells, but several other mesodermal progenitors have been shown to participate in this process under experimental conditions. These populations (called vascular associated progenitors (VAPs) for being mainly associated with the microvasculature) contribute to skm regeneration by direct differentiation in new myofibers and by improving neovascularization. Trying to identify molecules that can trigger skm repair through endogenous stem cell activation, reserch primarily focused on agents that induce VAPs’ proliferation and differentiation in vitro, such as nitric oxide (NO). NO is a key modulator of skm physiology and enhances the regeneration process via specific actions on satellite cells. NO also mediates vascular smooth muscle relaxation, leading to microvessel vasodilation and increased capillary permeability. In order to ascertain whether NO treatment increases VAPs’ contribution to skm in vivo we used a mouse line expressing an inducible Cre recombinase under the control of the endothelial specific Ve-Cadherin promoter. Crossing this mice to different reporter mice (R26RNZG or R26R-EYFP) and supplying tamoxifen during embryogenesis or in postnatal stages we could follow the fate of Ve-Cadherin expressing cells. The NO-donor drug molsidomine was given daily in the diet of pregnant mothers or adult mice (3 mg/kg bw) in order to evaluate VAPs’ contribution during skm development or remodeling after damage. We have shown that embryonic VAPs normally contribute to skm fibers in both foetal and perinatal stages, but not to satellite cells. NO treatment during embryogenesis effectively increases this contribution and we could detect myonuclei and satellite cells of endothelial origin. We also proved that in the adult, after acute damage, embryonic VAPs contribute to regenerating myofibers and that this contribution is dependent on circulating progenitors. NO-treated mice exhibit an accelerated regeneration, accompanied by a greater VAPs’ contribution to centronucleated myofibers and an enhanced neovascularization, with a marked increase in capillary number and caliber. Both these aspects are responsible for the improved muscle healing previously observed in NO-treated mice and we are now extending our analysis in order to define the optimal treatment conditions to achieve effective tissue repair.
57 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Gap junctional coupling with cardiomyocytes is necessary but not sufficient for cardiomyogenic differentiation of cocultured human mesenchymal stem cells Arti A. Ramkisoensinga, Daniël A. Pijnappelsa, Jim Swildensa,b, Marie José Goumansb, Willem E. Fibbec, Martin J. Schalija, Douwe E. Atsmaa, Antoine A.F. de Vriesa,b aDepartment of Cardiology, bDepartment of Molecular Cell Biology, cDepartment of Immunohematology and Blood Transfusion, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, the Netherlands. Rationale: Gap junctional coupling (GJC) is important for functional integration of transplanted cells with host myocardium. Accordingly, in this study, the role of connexin43 (Cx43), the major gap junctional protein of the heart, in the cardiomyogenic differentiation of human mesenchymal stem cells (hMSCs) was investigated. Methods: Lentiviral vectors (LVs) encoding Cx43 gene-specific short hairpin RNAs were used to inhibit Cx43 expression (Cx43↓) in naturally Cx43-rich fetal amniotic membrane (AM) hMSCs. Overexpression of Cx43 (Cx43↑) in inherently Cx43-poor adult adipose tissue (AT) hMSCs was accomplished with an LV coding for human Cx43. These hMSCs as well as hMSCs transduced with control LVs were exposed to cardiomyogenic stimuli by co-incubation with neonatal rat ventricular cardiomyocytes (nrCMCs). Cardiomyogenic differentiation was assessed by immunostaining and whole-cell current-clamping. A human lamin A/C-specific antibody was used to distinguish human from rat cells in the hMSC-nrCMC co-cultures. The Cx43↓ fetal AM hMSCs were transduced with an LV encoding human Cx45 to establish whether the effects of Cx43 knockdown could be reversed. Results: Cx43↓ fetal AM hMSCs contained ± 4-fold less Cx43 than control cells whereas overexpression of Cx43 in adult AT hMSCs resulted in a ± 18-fold increase in Cx43 level. Dye transfer experiments showed that knockdown of Cx43 expression in fetal AM hMSCs strongly reduced their functional coupling with co-cultured nrCMCs while the coupling efficiency between adult AM hMSCs and nrCMCs was increased several-fold by Cx43 overexpression in the adult AM hMSCs. Ten days after co-incubation not a single Cx43↓ fetal AM hMSC, control adult AT hMSC or Cx43↑? adult AT hMSC stained positive for -actinin, while control fetal AM hMSCs did (2.2±0.4%). Action potential recordings after pharmacological gap junctional uncoupling revealed that functional cardiomyogenic differentiation occurred only in control fetal AM hMSCs. Cx45 overexpression in Cx43↓ fetal AM hMSCs restored their ability to undergo cardiomyogenesis in co-culture with nrCMCs with 1.6±0.4% of the hMSCs showing α-actinin immunoreactivity. Conclusion: GJC is required for differentiation of fetal AM hMSCs into functional cardiomyocytes after co-incubation with nrCMCs. Heterocellular GJC thus plays an important role in the transfer of cardiomyogenic signals from nrCMCs to fetal hMSCs but is not sufficient to induce cardiomyogenic differentiation in adult AT hMSCs.
58 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Therapeutic potential of Askina® Gel in regenerative medicine Delle Monache Simona*, Giuliani Antonio§, Evtoski Zoran&, Sanità Patrizia&, Amicucci Gianfranco§ and Angelucci Adriano& *Department of Biomedical Sciences and Technologies; &Department of Experimental Medicine; §Department of Surgery Neovascularization is a critical determinant in wound-healing outcomes for several kinds of injuries. Tissue engineering is continuously searching for matrixes, scaffolds, cells and growth factors to create microenvironment able to promote tissue regeneration. Moreover, an adequate vascularization of the wound milieu is a necessary goal in regenerative medicine also in the case of transplanted cells utilization. A new focus of interest in regenerative medicine research is represented by the use of mesenchymal stem cells (MSCs). They are capable of self-renewal and multi-lineage differentiation and can be considered a potential autologous cell source. Adipose-derived stem cells (ASCs) represent an abundant source of MSCs easily accessible from subcutaneous and visceral adipose tissue. ASCs can be cultured as monolayers on tissue culture plastic loosing in this way cell-specific properties and so poorly reflect in vivo ASCs behaviour. We suggest that a sterile hydrogel (Askina® Gel) containing a modified starch polymer and commonly used in surgery, could serve as scaffold to improve neovascularization and cell regeneration. Therefore, we first evaluated the ability of this hydrogel to promote tubule formation in vitro using mouse endothelial cells (MS-1). We observed that when cultured on hydrogel, MS-1 form spontaneously capillary-like structures after 36-48 h from seeding. Conversely, MS-1 pretreated with an inhibitor of VEGFR2 (SU 1498) were not able to make tubules at the same times. In parallel, hydrogel samples were surgically implanted in the back of CD-1 mouse, and harvested at 7-10 days post-implantation. The analysis of hydrogel plugs cryosections by H&E staining and by immunofluorescence revealed a remarkable neovascularization in hydrogel plugs preloaded with growth factors. Finally, since ACSs can form three-dimensional spheroids with increased stemness capability when cultured on biomaterials, we tested the ability of ASCs isolated from human abdominal adipose tissue recovered from obese subjects (35-45 years old), to form spheroids on hydrogel. We demonstrated that hydrogel was effectively able to promote spherical cell aggregates formation that most of the cells into spheroids were viable. In conclusion, these preliminary results suggest that hydrogel, besides conventional use in surgery, could be used as good vehicle for the retention of transplanted stem cells at the cell graft promoting neo-angiogenesis and tissue regeneration.
59 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Engraftment responses of human bone marrow stromal cells are enhanced by Notch ligands in vitro Giulia Detela, Owen Bain, Anthony Mathur, Ivan Wall Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, United Kingdom. Bone marrow-derived mesenchymal stromal cells (MSCs) have been explored as a therapy for myocardial infarction, yet clinical trial results reveal only modest improvements in cardiac function and minimal engraftment of delivered cells. Little evidence exists regarding the molecular mechanisms that are required for long-term cell retention within the heart. Indeed, the identification of cell delivery methods that achieve sustained engraftment remains one of the primary research goals for cardiac cell therapy. Based on the central role of Notch in vascular development and in activation of adhesion receptors, we hypothesised that stimulation of Notch signalling would enhance engraftment capabilities of MSCs that might ultimately support vascular regeneration in ischemic myocardium. Our aim was to characterise the effect of Notch ligands Jagged 1 (Jag1) and Delta-like 4 (Dll4) on engraftment responses of human MSCs. Human MSCs (Stro1+/Thy1+/CD44+/CD146+/CD14-/CD19-) were expanded to passage 2 and functional characterisation of cell attachment and migration was assessed on extracellular matrix components fibronectin and type I collagen. Acute preconditioning of cells with the Notch ligands Jag1 or Dll4 for 45 minutes was conducted prior to performing the assays and cell responses were characterised in the presence or absence of these ligands. Additionally, we assessed formation of vessel-like structures using endothelial cell-MSC co-cultures on matrigel. Cell attachment assays revealed that MSCs attached more rapidly to fibronectin than to collagen (p<0.01) and Jag1 enhanced early cell adhesion on fibronectin (p<0.05) whereas Dll4 did not. In terms of migration, type I collagen facilitated more rapid cell migration towards chemotactic stimuli (FBS or SDF1) than fibronectin (p<0.05). Both Jag1 and Dll4 enhanced chemotactic cell migration across membranes coated with fibronectin and type I collagen (p<0.05). When co-cultured with endothelial cells in angiogenic conditions, MSCs preconditioned with Notch ligands marginally enhanced vessel branch point formation compared to untreated MSCs. In conclusion, our preliminary data supports the hypothesis that Notch ligands can enhance MSC engraftment potential and enhance functionality that supports vascular responses. Current studies in our lab aim to further understand the impact of preconditioning on improving cell function in vivo for the treatment of cardiovascular diseases.
60 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Effects of SOX2 overexpression on relative cell cycle distribution and cell migration of NT2/D1 cell clones Danijela Drakulic, Aleksandar Krstic, Andrijana Klajn, Milena Stevanovic Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, PO BOX 23, 11010, Belgrade, Serbia NT2/D1 is a widely characterized pluripotent embryonal carcinoma cell line that resembles early embryonic stem cells in morphology, antigen expression patterns, biochemistry, developmental potential and gene regulation. Although, this cell line is a well known system of ectodermal differentiation, results from literature suggest that NT2/D1 cells can activate genes of mesodermal lineages, including cardiac specific genes. SOX2, a universal marker of pluripotent stem cells, is a transcription factor with critical role in the regulation of genes required for self-renewal and pluripotency of stem cells. Furthermore, this transcription factor helps control embryonic development in vertebrates. Also, it was reported that the altered expression of SOX2 can influence cell cycle transition and cellular migration. In order to analyze relative distribution of cells in cell cycle phases and migratory activity of SOX2-overexpressing NT2/D1 cell clones we performed flow cytometry analysis and wound-healing assay, respectively. Flow cytometry analysis by propidium iodide nuclei staining revealed that the relative distribution of cells in cell cycle phases was not altered in SOX2-overexpressing NT2/D1 cell clones when compared to parental NT2/D1 cells. No increase in population of cells undergoing apoptosis was detected in these cell clones. In cell clone with increased SOX2 expression by approximately 3.1 fold when compared to the parental cell line, increased migratory activity was detected by wound-healing assay. Taken together, we demonstrated that SOX2 overexpression resulted in altered migration potential and unaltered cell cycle transition of NT2/D1 cell clones.
61 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Effects of SOX2 over-expression on pluripotency, proliferation and differentiation of embryonal carcinoma stem cells NT2/D1 Milena Stevanovic, Danijela Drakulic, Aleksandar Krstic and Andrijana Klajn Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, PO BOX 23, 11010, Belgrade, Serbia Transcription factors SOX2, Oct4 and Nanog are recognized as master regulators that orchestrate mammalian embryogenesis as well as the self-renewal and pluripotency of embryonic stem (ES) cells. These factors play essential roles in determining the fate of ES cells, regulating two distinct and opposing functions: self-renewal and differentiation. In addition, SOX2 is one of the key transcription factors involved in reprogramming of somatic cells and production of induced pluripotent stem cells (iPS). Considering the critical role of the SOX2 transcription factor in the regulation of genes required for self-renewal and pluripotency, we developed and characterized stable SOX2 over-expressing cell clones using embryonal carcinoma stem cells NT2/D1. NT2/D1 pluripotent cells closely resemble early ES cells in morphology, antigen expression patterns, biochemistry, developmental potential and gene regulation. Southern blot confirmed the integration of exogenous SOX2 copy, while semi-quantitative RT-PCR revealed SOX2 over-expression in two NT2/D1 cell clones. Our results demonstrated that SOX2 over-expression in NT2/D1 cell clones resulted in altered expression of key pluripotency genes OCT4 and NANOG. It was previously shown that constitutive expression of Sox2 in neural progenitors results in maintenance of proliferation and self-renewal, as well as in blocking of neuronal differentiation. However, SOX2-overexpressing NT2/D1 cell clones entered into retinoic acid-dependent neural differentiation, even in the presence of elevated SOX2 expression. After 21 days of induction by retinoic acid, expression of neural markers (neuroD1 and synaptophysin) was higher in induced cell clones than in induced parental cells. One of the cell clones with SOX2 over-expression has an approximately 1.3 fold higher growth rate, compared to parental cells. Taken together, we developed two SOX2-overexpressing cell clones that retained constitutive SOX2 expression after three weeks of retinoic acid treatment. SOX2 over-expression resulted in altered expression of pluripotency-related genes, increased proliferation, and altered expression of neural markers.
62 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Effect of extracellular matrix on human embryonic stem cell behavior Kevin Dzobo and M.I. Parker International Centre for Genetic Engineering and Biotechnology, Cape Town Component, Cape Town, South Africa The activity of stem cells is governed by the micro-environment, or `niche’, consisting of the basement membrane of the myofiber, resident supporting cells, vascular system and the extracellular matrix (ECM). The ECM surrounding cells consists of a network of glycoproteins like collagens, laminin and fibronectin organized by proteoglycans such as heparin sulfate. The cellular response to the ECM is predominantly mediated through a class of heteromeric transmembrane glycoproteins, the integrins. These proteins consist of an α and a β subunit which combine and form a multitude of functional protein complexes that transmit signals for activation of cellular processes like migration, proliferation, survival and differentiation. The activity of stem cells is governed by the micro-environment, or `niche’, consisting of the basement membrane of the myofiber, resident supporting cells, vascular system anApparently, the ECM is important for cell identity and behavior and provides a suitable environment to sustain the self-renewal and differentiation capacity of primary stem cells. Therefore, by using the ECM components we can investigate differentiation and regulation of signaling pathways involved in stem cells differentiation. We used the muscle extracellular matrix proteins collagen type I, fibronectin, laminin, and also gelatin and Matrigel as surface coatings of tissue culture plastic to resemble normal extracellular matrix. In this study, we examine the ECM components collagen, laminin, fibronectin, gelatin and Matrigel for their capacity to improve the proliferation and differentiation capacities of cultured human embryonic stem cells.
63 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Functional screening identifies microRNAs inducing cardiac regeneration Ana Eulalio, Miguel Mano, Matteo Dal Ferro, Lorena Zentilin, Serena Zacchigna and Mauro Giacca Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy In mammals, enlargement of the heart during embryonic development is primarily dependent on the increase in cardiomyocyte number, but shortly after birth cardiac myocytes stop proliferating and further growth of the myocardium occurs through hypertrophic enlargement of existing myocardial cells. As a consequence of the limited proliferation capacity of adult cardiomyocytes, the ability of the mammalian heart to regenerate itself following injury is very restricted. Despite extensive research, the exact molecular mechanisms controlling proliferation and differentiation of cardiac myocytes remain largely unknown. A novel, exciting possibility is that microRNAs - evolutionarily conserved small noncoding RNAs that regulate gene expression at the post-transcriptional level - may actively control cardiomyocyte proliferation. Several microRNAs have been implicated in different aspects of heart function and dysfunction, though only a few of them have so far clearly been shown to take part in the control of cardiomyocyte proliferation, all exerting a negative role. Taking into consideration the vast number of microRNAs and the impact that the few examined so far have in heart development and disease, it is clear that further functions of microRNAs in cardiac function are awaiting discovery. To systematically identify microRNAs able to promote cardiomyocytes proliferation, we performed a high-content, fluorescence microscopy-based high-throughput screening in rat neonatal cardiomyocytes using a library of microRNA mimics corresponding to all the annotated microRNAs (~1000 microRNAs). Proliferation was assessed using the combination of different read-outs: EdU incorporation, Ki-67 expression, histone H3 phosphorylation and AuroraB-kinase localization to mid-bodies. We identified 38 microRNAs able to increase cardiomyocyte proliferation by at least 3-fold, as well as microRNAs able to completely block proliferation. Two of the microRNAs increasing cardiac myocyte proliferation were tested in vivo by both injecting the synthetic microRNA intracardiacally into the heart of newborn rats and by delivering their coding sequence using AAV9 vectors, which were injected intraperitoneally into neonatal mice. Both microRNAs were found to significantly induce proliferation of cardiomyocytes in vivo. Taking into consideration the increase in cardiomyocyte proliferation observed upon treatment with selected microRNAs in vitro and in vivo, we assessed the beneficial effect of these microRNAs on cardiac function after myocardial infarction. AAV9 vectors expressing the two selected microRNAs were injected into the infarct border zone after ligation of the coronary artery in mice, followed by histological, morphological and echocardiographic evaluation of cardiac function. The treated animals showed markedly reduced infarcted areas and improved cardiac function. Overall, our results indicate that the induction of cardiomyocyte proliferation in vivo by modulation of specific microRNAs might be the basis for the development of novel, exciting therapeutic approaches against ischemic cardiomyopathy and heart failure.
64 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Soluble VEGFR-1 interaction with α5β1 integrin triggers an endothelial cell dynamic and angiogenic phenotype Angela Orecchia1,2, Amel Mettouchi1, Paolo Uva3, Glenn Simon4, Diego Arcelli5, Simona Avitabile2, Gianluca Ragone5, Guerrino Meneguzzi1, Karl Pfenninger4, Giovanna Zambruno2, and Cristina Maria Failla2 1U634 Inserm, France; 2Molecular and Cell Biology Lab.,5Molecular Oncology Lab., IDI-IRCCS, Italy; 3CRS4 Bioinformatics Laboratory, Polaris Science and Technology Park, Italy; 4Department of Pediatrics, University of Colorado, USA Soluble vascular endothelial growth factor receptor-1 (sVEGFR-1) acts both as a decoy receptor for VEGFs and as an extracellular matrix associated protein binding to α5β1 integrin. A sVEGFR-1-derived peptide, named peptide 12, that interacts with and sustains α5β1 integrin clustering promotes in vitro and in vivo angiogenesis. However, canonical signal downstream integrin activation is not induced, resulting into impaired focal adhesion assembly and delay in cytoskeleton reorganization. In fact, upon endothelial cells (ECs) adhesion to sVEGFR-1 or peptide 12, phosphorylation of FAK and Shc is not induced, and Erk1/2 is phosphorylated only in the concomitant presence of VEGF-A. To address the mode of action of sVEGFR-1/α5β1 integrin interaction in the induction of angiogenesis, we analyzed gene expression in ECs adhering to sVEGFR-1, to fibronectin (FN), or in non-adhering conditions. We found that a few biological pathways, specifically "adhesion-dependent regulation", "cytoskeleton rearrangement" and "angiogenesis", were differently modulated in ECs adhering to sVEGFR-1 versus FN. Three protein kinase-C (PKC) substrates belonging to these pathways, i.e. adducin, myristoylated alanine-rich C-kinase substrate (MARCKS), and radixin, were differently phosphorylated. We detected similar levels of phosphorylated PKCα in sVEGFR-1 and FN adhering ECs, but adducin and MARCKS were less phosphorylated, even if total protein amounts were higher. Conversely, radixin was substantially phosphorylated in sVEGFR-1 adhering ECs and activation of the small GTPase Rac1 was detected in the absence of a concomitant Cdc42 or RhoA activation.
We also showed the activation of a pathway involving the heterotrimeric G protein α13. These data indicate that ECs adherent to sVEGFR-1 acquire a highly motile phenotype, confirmed by the protracted presence of dynamic adhesions instead of focal adhesions. Moreover, we demonstrated that radixin is responsible for the angiogenic effect of sVEGFR-1/α5β1 integrin interaction, and that this outcome depended on an activated VEGFR-2. Overall, our data indicate that an initial presence of sVEGFR-1 in the EC microenvironment is necessary to shift α5β1 integrin signaling from the “classical” adhesion pathway to that which induces the acquisition of a dynamic, more motile phenotype
65 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Open chromatin conformation at Notch1 target genes sustains neonatal cardiomyocyte proliferation Giulia Felician, Chiara Collesi, Marina Lusic, Matteo Dal Ferro, Lorena Zentilin and Mauro Giacca Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano, Trieste, Italy, 34149 Notch signaling is critical for proper myocardial development. Our previous work has shown that Notch1 plays a pivotal role in the regulation of cardiomyocyte proliferation during the perinatal age; in particular, loss of Notch1 signaling after birth correlates with withdrawal of cardiomyocytes from the cell cycle, both ex vivo and in vivo. We therefore wanted to analyze the chromatin conformation at Notch-1 responsive promoters (Notch1 itself, Hey1 and Hes1) at different time points in both replicating and differentiated neonatal rat cardiomyocytes. We found that, after birth, the exit of cardiomyocytes from the cell cycle correlates with the progressive reduction of transcription of these genes, concomitant with decreased modification of the active histone 3 (H3) acetylation and H3K4 methylation markers, increased levels of H3K27 trimethylation, a marker of repressive chromatin, and promoter association with Ezh2, a component of the PolyComb complex. Epigenetic marks of repressive chromatin were also detected in adult rat cardiomyocytes. Next we tried to activate Notch signaling by transduction with an Adeno-Associated-vector coding for either the constitutively activated Notch1 intracellular domain (AAV6-N1ICD), or the soluble form of the Jagged1 extracellular Notch ligand (AAV6-sJ1). Both treatments determined a strong increase in neonatal cardiomyocyte proliferation. This coincided with the maintenance, at the Notch1 responsive genes, of a chromatin profile similar to that detected immediately after birth in proliferating cardiomyocytes. Finally, we wanted to assess the effect of activating Notch1 signaling in adult hearts after myocardial infarction (MI). Infarcted mice were injected intramyocardially with AAV9-sJ1 and functional cardiac parameters were evaluated over time by echocardiography. At 30 and 60 days after MI, the AAV9-sJ1 transduced animals showed improved left ventricular ejection fraction, fractional shortening and reduced chamber dilatation compared to animals injected with a control AAV9 vector. In conclusion, the maintenance of Notch1 signaling after birth extends the proliferative potential of neonatal cardiomyocytes, while reactivation of the Notch pathway after MI improves functional outcome, by possibly expanding the ineffective cardiomyocyte proliferation normally occurring after myocardial damage.
66 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
The role of Ccbe1, a novel gene coding for an EGF-like domain protein, in the process of organogenesis of the heart João Furtado1,2*, Margaret Bento1,2, and José A. Belo1,2 1 Regenerative Medicine Program, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, Portugal. 2 IBB-Institute for Biotechnology and Bioengineering, Centro de Biomedicina Molecular e Estrutural, Universidade do Algarve Establishment of the biochemical and molecular nature of cardiac development is essential to understand the relationship between genetic and morphological aspects of heart formation. The need to identify genes that are differentially expressed between cell populations within the embryo and within the embryonic heart is critical to elucidate the complex processes of heart development. With this in mind, our laboratory focused on the identification and study of novel genes expressed and involved in the correct development of the vertebrate heart/hemangioblast precursor cell (HPC) lineages. A differential screening using Affymetrix GeneChip Chick system was performed, leading to the identification of several new genes expressed in the haematopoiesis, angiogenesis or cardiogenesis precursor lineages. One of these novel genes, was initially denominated Ccbe1 (collagen and calcium-binding EGF-like domain 1) that encodes for a novel secreted protein that contains a Calcium-binding EGF-like domain. In situ Hybridization analyses have shown that Ccbe1 is expressed since very early in cardiac mesoderm precursor cells (HH4) and later in the secondary heart field (SHF; HH12). Ccbe1 contains calcium binding EGF domains that have been shown to be involved in the control of proliferation. Since Ccbe1 is expressed in the SHF precursors and cell derivatives, we hypothesise that Ccbe1 signalling pathway might be required for appropriate SHF development. Indeed, knockdown of Ccbe1 using morpholino antisense oligonucleotides resulted in aberrant heart formation, in which the fusion of the heart fields was incomplete or failed to fuse. The potential role of Ccbe1 in the heart morphogenesis is currently being studied and the results will be discussed.
67 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Analysis of molecular profiling data to understand the whole-genome effects of HGF stimulus in the heart Stefano Gatti1, Christian Leo1, Simona Gallo1, Valentina Sala1, Enrico Bucci2, Alice Poli2, Daniela Cantarella3, Enzo Medico3, Tiziana Crepaldi1 1 Dep. of Anatomy, Pharmacology and Forensic Medicine, University of Turin, Italy 2 BioDigitalValley, Pont Saint Martin (Ao), Italy 3Institute for Cancer Research and Treatment, University of Turin, Italy The Hepatocyte Growth Factor (HGF) is a pleiotropic cytokine involved in many physiological processes, including skeletal muscle, placenta and liver development. However, the HGF downstream effectors and its effects on whole-genome transcription are poorly understood. To investigate the genes transcriptionally regulated by HGF in the heart we used the hearts of neonatal transgenic mice with cardiac-specific and tetracycline-suppressible expression of HGF for a full genome DNA microarray analysis. When comparing HGF-expressing versus control hearts we found a total of 249 transcripts significantly differentially expressed more than 1.7-fold (210 upregulated and 39 downregulated). To analyze our transcriptional data we used different bioinformatics tools and softwares. Combining these results, we finally selected 70 biological process, 35 molecular function and 9 KEGG pathways clusters that were significant in at least two softwares. The gene groups lead to identify the molecular mechanism that are more involved in the HGF stimulus in the heart. These analysis, in fact, showed that the transcripts modulated by HGF were enriched for metabolic functions including regulation of transcription, regulation of muscle development, protein translation, purine nucleotide byosynthesis, intracellular transport, polyamine metabolism, fatty acid catabolic process. Subsequently we performed further informatic meta-analysis based on literature knowledge, which allowed to create a co-occurrence network oriented to the positive regulatory role of Myc and Notch1 in controlling some of the genes downstream to HGF. These in silico data are supported by in vivo increasing cell proliferation in neonatal hearts of HGF-expressing mice. In conclusion our bioinformatics analysis of global gene expression may be useful for understanding the orchestrated processes of postnatal development in the cardiac environment, and show that HGF has an important role during the heart development since it prolongs the plastic and proliferative phase.
68 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Characterization of the c-Kit receptor function in cardiac regeneration by transgenic mouse models Gimmelli R.1, Di Siena S.1, Barbagallo F.1, Campolo F.2, Dolci S.2, Nori S.3, Isidori A.4, Lenzi A.4, Naro F.1, Pellegrini M.1 1 DAHFMO, "La Sapienza" Univ. of Rome 2 Dep. of Public Health and Cell Biology, "Tor Vergata" Univ. of Rome 3 Dep. of Pharmaceutical and Biomedical Sciences, Univ. of “Salerno”, Fisciano 4 Dep. of Experimental Medicine, "La Sapienza" Univ. of Rome Background: Cardiac stem cells expressing the tyrosine kinase receptor c-kit have been recently used in in vivo and in vitro cardiac regenerative studies. However, it remains to be clarified whether the c-kit receptor itself plays a critical role in the process of cardiac regeneration. In order to clarify this point, we will explore whether c-Kit receptor affects cardiac stem cells proliferation, survival, migration and differentiation after heart injury. Methods and Results: We have generated transgenic mice in which an activatory point mutation (c-KitD814Y mice) has been introduced in the kinase domain of the c-kit gene. Initially, we have analyzed c-kit expression in tissues and organs at different stages of embryonal and post-natal development through immunohystochemical and biochemical analyses. We have found that in two transgenic lines the receptor is highly expressed and activated in heart, testis and cerebellum, compared to wild type mice. In order to follow the fate of the c-Kit transgenic stem cells we crossed c-KitD814Y mice with mice expressing GFP under c-Kit regulative sequences control. By cytofluorimetric and fluorescence microscopy analyses, we observed a 2 fold of increase in the number of c-kit positive cells on heart samples from double transgenic mice at different ages. To verify the c-kit role in cardiac regeneration we performed a necrotic heart damage in vivo and monitored cardiac repair in transgenic mice versus wild-type mice. After 9 days the wounded hearts of transgenic mice presented a larger connectival tissue area compared to wild-type mice. On the contrary, after 45 days a consistent reduction of fibrotic area was observed in transgenic mice. These preliminary results suggest a faster repair of damaged heart area that contain stem cells with an activated c-kit receptor. Further in vitro and in vivo experiments will be performed to assess whether transgenic c-kit cells directly transdifferentiate into cardiomyocytes or whether they act in a paracrine manner. In summary, the generation of transgenic mice carrying a constitutively activated c-kit in cardiac stem cells, will allow to investigate the role of the receptor and to highlight the molecular mechanism underlying heart regeneration.
69 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
AAV2.9 Thymosin beta 4 gene therapy: requirements for therapeutic neovascularization R. Hinkel1, T. Trenkwalder1, F. Gesenhues1, A. Pfosser1, G. Stachel1, F. Götz1, C. Lebherz1, I. Bock-Marquette2, E. Hannappel3, C. Kupatt1 1Internal Medicine I, Klinikum Großhadern - LMU, München; Germany 2UT Southwestern Medical Center, Dallas, USA; 3Institut für Biochemie, Universität Nürnberg-Erlangen, Erlangen, Germany Thymosin beta4 (TB4), a vasoactive peptide, has been shown to promote angiogenesis in the developing heart. For therapeutic use, however, conductance vessel growth is viewed as inevitable, which is not provided by all vessel growth factors at the same rate. For this process, microvessel maturation and smooth muscle cell proliferation of collaterals appear necessary. We investigated the potential of TB4 to induce therapeutic neovascularization. Methods: In vivo, rabbits (n=5) underwent femoral artery excision on day0 and 5x1012 AAV2/9 expressing Tb4, Ang-2 or Lac-Z were injected intramuscular. Application of Tb4 was performed over the whole limb or in the upper and lower limb separately. Injection of AAV2/9-Ang-2 was performed selectively into the lower limb. In an additional group L-NAME, an unselective NO inhibitor was applied. Quantification of collaterals was measured via angiography on day7 and day35 (%d7). Regional perfusion was assessed by application of fluorescent microspheres (%d7). HPLC-Anaylsis of Tb4 protein levels and Immunohistochemistry for PECAM-1 positive cells (capillaries/muscle fiber = c/mf) and vessel maturation (NG-2 positive pericytes/capillary = p/c) were quantified. Results: Overexpression of Tb4 in chronic ischemia increased capillary density (1.96±0.1 vs. control 1.37±0.1c/mf), collateral growth (175±15% vs. 95±6 %d7 in controls) and blood perfusion (187±5%d7 vs. control 110±6%d7). Furthermore, pericyte coverage was stimulated in the Tb4-treated group (0.69±0.1p/c) compared to control (0.46±0.1 p/c). Transduction of Tb4 only into the lower limb was sufficient to induce collateral growth (177±4%d7) and blood perfusion in the upper limb (207±8%d7), whereas selective injection into the region of collateral formation in the upper limb resulted only in a slight improvement of arteriogenesis (150±3%d7) and blooflow (142±6%d7). Furthermore, nitric oxide mediated increases in flow and shear stress were necessary for collateral growth, since the unselective NO-inhibitor L-NAME prevented arteriogenesis (118±8%) and perfusion gain (123±13%) after LL-TB4 transduction. Conclusion: TB4 induces arteriogenesis of conductance vessels in the upper limb, even when applied remotely in the lower limb. The mechanisms include microcirculatory maturation, i.e.pericyte coverage, and backward signaling via shear stress and nitric oxide formation.
70 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Expression profile of striated muscle signaling protein Ankrd2 in neonatal rat cardiomyocytes and its putative role in heart function and development Snezana Kojic1, Valentina Martinelli2, Jovana Jasnic-Savovic1, Ljiljana Rakicevic1, Aleksandra Nestorovic1, Dragica Radojkovic1 and Georgine Faulkner3 1 Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Serbia 2 International Centre for Genetic Engineering and Biotechnology, Trieste, Italy 3 CRIBI, University of Padova, Italy Ankrd2 protein (ankiryn repeat protein 2) belongs to the MARP family together with cardiac specific regulator Ankrd1/CARP. Both proteins, apart from the mechanosensory function, have an important role in transcriptional regulation, myofibrillar assembly, cardiogenesis and myogenesis. In skeletal muscle, Ankrd2 has a structural role as a component of a titin associated stretch-sensing complex in the myofibril and a regulatory function as co-factor of transcription in the nucleus. Its role in myogenic differentiation and coordination of myoblast proliferation has been also demonstrated. Although Ankrd2 is expressed in heart, its role and molecular mechanisms that regulate its expression in the cardiac muscle are completely unknown. There is some evidence that supports the importance of Ankrd2 in heart muscle: i) its expression is potentially regulated by the cardiac specific regulatory factors Nkx2.5, HAND2 and CARP/Ankrd1 and ii) hypertrophic and dilated cardiomyopathy pathways are altered upon Ankrd2 silencing in human myotubes. In order to decipher the role of Ankrd2 in heart function and development, we have initiated studies on cardiac Ankrd2 using neonatal rat cardiomyocytes. Here, we demonstrate, for the first time, the expression profile of Ankrd2 in rat cardiac cells (both cardiomyocytes and fibroblasts) obtained by Western blot analysis using a panel of anti-Ankrd2 antibodies raised against the human recombinant protein. The pattern of Ankrd2 expression in neonatal rat heart is compared to that of adult rat and adult human heart. Extract of human skeletal muscles was used as a control. We also performed the localization studies of endogenous Ankrd2 protein in neonatal rat cardiomyocytes using immunofluorescense and confocal microscopy. Our results demonstrate, for the first time, that endogenous Ankrd2 is expressed in rat neonatal cardiomyocytes and could be detected by antibodies directed against human protein. It is found both in cytoplasm and nucleus, similar to its localization in myoblasts. Thus, the neonatal rat cardiomyocytes represent a suitable model system for further investigation of Ankrd2 protein in the heart.
71 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Effect of sterilization by gamma-irradiation on biocompatibility of starch-based polymers and composites Abdel Wahab El Naggar, Magdy M Senna, Soheir Korraa and Sanaa Refaay National Center for Radiation Research and Technology Atomic Energy Authority Studies with biodegradable starch-based polymers have recently demonstrated that these materials have a wide range of properties, which make them suitable for use in several biomedical applications, ranging from bone plates and screws to drug delivery carriers and tissue engineering scaffolds. The aim of this study was to screen the cytotoxicity and evaluate starch-based polymers and composites after being sterilized by ionizing radiation. Also, the effect of low level laser energy (LLLI) on enhancing the proliferation of cells on such scaffolds, were examined. The biocompatibility of three different blends of (100 % starch, starch mixed with 10 % cellulose acetate & 10 % acrylic acid & 5 % carboxy methyle cellulose and starch mixed with 10 % cellulose acetate & 10 % carboxy methyle cellulose. Scaffolds were sterilized by ionizing radiation and cells were allowed to grow on the designed scaffolds. After 48 hours cytotoxicity and cell adhesion properties were assessed. The MTT assay was performed with the extracts of the materials in order to evaluate the short-term effect of the degradation products. The cell morphology of endothelial cell line was also analyzed after direct contact with polymers and composites for 48 hours time periods and the number of cells adhered to the surface of the polymers was determined by quantification of the cytosolic lactate dehydrogenase (LDH) activity. Both types of starch-based polymers exhibit a cytocompatibility that might allow for their use as biomaterials. starch mixed with 10 % cellulose acetate & 10 % carboxy methyle cellulose.blends were found to be the less cytotoxic for the tested cell line, although cells adhere better to starch cellulose acetate surface. Scanning electron microscopy (SEM) analysis showed that cells were much more spread on the blend 1, blend 2 blend 3r. Measurements showed a higher number of cells on starch cellulose acetate surface. Exposure to 2 J/cm2 He:Ne laser greatly enhanced cell proliferation on all of the given scaffolds.
72 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Hyperpolarized 13C-pyruvate to study carbohydrate metabolism in beating heart by magnetic resonance imaging. V. Lionetti a, G. D. Aquaro b, F. Frijia b, L. Menchetti c, F. Santarelli c, V. Positano b , S. L. Romano a, G. Giovannetti c, L. Landini c, M. Lombardi b, F.A. Recchia a, d (a) Scuola Superiore Sant’Anna, Pisa, Italy; (b) Fondazione Toscana “G. Monasterio”, Pisa, Italy; (c) Institute of Clinical Physiology, CNR, Pisa, Italy;(d) Dpt. Physiology, Temple University, USA Tracking biochemical events in vivo is one of the most ambitious goals of modern physiology. Cardiac magnetic resonance (MRI) with hyperpolarized 13C-pyruvate is a promising new technique for the assessment of myocardial energy substrate metabolism, in intact hearts. We tested the effectiveness of this technique to quantify myocardial pyruvate uptake and the formation of its metabolites lactate (reduction) and CO2/bicarbonate (full oxidation in the Krebs cycle) in a pig model of acute ischemia/reperfusion. Seven pigs were chronically instrumented with a pneumatic occluder placed around the left anterior descending coronary artery. After recovery, they were subjected to MRI with a 3T scanner and a 13C quadrature birdcage coil. 20 ml of 13C-pyruvate (230 mM) were hyperpolarized using Dynamic Nuclear Polarization and injected intravenously at rest, during coronary occlusion (10 minutes) and at 5 minutes of reperfusion. During occlusion, we found a 17% decrease of 13C-lactate and a 62% decrease of 13C-bicarbonate in the area at risk as compared with remote myocardium. In the same area, the 13C-lactate signal increased by 27% during reperfusion, consistent with the enhanced pyruvate reduction in the post-ischemic phase, while 13C-bicarbonate was found persistently reduced, consistent with the expected presence of post-ischemic depression of the oxidative metabolism. In conclusion, 13C-pyruvate MRI is sufficiently sensitive to detect transient changes in regional myocardial metabolism in an in vivo model of ischemia-reperfusion.
73 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Identification of novel genes regulating AAV transduction of cardiomyocytes and other post-mitotic cells by high-throughput, genome-wide siRNA screening Miguel Mano, Jasmina Lovric, Rudy Ippodrino, Lorena Zentilin and Mauro Giacca Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy Over the last few years, viral vectors based on AAV have gained increasing popularity due to several advantageous characteristics, including their excellent safety profile, lack of inflammatory response, prolonged transgene expression, relative genetic simplicity and high efficiency of transduction of post-mitotic tissues, such as muscle, heart, brain and retina. Given the molecular simplicity of AAV vector particles, all the determinants of permissivity to vector transduction appear to reside among the molecular features of the host cell. Here we show that the cellular proteins of the DNA Damage Response (DDR) are negatively regulators of AAV transduction in cell culture and in vivo. We observed that cardiomyocyte and skeletal muscle permissivity to AAV transduction correlates with the loss of expression of Mre11, Rad50, Nbs1 (the MRN complex), which become downregulated upon terminal cell differentiation. In particular, the levels of these proteins, which bind the incoming AAV genomes and block its processing, fades in cardiomyocytes at approximately two weeks after birth, coinciding with increased AAV permissivity. Consistent with this information, in vivo treatment of relatively poorly permissive tissues such as the juvenile liver, with siRNAs against members of MRN markedly increases AAV permissivity of these tissues. To extend these observations and systematically identify novel host cell factors involved in the internalization, intracellular trafficking, AAV genome processing and AAV gene expression, we also performed a high-throughput screening using a genome-wide siRNA library (18,175 human gene targets) using a recombinant ssAAV2 vector expressing the firefly luciferase reporter gene. Using this approach, we identified 1528 genes that affect transduction of AAV vectors more than 4-fold (184 genes by more than 8-fold). Of these genes, 993 are inhibitors of AAV transduction, whereas 535 are required for efficient transduction by AAV vectors; most of these genes have not been previously associated with AAV transduction. Analysis of the inhibitor genes revealed a clear overrepresentation of genes related to DNA recombination and repair and cell cycle control. Of note, the 10 siRNAs most effective for AAV2 also significantly increased AAV transduction by other serotypes (e.g. AAV1, AAV5, AAV6, AAV9) and in various cell types, suggesting that the regulation of AAV permissivity mainly acts at a step subsequent to receptor binding and virion internalization. Transient inhibition of cellular factors repressing AAV transduction in vivo can be exploited to improve vector transduction to a clinically applicable level and extend vector efficacy to poorly permissive tissues.
74 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Functional characterization of stem cell-derived cardiomyocytes. Irene C. Marcu1, Pernilla Hoffmann2, Marisa Jaconi2, Nina D. Ullrich1 1 Department of Physiology, University of Bern, Switzerland; 2 Department of Pathology and Immunology, University of Geneva, Switzerland. The prospect of using stem cell-derived (SC-) cardiomyocytes in cardiac regenerative medicine provides new hope for the treatment of myocardial infarction (MI) and the prevention of heart failure. Despite of recent studies using SC-cardiomyocytes for preclinical and clinical trials, the functional properties of these newly developed cells have not yet been sufficiently described to ensure successful integration into the damaged myocardium. In this study, we characterized the electrophysiological properties of single SC-cardiomyocytes and signal transmission between cells. The aim of this project was to evaluate the functional properties of SC-cardiomyocytes and compare them with native neonatal and cultured cardiac HL-1 cells. Using the “hanging drop” method, murine embryonic stem cells (mESCs) were directed into the cardiogenic lineage and formed spontaneously beating embryoid bodies. Beating areas were enzymatically dissected to obtain single and small groups of cells. Using the whole-cell patch clamp technique, cells were voltage-clamped and examined for cardiac ion channels. In the current-clamp mode, action potentials were measured. Simultaneously, Ca2+ signals were recorded using fluo-3 and confocal laser-scanning microscopy. The gap junction permeant fluorescent dye Calcein was used to study intercellular communication between neighboured myocytes, and expression of gap junction proteins was confirmed in immunocytochemical assays. Our results show that two types of cardiac cells can be distinguished, spontaneously active and silent but excitable SC-cells, as reflected in the shape of their action potentials. SC-cardiomyocytes functionally express the cardiac voltage-dependent Na+, L- and T-type Ca2+ channels and the pacemaking HCN channel in spontaneously active cells. Current properties and pharmacology are comparable with native cardiomyocytes. Immunocytological examination revealed strong expression of the major cardiac gap junction protein connexin-43. Functional tests of intercellular coupling confirmed electrical coupling between SC-cardiomyocytes. Permeation kinetics of calcein dye transfer in SC-cardiomyocytes were similar to HL-1 cells, but significantly reduced in comparison with neonatal cells. In conclusion, we provide a clear functional profile of single and coupled SC-cells and their similarities with native cardiomyocytes and HL-1 cells, which makes SC-cardiomyocytes realistic candidates for cell therapy.
75 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Controlled VEGF expression in a cardiac patch improves vascularization and cardiac function in a myocardial infarction model Marsano A1,2; Luo J1; Boccardo S3; Maidhof R1; Morales A1; Fujikara K1; Konofagou E1; Banfi 2A * and Vunjak-Novakovic G1* (*equally contributing authors) 1Department of Biomedical Engineering, Columbia University, NY, USA; 2Departments of Biomedicine and of Surgery, University Hospital Basel, CH; 3Department of Robotics, Brain & Cognitive Sciences, Italian Institute of Technology, Genova, I Introduction Vascularization of engineered cardiac grafts remains a critical issue to treat the sequelae of cardiac ischemia. In this study we investigated a cardiac patch, generated from naïve rat cardiomyocytes (RCM) and mouse skeletal myoblasts (SM) engineered to express a specific, safe and effective VEGF level, in a nude mouse model of cardiac ishemia. We tested the hypothesis that controlled levels of VEGF, released by retrovirally transduced SM cells, could induce a stable and functional vascular network and thereby support the survival of both cell types and patch function.
Methods SM were transduced with retroviral vectors expressing either mouse VEGF164 linked to a truncated version of the mouse CD8, or CD8 alone as a control. SM expressing a specific level of VEGF were then FACS-purified based on their intensity of CD8 expression (Misteli, Stem Cells, 2010). Nude mice underwent myocardial infarction by ligation of the left anterior descending coronary artery. After 1 week, heart failure was confirmed by high resolution echocardiography (ECHO) and mice were implanted with patches made of polyglycerol sebacate (PGS) scaffold seeded with cells and cultured for 5 days in a perfusion bioreactor. Four groups of patches were tested: (i) RCM, (ii) co-culture of RCM and CD8-expressing SM (CD8), (iii) RCM and VEGF-expressing SM (VEGF), (iv) cell-free scaffolds (PGS). Cardiac function was assessed at day 0 as baseline, 1 week after myocardial infarction and 4 weeks post-implantation by ECHO, followed by end-point histological assays. Results Efficient normal angiogenesis was induced only by VEGF-expressing cells, both inside the patch and, interestingly, also in the underlying myocardium. Consistently, the RCM and SM survived only in the VEGF group, leading to a robust generation of differentiated tissue that was integrated with the underlying myocardium. ECHO showed that only the treatment with VEGF-expressing patches significantly improved cardiac function. Conclusions Controlled VEGF expression in a cardiac patch is effective in improving cardiac contractility after infarction by a dual mechanism, i.e. driving effective vascularization both of the engineered patch, leading to implanted cell differentiation and function, and of the underlying ischemic myocardium. Acknowledgments This work was supported by the NIH-R21 grant HL089913 to GVN and AB and the Swiss National Science Foundation.
76 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Controlled VEGF expression ensures safe angiogenesis and functional improvement in a model of myocardial infarction Melly L1,2, Marsano A1, Helmrich U1, Heberer M1, Eckstein F2, Carrel T3, Cook S4, Giraud MN4, Tevaearai H3, Banfi 1A 1 Cell and Gene Therapy and 2 Cardiac Surgery, Basel University Hospital 3 Department of Cardiovascular Surgery, Inselspital, Bern University Hospital 4 Cardiology, University of Fribourg, Switzerland Introduction. VEGF can induce normal or aberrant angiogenesis depending exclusively on the amount secreted in the microenvironment around each cell. To make this concept clinically applicable, we developed a FACS-based technique to rapidly purify transduced progenitors that homogeneously express a specific VEGF level from a heterogeneous primary population. Here we aim at inducing safe and efficient angiogenesis in the heart by cell-based expression of controlled VEGF levels. Methods and Results. Human adipose-tissue stem cells (ASC) were transduced with retroviral vectors expressing either rat VEGF linked to the FACS-quantifiable surface marker CD8, or CD8 alone (CD8) as control. VEGF-expressing cells were then FACS-purified to generate populations producing either a specific (SPEC) or heterogeneous (ALL) VEGF levels. In a non-ischemic study, 10^7 cells of each treatment group (CD8, SPEC, ALL) were injected into the myocardium of nude rats. After 4 weeks, vessel density was increased 2-3 fold by both VEGF-producing groups. However, ALL cells caused the development of numerous aberrant angioma-like structures, while SPEC cells induced only normal and mature microvascular networks. To determine the safety and functional efficacy in cardiac ischemia, 70 nude rats underwent myocardial infarction by coronary artery ligation. Two weeks later, animals received at the infarction border either 10^7 cells of one of the 3 treatment groups or PBS. Four weeks post-treatment, the ejection fraction was significantly worsened by treatment with either ALL VEGF (-13.2%) or control (CD8) cells (-6.4%) as well as the PBS group (-8.1%) compared to SPEC VEGF cells (+1.1%). Similar trends in cardiac function were confirmed by Pressure Volume loop analysis performed at the time of sacrifice. Histological analysis confirmed the induction of aberrant structures in the ALL group, which were completely prevented by SPEC cells similarly to the results in non-ischemic tissue. A positive remodeling effect was observed in the SPEC group, with significantly reduced fibrosis in the infarcted area compared to all other groups (CD8, ALL, PBS). Conclusions. Controlled VEGF delivery by FACS-purified transduced ASC is effective to reliably induce only normal vascular growth in the myocardium and is a promising novel strategy to achieve safe and therapeutic angiogenesis to treat chronic cardiac ischemia.
77 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Carbon nanotubes as a synthetic substrate for in vitro physiological heart tissue engineering Valentina Martinelli,1a, Giada Cellot,2,3a, Francesca Maria Toma,4 Carlin S. Long,5 John H. Caldwell,6 Lorena Zentilin,1 Mauro Giacca,1 Antonio Turco,4 Maurizio Prato, 4 Laura Ballerini,2 and Luisa Mestroni 5 1 International Centre for Genetic Engineering and Biotechnology, Trieste, Italy 2 Life Science Department, B.R.A.I.N. and 4 Department of Chemical and Pharmaceutical Sciences, University of Trieste, Trieste, Italy 3 Department of Neurobiology, International School for Advanced Studies (SISSA), Trieste, Italy 5 University of Colorado Cardiovascular Institute and 6 Department of Cell and Developmental Biology, University of Colorado, Denver, Aurora, Colorado 80045, United States a These authors contributed equally to this work Cardiac tissue engineering represents an attracting possibility to provide surrogate heart muscle for in vitro models and for myocardial repair. In this respect, the development of appropriate scaffolds as essential structural components in tissue engineering is drawing increasing interest. Among others, carbon nanotubes have been already applied in several areas of nerve tissue engineering to study cell behavior or to instruct the growth and organization of neural networks. Recently, we have demonstrated that defunctionalized multi-wall carbon nanotubes are also suitable for growth and expansion of neonatal cardiomyocytes. Using electron transmission microscopy we showed that cultured neonatal rat ventricular myocytes interact with carbon nanotubes by forming tight contacts; these cells displayed increased viability and proliferation with respect to cells growing on gelatin, and progressive changes in electrophysiological properties towards an adult mature phenotype. Furthermore, combining microscopy and molecular biology techniques, we demonstrated that, in cultures of cardiac myocytes, carbon nanotube substrate is able to induce an enhancement of connexin-43 expression with development of functional syncytia and increased number and extension of gap junctions. These modifications are paralleled by the induction of a gene expression profile characteristic of terminal differentiation and physiological hyperthrophic growth. Collectively, our observations demonstrate that carbon nanotubes are a convenient substrate to promote optimal expansion and survival of cardiomyocytes in culture and to guarantee their differentiation towards a mature phenotype. These features have remarkable potential for innovative clinical, pharmacological and tissue engineering applications in the currently vexing field of regenerative medicine applied to the heart.
78 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Role of Cyclin-Dependent Kinase 9 in cardiac injury-recovery in the zebrafish embryo G. Matrone, K.S. Wilson, J.J. Mullins, C.S. Tucker & M.A. Denvir Centre for Cardiovascular Science, The Queen’s Medical Research Institute, College of Medicine and Veterinary Medicine, The University of Edinburgh, 47 Little France Crescent, Edinburgh, EH16 4TJ. Injured mammalian hearts do not regenerate, replacing necrotic tissue with scar tissue and hypertrophic cardiomyocytes; interestingly zebrafish fully regenerate and restore cardiac function after partial cardiac ablation. Although many of the pathways of the hypertrophic response are associated with cell division, the underlying mechanisms that reactivate the cell cycle, switching from hypertrophy to proliferation, are unclear. The P-TEFb complex is involved in mRNA increase during cardiac cell growth, together with the hyperactivation of CDK9 which is related to cardiac hypertrophy. This study investigates the role of CDK9 in the zebrafish (Danio rerio) embryo heart during normal development and recovery from ventricular laser-induced injury. Tg(cmlc2:EGFP) zebrafish embryos (n=12, 2 experiments) were exposed to 3µM Flavopiridol, a selective CDK9 inhibitor, from 24 to 120 hours post fertilization (hpf); at 72hpf embryos underwent ventricle laser injury. Ventricle Ejection Fraction (EF) was measured in beating hearts under mild anaesthesia. Whole ventricle cardiomyocyte number (VCm) was assessed (DAPI stained nuclei counts) in surgically isolated hearts. Gene expression were measured by qPCR, analysis was performed pre-laser and 3, 24 and 48hour post-laser injury, with additional time-points at 10 minutes and 1 hour post-laser for gene expression. Data are expressed as mean±SEM and analysed by ANOVA. Flavopiridol had no effect on EF prior to laser but produced a statistically significant VCm reduction, at all time points, compared to controls. Laser injury alone caused a reduction in both EF (-29%, p≤0.001) and VCm (-18%, p=0.078) vs. controls at 2 hours, recovering to control levels at 24 and 48 hours post-laser. In embryos pre-treated with flavopiridol, laser injury caused a further reduction in EF (-46%, p≤0.001) without additional reduction in VCm (-11%, p=0.17) vs. controls. All embryos pretreated with flavopiridol showed no recovery in either EF or VCm at 24 and 48 hours. Additionally, laser-injury produced a 120% increase in CDK9 gene expression as early as 10 min post-laser compared to control; this will be compared with Western Blot analysis. Flavopiridol inhibition of CDK9 activity reduces cardiomyocyte proliferation in the developing zebrafish ventricle resulting in globally reduced contractile function. Furthermore, CDK9 inhibition impairs functional recovery following laser injury.
79 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
VEGF and TNF up-regulate, NSAID down-regulate SOX18 protein level in HUVEC Milivojevic Milena,1 Petrovic Isidora,1 Nikcevic Gordana,1 Zaric Jelena,2 Ruegg Curzio,2 Stevanovic Milena1 1 Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, PO BOX 23, 11010 Belgrade, Serbia 2 University of Fribourg, Faculty of science, Ch. du Musée 8, CH-1700 Fribourg, Switzerland Angiogenesis, the process of generating new blood vessels, is essential to embryonic development, organ formation, tissue regeneration and remodeling, reproduction and wound healing. Also, it plays an important role in many pathological conditions, including chronic inflammation and cancer. Angiogenesis is regulated by a complex interplay of growth factors, inflammatory mediators, adhesion molecules, morphogens and guidance molecules. Transcription factor SOX18 is transiently expressed in nascent endothelial cells during embryonic development and postnatal angiogenesis, playing a role in endothelial cell specification or differentiation, angiogenesis and atherogenesis. Accordingly, the aim of this study has been to investigate whether pro-angiogenic molecules and pharmacological inhibitors of angiogenesis modulate SOX18 expression in endothelial cells. Therefore, we treated human umbilical vein endothelial cells (HUVEC) with angiogenic factors, extracellular matrix proteins, inflammatory cytokines and nonsteroidal anti-inflammatory drugs (NSAID) and monitored SOX18 expression. We have observed that the angiogenic factor VEGF and the inflammatory cytokine TNF increase, while the NSAID ibuprofen and NS398 decrease the SOX18 protein level. These results for the first time demonstrate that SOX18 expression is modulated by factors and drugs known to positively or negatively regulate angiogenesis. This opens the possibility of pharmacological manipulation of SOX18 gene expression in endothelial cells to stimulate or inhibit angiogenesis.
80 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
The role of transcription factors ZBP-89, SP3, NF-Y and EGR1 in the regulation of the SOX18 promoter activity Petrovic Isidora, Kovacevic-Grujicic Natasa, Milivojevic Milena, Stevanovic Milena Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, PO BOX 23, 11010 Belgrade, Serbia Sox18/SOX18 gene is an important regulator of vascular development playing a role in endothelial cell specification or differentiation, angiogenesis and atherogenesis. However, little is known about its transcriptional regulation. We have performed comprehensive functional characterization of the human SOX18 promoter with an emphasis on angiogenesis-related transcription factors involved in its transcriptional regulation. Analyses were performed in HeLa cells, representing a tumor cell line, and in EA.hy926 cells used as an endothelial model system. Combining in silico analysis, in vitro binding assays and functional analyses we have shown that transcription factors ZBP-89, Sp3, NF-Y and EGR1 play important functional roles in the regulation of the SOX18 promoter activity. In particular, we have shown that ZBP-89 and Sp3 act as repressors, while NF-Y and EGR1 act as activators of SOX18 promoter activity. We have focused our interest on the transcription factor EGR1, since this factor has the pivotal role in the transcriptional response of endothelial cells to angiogenic growth factors involved in angiogenic switch. We have identified the presence of essential regulatory element within the SOX18 promoter positioned -89 to +29 relative to tsp, which harbors cluster of three EGR1 binding sites. By functional analyses we have shown that these three putative binding sites are functionally relevant and sufficient for EGR1-induced SOX18 transcription. Mutations of these binding sites significantly impair activity of the SOX18 promoter, particularly in EA.hy926 cells, indicating the importance of these regulatory elements for SOX18 promoter activity in endothelial setting. Finally, overexpression of EGR1 protein in both HeLa and EA.hy926 cells led to increase of SOX18 expression on both mRNA and protein level. By our data, we have established SOX18 as a novel target gene regulated by transcription factor EGR1, thus providing the first functional link between two transcription factors previously shown to be involved in the control of angiogenesis.
81 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
The G-protein coupled receptor APJ keeps the balance between proliferation and cardiovascular differentiation of multipotent mesodermal progenitors in Embryonic Stem Cells. Cristina D’Aniello1, Alessandro Fiorenzano1, Salvatore Iaconis1, Gennaro Andolfi1, Gilda Cobellis2, Anna Lisa Fico1, Gabriella Minchiotti1 1 Stem Cell Fate Laboratory, Institute of Genetics and Biophysics “A. Buzzati-Traverso”, CNR, Naples, Italy 2 Department of General Pathology, Second University of Naples, Naples, Italy Mammalian cardiomyogenesis starts early during embryogenesis and requires different extracellular signals that are tightly regulated in time and space, intersecting with intracellular genetic programs that determine the cellular response. Unmasking the essential signals for cardiac lineage specification in mammals has great importance for development and regenerative medicine. Pluripotent stem cells represent a powerful model system to study the early steps of cardiac specification and differentiation for which the molecular control is still largely unknown. We have recently shown that the G-protein-coupled receptor APJ and its ligand Apelin are key regulators of embryonic stem cell (ESC) cardiomyogenesis. Here we used both loss and gain of function approaches in mESCs to get mechanistic insights into the role of APJ in the early events of cardiomyogenesis. Loss of function experiments revealed a crucial role for APJ in the gene regulatory cascade promoting mesoderm patterning and cardiac specification and differentiation. Notably, while expression of the early mesoderm marker Brachyury and the pre-cardiac marker Mesp1 was delayed in APJ Knock Down (APJ KD) ESCs, expression of early and late cardiac differentiation markers was dramatically reduced or absent and never recovered even after prolonged in vitro culture. Interestingly, proliferating mesodermal progenitor cells failed to exit from the cell cycle and eventually differentiate into cardiomyocytes. Accordingly, expression of the cyclin-dependent kinase inhibitors p57Kip2 and p21Kip2 was dramatically reduced. Most remarkably, we found that expression of the cardiac inducer Cerberus was almost absent in APJ KD EBs. Conversely, a time –controlled APJ overexpression, i.e. in the early time window (T0-4) of cardiac differentiation, results in reduced proliferation, and increased cardiomyogenesis. All together our data point to a central role for APJ in maintaining the balance between proliferation and differentiation of multipotent mesodermal progenitors in ESC cardiomyogenesis. Moreover, we propose a mechanism by which APJ regulates mesodermal progenitor cell proliferation/differentiation, at least in part, through the BMP and Nodal inhibitor Cerberus.
82 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Isolation and propagation of mesenchymal stem cells from human cord tissue and blood in Iraq Farooq I Mohammad; Majeed Irsheed Sabah; Ali Hayder; Shaimaa Yusif Abdualfatah; Ban Sabah and Baraa Abdualhadi Biotechnology Research Center/ Al Nahrain University/ Iraq For a first time in Iraq we success to isolate and propagate MSCs from cord blood and cord tissue by using simple facility and equipments, we used different method for MSCs isolation like scrapping, tissue fragmentation and fixing enzymatic dissociation we found the tissue fragmentation and fixing is the better way to isolate and propagate the cells from cord tissue, the cells expands after one week of culture, for blood we used the ficoll method the expanded cells appeared second day of isolation and culturing the cells and the cells lasts for more than 3 months in culture then we store it in deep freezer -80°C to check the freezing and thawing ability and viability of the cells and the viability was more than 90%. We studied the morphology, growth curve, and effect of different cell culture media on the morphology and doubling time of the cells we tested MEM alpha, M199, RPMI 1640 and DMEM we found MEM alpha and DMEM the better media for cell proliferation and growth
83 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Ultrastructural characteristics of myogenic cells in hypertrophying myocardium of rodents. Marta Novotová and Ivan Zahradník Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Bratislava, Slovak Republic Maturation of mammalian myocardium is accompanied by transformation of myocytes to a fully differentiated state characterized by well organized cellular architecture and no signs of proliferation. Recent studies provide increasing evidence on ongoing neomyogenesis by techniques of molecular biology that, however, lack information on the ultrastructural organization of the new myocytes that is crucial for their proper functionality. Here, with the use of electron microscopy, we describe atypical muscle-like cells in the hypertrophying myocardium of normal 21 days old mice (the phase of slow hypertrophy, Leu et al., Anat Embryol (Berl) 204, 217-224, 2001) and in isoproterenol-treated rats (myocardial injury-induced hypertrophy, Mikušová et al., Can J Physiol Pharmacol 87: 641-651, 2009). In the phase of slow cardiac hypotrophy, the atypical myocytes formed small bundles in the interstitium of the endocardium surrounded by invasive cells. In contrast to ventricular myocytes, they were of a small diameter and not fully differentiated. Their contractile proteins were not well organized into myofibrils, mitochondria were small and numerous, and the network of t-tubules and of sarcoplasmic reticulum was absent. Altogether, these atypical myocytes displayed well developed myogenic character. Similar bundles of myogenic cells were observed in the hypertrophied myocardium of rats treated with isoproterenol. In contrast to myocardium of untreated rats, the interstitium contained small bundles of miniature cells shoving signs of developing cardiomyocytes. In some of these cells the myofilaments were not organized into myofibrils or, if present, the myofibrils were disorganized. The membrane network system was lacking, instead small vesicles between myofilaments were occasionally observed. The mitochondria were often swollen or lacked cristae. The intercalated discs were extensive. Altogether, these observations reveal that the interstitium of hypertrophying myocardium represents a substrate for homing and developing of myogenic cells. Supported by grants VEGA 2/0116/12, VEGA 2/0203/11, and APVV-0721-10.
84 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Cell-free cryopreserved arterial allografts from multiorgan donors: a new strategy to fabricate artificial blood vessels suited for peripheral vascular surgery F. Papadopulos*, B. Weber°, M. Buzzi", M. Gargiulo*, G. Pasquinelli^ *Dep.of Specialistic Surgery and Anesthesiological Sciences, ^Surgical Pathology, "Cardiovascular Tissue Bank S. Orsola-Malpighi Hospital, Bologna °SCRM, University of Zurich, Switzerland, Introduction Critical limb ischemia is a severe blockage in peripheral arteries, which reduces blood-flow. A common approach is infrainguinal bypass that permit a functional reestablishment of blood flow by replacing the diseased segment and redirecting blood flow around the blocked portion of the artery. Synthetic vascular prosthesis, commonly used, are good conduits in high-flow low-resistance conditions but have difficulty in their performance as small diameter vessel grafts. A new approach could be the use of native decellularized vascular tissues. Cell-free vessels are expected to have improved biocompatibility when compared to the synthetic one and are optimal natural 3D matrix templates for driving stem cell growth and tissue assembly in vivo. Decellularization of tissues and their integration with tissue engineering represent a promising field for regenerative medicine, with the aim to develop a methodology to obtain small-diameter-artery allografts to be used as a natural scaffold suited for in vivo cell growth and pseudo-tissue assembly, eliminating failure caused from immune response activation. Material and methods Umbilical cord-derived mesenchymal cells were isolated from human umbilical cord tissue and expanded in advanced DMEM. Immunofluorescence and molecular characterization revealed a stem cell profile. A non-enzymatic protocol, that associate hypotonic shock and low-concentration ionic detergent, was used to decellularize segments of saphenous artery. Cells were seeded on the cell-free scaffolds using a compound of fibrin and thrombin and incubated in DMEM, after 4 days of static culture they were placed for 2 weeks in a flow-bioreactor, which mimics the physiological cardiovascular pulsatile flow. At the end of the dynamic culture, samples were processed for histological, biochemical and ultrastructural analysis. Results and discussion Histology showed that the dynamic culture cells initiate to penetrate the extracellular matrix scaffold and to produce components of the ECM, as collagen fibres. Sirius Red staining showed layers of immature collagen type III and ultrastructural analysis revealed 30 nm thick collagen fibres, presumably corresponding to the immature collagen. These data confirm the ability of cord-derived cells to adhere and penetrate a natural decellularized tissue and to start to assembly into new tissue. This achievement makes natural 3D matrix templates prospectively valuable candidates for clinical bypass procedure.
85 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Defining the role of Neuropilin1 in the cardiac neural crest Alice Plein, Alex Fantin, Christiana Ruhrberg Institute of Ophthalmology, University College London (UCL) The neuropilins NRP1 and NRP2 co-operatively control outflow tract septation and great vessel remodelling during heart development. These processes rely on the regulated interaction of vascular endothelial and cardiac neural crest cells, two cell types known to express neuropilins. We have compared the requirement for neuropilins in endothelial versus neural crest cells during outflow tract septation as well as pharyngeal arch artery formation, coverage with cardiac neural crest-derived smooth muscle and remodelling into the great vessels. In addition, we further examined the genetic requirement for two distinct classes of neuropilin-binding guidance cues in PAA and outflow tract development, the class 3 semaphorins and the VEGF164 isoform of the vascular endothelial growth factor VEGF to determine the signalling pathway responsible for proper great vessel remodelling.
86 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Human umbilical cord blood progenitors coordinate a regenerative angiogenic microenvironment in ischemic tissues David M. Putman, Kevin Y. Liu, Heather C. Broughton, Gillian I. Bell, David A. Hess Department of Physiology and Pharmacology, The University of Western Ontario Krembil Centre for Stem Cell Biology, Robarts Research Institute Understanding the role of somatic progenitor cells in angiogenesis is integral to the development of effective cell-based therapies to treat ischemic disease. Human umbilical cord blood (UCB) is a readily available source of angiogenic hematopoietic and endothelial colony forming cells implicated in the support of vascular regeneration. Using high aldehyde dehydrogenase activity (ALDHhi), a conserved stem cell function, we simultaneously isolate a mixed population of pro-angiogenic hematopoietic and endothelial progenitors. We characterized the pro-angiogenic functions of UCB ALDHhi cells in co-culture with human umbilical vein endothelial cells (EC). Under growth factor-free/serum-free conditions EC invariably died. Under these conditions, supplementation of UCB ALDHhi cells in transwells promoted the survival of EC for 72 hours (n=5, p<0.05). Furthermore, on growth factor reduced matrigel, supplementation with UCB ALDHhi cells significantly increased EC tubule formation (n=5, p<0.05). Compared to injection of human ALDHlo cells or saline control, intravenous transplantation of UCB ALDHhi cells enhanced recovery of perfusion in the ischemic limb after induction of hindlimb ischemia by femoral artery ligation (p<0.01). Furthermore, ALDHhi cell transplantation prevented loss of vWF+ and CD31+ blood vessel density in the ischemic hindlimb of mice as observed in controls (p<0.01). Although, augmented recovery of blood flow was not associated with long-term human cell engraftment in the ischemic hindlimb, direct intramuscular injection of UCB ALDHhi cells in the ischemic muscle accelerated the recovery of perfusion despite similarly low long-term engraftment. Thus, UCB ALDHhi cells promote vascular repair via paracrine activities. Preliminary results indicate that transplantation of the endothelial colony forming cells present in UCB alone is insufficient to augment recovery of blood flow in the ischemic hindlimb. Experiments are underway to investigate the contributions of culture-expanded hematopoietic progenitors. We propose that hematopoietic and endothelial progenitors may cooperate synergistically to promote a regenerative niche at the site of acute ischemic injury whereby the survival of endothelial cells in ischemic vessels is augmented and an angiogenic program is initiated. Future studies will identify the specific cellular constituents and paracrine pathways that co-ordinate this putative mechanism for functional vascular regeneration.
87 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Cardiac effects of modified embryonic mesoangioblasts expressing PlGF, MMP9 or both after myocardial infarction in the mouse Giuseppe Ristagno, Beatriz G. Galvez, Salman Afroze Azmi Mohammed, Giovanna Balconi, Noeleen De Angelis, Fabiola Molla, Lidia Staszewsky, Roberto Latini, Giulio Cossu Laboratory of Cardiovascular Clinical Pharmacology, “Mario Negri” Institute for Pharmacological Research, Milan, Italy; Division of Regenerative Medicine, San Raffaele Scientific Institute, Milan, Italy Benefit of stem cells (SC) therapy after myocardial infarction (MI) is known. However, it is debated if regeneration of myocardium is related to SC differentiation or SC-released factors. We investigated effects of embryonic mesoangioblasts (MABs) infected with lentiviral vectors expressing placental growth factor (PlGF), or matrix metallopeptidase 9 (MMP9), or both (MIX) in a MI model. C57BL/6 mice were used. 70 underwent permanent coronary artery ligation (CAL), while 53 were subjected to 45 min ischemia followed by reperfusion (IR). MABs expressing PlGF, MMP9, MIX or PBS were injected either into the left ventricular (LV) wall (IW) or into the LV chamber (IC). Echocardiography was performed 4 wk later. After IR, 74% of mice survived for 4 wk, with a greater survival in IW MAB mice, 95%, than IC MAB ones, 62%. After CAL, 94% of mice survived for 4 wk. Survival was 92% in the IW MAB mice and 100% in the IC MAB ones. However, no survival differences were observed among MAB-PIGF, -MMP, -MIX, or -PBS mice. Echocardiography showed LV dilatation and decreased systolic function in MAB-PBS mice compared to sham ones. After IR, no significant differences were observed in myocardial function among MAB treated groups, independently of IW or IC injection. Slightly reduced LV end diastolic diameter and increased LV shortening fraction (SF) were observed in IW MAB-MMP9 group compared to IW MAB-PBS one. After CAL, instead, IW MAB-MMP9 mice showed improvements in LVSF and ejection fraction (17% and 14% respectively) compared to IW MAB-PBS ones. IW MAB-MMP9 mice presented also a smaller LV end systolic diameter compared to other IW MAB-treated animals. Following IC MAB injection, no differences in myocardial function was found among groups. Histopathology was conducted only on mice receiving IW MAB injection after CAL. Infarct size (IS) was 23% smaller in MAB-PlGF mice and 30% in MAB-MMP9 mice, compared to MAB-PBS ones. Capillary density was reduced from 2743 cap/mm2 in sham animals to 1744 cap/mm2 in MAB-PBS ones. Capillary density increased to 2003 cap/mm2 after MAB-MMP9, while it remained low after MAB-PlGF and MAB-MIX. Cross sectional area (CSA) showed a significant reduction only in MAB-MMP9 mice compared to MAB-PBS ones. IW injection of MAB-MMP9 after CAL ameliorated LV remodeling and reduced IS and myocyte hypertrophy, while improved capillary density, compared to MAB-PBS injection. However, these benefits were not significant after IR and after MAB-MIX.
88 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Nuclear architecture dynamics during human myoblast in vitro differentiation analyzed by 2D and 3D FISH. Natalia Rozwadowska(1), Tomasz Kolanowski(1), Piotr Pawlak(2), Marcin Siatkowski(3), Ewa Wiland(1), Marta Olszewska(1), Maciej Kurpisz(1) (1) Institute of Human Genetics,Poznań, Poland, (2) Poznań University of Life Sciences, Poland, (3) IBIMA, Medical Faculty, University of Rostock, Germany Objectives: For several years muscle-derived stem cells were intensively investigated in order to play crucial role in stem cell-based therapies. Therefore, there is a need to characterize these cells during differentiation process. Moreover, little is known about the nuclear architecture and its changes during differentiation process of human myoblasts. Therefore the aim of our study is to observe the selected centromers position during in vitro differentiation of human skeletal muscle stem cells. Methods: Analysis was performed by two- and subsequently by three-dimensional fluorescent in situ hybridization (2D & 3D FISH) together with in silico analysis of obtained results. Results: Comparison of nuclei morphology during differentiation process revealed diminution of the mean nucleus size and flattering of the shape. For analysis of chromosome centromers, nucleus was divided into 5 regions based on radial distance starting from the central point. So far, the most crucial chromosomes, including 1, 2, 3, 7, 11, 12, 16, 17 and X, were examined by 2D or/and 3D FISH in cells pre- and after differentiation in vitro. Change of most chromosome centromers position was observed, directed them toward nuclear periphery. Only chromosome 11 position was not altered during this process. Conclusion: To summarize, analysis of morphological changes in nuclei of skeletal muscle stem cells during differentiation process indicated a tendency for diminishing their size, moreover changes in centromers position are suggested to be connected with gene expression profile alterations. The following step will be examination of the exact localization of crucial genes aimed by gene-specific probes.
89 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Identification of novel cardioprotective factors by in vivo gene selection using AAV vectors Giulia Ruozi, Serena Zacchigna, Matteo Dal Ferro, Francesca Bortolotti, Antonella Falcione, Lorena Zentilin and Mauro Giacca Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano, Trieste, Italy, 34149 In our efforts to identify novel genes controlling cardiomyocyte proliferation and survival, which might be exploited therapeutically, we devised an innovative functional selection strategy (FunSel), based on in vivo gene transfer of a collection of secreted factors (>1600 genes) cloned into AAV vectors. According to FunSel, pools of AAV vectors are directly selected in vivo under cell damaging conditions, and become progressively enriched for potentially therapeutic genes upon subsequent rounds of delivery and recovery. Before starting the in vivo implementation of the FunSel protocol, we first confirmed the theoretical feasibility of the iterative selection approach and demonstrated its efficiency. In particular, AAV pools were tested for their ability of packaging, in vivo transduction, persistence and level of expression, in order to guarantee a faithful maintenance of AAV vector genome abundance during pooled production. After extensive preliminary validation analysis, we applied FunSel for the selection of protective genes after ischemia or isoproterenol-induced myocardial damage using a pool of 100 AAV vectors expressing various growth factors, hormones and cytokines. In the first screen, after three rounds of in vivo selection, we observed a marked enrichment for AAVs expressing Ghrelin, a neurohormone involved in endocrine metabolism. A single cycle of cardiac toxic damage instead led to the selection of vectors expressing Insulin-Like peptide 6 (INSL6), a poorly characterized member of the insulin gene family. The cardioprotective potential of these two molecules was further assessed by a series of morphological and functional assays. The persistent AAV9-mediated expression of these factors in the heart preserved cardiac function and reduced infarct size upon myocardial infarciton, as assessed by echocardiography, histological and morphometric analysis. In animals transduced with AAV9-Ghrelin or AAV9-INSL6, the marked improvement in cardiac contractility was paralleled by a decreased number of apoptotic cells at early time points after infarction and reduced inflammatory cells infiltration in the peri-infarctual region. Moreover, ghrelin counteracted the induction of markers of cardiac damage (e.g. miR21 and MMP-2) and prevented the deregulated expression of markers of pathological left ventricle remodelling, such as bMHC, BNP and others. These results indicate the feasibility of the in vivo, FunSel approach for cardioprotective factors and identify Ghrelin and INSL6 as novel and powerful molecules providing benefit after cardiac damage. (This work was supported by the European Research Council (ERC) Advanced Grant 250124 “FunSel”)
90 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Dose- and time-dependent angiogenesis by controlled delivery of matrix-bound growth factors Sacchi Veronica1, Martino Mikael3, Morton Tatjana2, Hofmann Anna2, Groppa Elena1, Gianni-Barrera Roberto1, Hubbell Jeffrey3, Redl Heinz 2 and Banfi Andrea1 1 ICFS, Basel University Hospital, Basel (Switzerland) 2 LBI Trauma, Vienna (Austria) 3 EPFL, Lausanne (Switzerland) Vascular endothelial growth factor (VEGF) is the key factor to induce therapeutic angiogenesis. The induction of safe and stable angiogenesis requires sustained VEGF delivery for at least 4 weeks at homogeneous microenviromental doses. Further, we previously found that cell-based co-expression of platelet derived growth factor- BB (PDGF-BB) with VEGF at a fixed molar ratio (1:3) prevents VEGF-induced aberrant angiogenesis and promotes faster vessels stabilization already at 2 weeks, despite heterogeneous and uncontrolled VEGF expression levels. Continuous release of matrix-bound growth factors is a convenient approach for clinical translation of these biological concepts, and provides also an easily adaptable system to further investigate the angiogenic process in vivo. Therefore, here we aim to determine the requirements to induce normal and stable angiogenesis by controlled release of angiogenic growth factors from a state-of-the-art matrix-bound system, based on transglutaminase (TG) reaction to bind the modified recombinant factor into fibrin gels. Since both VEGF dose and duration are function of degradation rate, we first determined the optimal gel composition to ensure sufficient in vivo persistence. Then, to determine VEGF therapeutic window, different VEGF concentrations were tested combined with various concentrations of aprotinin, which inhibits gel degradation by plasmin. An aprotinin concentration of 56μg/ml ensured both sufficient persistence and an adequate VEGF release rate. In this condition, after 9 days a VEGF concentration of 25μg/ml triggered the switch from aberrant to predominantly normal angiogenesis. A lower VEGF dose of 5μg/ml almost eliminated any aberrant angiogenesis by 9 days and showed only normal and well perfused capillaries by 4 weeks. Co-delivery by fibrin gel of PDGF-BB and VEGF on a ratio 1:3 after 2 weeks showed normalization of aberrant angiogenesis induced from a high VEGF dose. In conclusion, in vivo release of TG-VEGF from fibrin hydrogels can be precisely tuned under optimized conditions to efficiently induce dose-dependent and stable angiogenesis. Further, controlled co-delivery of TG-VEGF and PDGF-BB proteins is a novel approach to expand the range of VEGF doses with which normal angiogenesis can be reliable induced. The therapeutic potential of these approaches is being determined in a mouse model of hind limb ischemia. We acknowledge support by the EU FP7 Project ANGIOSCAFF (CP-IP 214402).
91 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Gene expression profile of the early chick hemangioblast Ana Teresa Tavares (1,2), Vera Teixeira (2), Antonio Duarte (1,2) (1) CIISA, Faculdade de Medicina Veterinaria (UTL), Av. da Universidade Tecnica, 1300-477 Lisboa, Portugal; (2) Instituto Gulbenkian de Ciencia, Rua da Quinta Grande, 2780-156 Oeiras, Portugal During early vertebrate embryogenesis, there is a close developmental relation between hematopoiesis and vasculogenesis. Primitive hematopoietic and endothelial lineages both derive from aggregates of mesodermal cells that form the blood islands in the extraembryonic yolk sac. This observation led to the hypothesis that both cell types originate from a common precursor known as the hemangioblast. Chick Cerberus gene (cCer) codes for a secreted factor expressed in the anterior mesendoderm that gives rise to blood islands, among other tissues. During the study of cCer transcriptional regulation, we isolated a short cis-regulatory region that is able to drive reporter gene expression specifically in blood island precursor cells or hemangioblasts. In the present study, we have used this hemangioblast-specific reporter (Hb-eGFP) to analyze the gene expression profile of the early hemangioblast. For this, Hb-eGFP+ and control pCAGGS-RFP+ cells were sorted from electroporated HH5-6 chick embryos and their expression profiles compared using Affymetrix microarray analysis. Out of 32,773 transcripts, 1,015 were differentially expressed (> 1.5 fold change), including 718 known genes (264 upregulated and 454 downregulated in hemangioblasts). Out of 33,457 transcripts, 658 were differentially expressed (fold change > 1.7), including 476 known genes. As expected, some of the known hemangioblast markers are enriched in the Hb-eGFP-positive population, such as Lmo2 (+3.73 fold) and Scl/Tal1 (+3.62 fold). Here, we will present the gene lists as well as the outcome of the data analysis (using Ingenuity Pathway Analysis).
92 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Ultrastructural characteristics of myogenic cells in failing myocardium of a patient on chemotherapy Ivan Zahradník1, Jozef Bartunek2, Marc Vanderheyden2, Marta Novotová1. 1Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Bratislava, Slovakia, 2Cardiovascular Center, OLV Hospital, Moorselbaan 164, 9300 Aalst, Belgium. Chemotherapy is associated with potential cardiac toxicity and risk of heart failure. Ultrastructural changes underlying the chemotherapy-induced cardiomyopathy are not well understood. Here, we report electron microscopy analysis of a left ventricular biopsy obtained from a 50-year old female patient with congestive heart failure after preceding chemotherapy. Left ventricular biopsy was procured at the time of the biventricular pacemaker implantation. The biopsy was transferred first to calcium-free Tyrode solution to prevent hypercontraction of myocytes. Subsequently, after 5 to 10 minutes, the sample was prefixed with 2% glutaraldehyde for 30 minutes, then divided into about 1 mm cubes and processed for electron microscopy. The structure of myocytes was assessed in 60 nm ultrathin sections under a Jeol 200B electron microscope and micrographs were taken with a 2_megapixel Gatan camera at a magnification of up to 60 000×. As expected, the myocytes displayed hypertrophic ultrastructural features including plasma membrane changes, vesiculation of t-tubules, increased content of cytosol and mitochondria. Other cardiomyocytes showed features of ischemic damage such as cytoplasmic edema with enormous amount of mitochondria and degradation of the contractile myofibrils. Notably, in regions of increased mass of interstitium, we have identified small islands composed of a few minute cells with some characteristics of myocytes. They contained myofilaments that were not well organized into myofibrils. Occasionally, small stretches of endoplasmic reticulum and a population of ribosomes could be observed in their cytoplasm beneath the plasma membrane. This myogenic like cells communicated with each other by simple intercalated discs. They lacked a system of t-tubules and membranes of sarcoplasmic reticulum. Their cytoplasm contained sparse vesicles. We conclude that ultrastructural changes in the damaged myocardium due to cardiotoxic therapy might be accompanied by emergence of newly formed myogenic cells in the interstitium. Supported by grants VEGA 2/0116/12, VEGA 2/0203/11, and APVV-0721-10.
93 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Induction of cardiomyogenic markers in murine and human adult stem cells isolated from different sources upon electric stimulation Zamperone A.1, Pietronave S.1, Pavesi A.2, Oltolina F.1, Consolo F.3, Novelli E.4, Diena M.4, Fiore G.B.3, Soncini M.3, Radaelli A.3 and Prat M.1 1Department of Health Sciences, Università del Piemonte Orientale “A. Avogadro” Novara. 2Politecnico di Milano. 3Politecnico di Torino. 4Division of Cardiosurgery, Clinica S.Gaudenzio, Novara Introduction and Aims. Many studies have shown that exogenous stimuli, such as physical stimuli (mechanical or electric), enhance stem cells commitment towards a cardiac phenotype. However, the effects of pulsed EF involved in myogenic commitment are still poorly understood. The aim of this work was to analyze the effects of mono and biphasic electrical stimulation on an adipose-derived mouse stem cell line (m17.ASC) and on adult stem cells isolated from human subcutaneous adipose tissue and auricula. Methods. Device: the culture system composed of a chassis carrying an electrical wiring system is equipped with housings for multiple PDMS culture chambers, with their own embedded stainless steel electrodes. This allows simultaneous stimulation of multiple cell cultures with independent electrical patterns. The bioreactor is driven by a dedicated graphical-interface software, where the operator can set and control the stimulation patterns delivered to the electrodes. Biological experiments: m17.ASC cell line and human adult stem cells were dropwise seeded on glass slides and 24 hours later the EFs (square mono-phasic 2ms, 1Hz, 8V amplitude or bi-phasic 2ms, 1 Hz, 4 V amplitude) were applied for different times (3, 8, 24, 72 hours). Cells were analyzed for proliferation by crystal violet staining and for differentiation on the basis of the expression of the early cardiac markers Connexin 43 (Cx-43) and Gata-4 by IF. Results. m17.ASCs upon both electrical stimulation protocols showed proliferation rates similar to those of not-stimulated cells. Three days of biphasic stimulation promoted high expression of Cx-43. Similar results were observed for both human cell types. In the case of cardiac human progenitors bi-phasic stimulation induced also the up-regulation of GATA-4. Conclusions. These results suggest that the bi-phasic EF can be an appropriate signal to induce myogenic commitment in mesenchymal stem cells. Experiments which combine this stimulus with stiffness and topography in the culture substrates are in progress. (Supported by Regione Piemonte and Cariplo Foundation, grant # 2008-2459)
94 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Post-translational modification by acetylation regulates VEGFR2 activity Annalisa Zecchin1, Lucia Pattarini1, Maria Ines Gutierrez1, Miguel Mano1, Mike P. Myers2, S. Pantano3 and Mauro Giacca1 1Molecular Medicine Laboratory and 2Protein Networks Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy 3Institute Pasteur of Montevideo, Uruguay The tyrosine kinase receptor Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) is a key regulator in developmental angiogenesis, by triggering proliferation, migration and survival of endothelial cells. Activity of the receptor follows interaction with its cognate ligands, primarily the members of the VEGF family, and involves phosphorylation of various tyrosine residues in the intracytoplasmic portion of the receptor. By combining biochemical and proteomics studies, here we provide the first evidence that VEGFR2 activity is essentially regulated by acetylation: in both endothelial and non-endothelial cells, membrane-associated VEGFR2 was found to be acetylated in at least four lysine residues that form a dense cluster in the kinase insert domain, and in a single lysine located in the activation loop. The enzyme responsible for these modifications is p300/CBP, a well-characterized acetyltransferase. Members of the class II histone deacetylase family are able to interact with and deacetylate the receptor in endothelial cells. A computational model of the VEGFR2 activation loop revealed that acetylation of the lysine located at this position contributes to the transition to an open active state, in which tyrosine phosphorylation is favored by better exposure of the kinase target residues. In addition, we found that VEGFR2 acetylation essentially regulates receptor stability, since a mutant in which the five lysines identified as acetylated were substituted with arginines, was significantly less stable than the wild type protein. Furthermore, we observed that over-expression of p300 dramatically increased the levels of receptor tyrosine autophosphorylation upon ligand binding. Cells expressing VEGFR2 mutants that were not acetylated showed reduced levels of receptor phosphorylation and their migratory capacity was impaired. Finally, receptor acetylation was found to counteract the process of receptor desensitization following VEGF stimulation. Upon hyper-acetylation, high levels of functionally active, phosphorylated VEGFR2 were maintained on the cell membrane over time and sustained intracellular signaling upon prolonged ligand stimulation. Taken together, these findings indicate that post-translational modification by acetylation is a critical mechanism that directly affects VEGFR2 receptor biology and function.
95 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Abstract Author Index
A Bartunek J. 93 Baruscotti M. 53 Abdualfatah S. Y. 83 Baykal A. T. 46 Abdualhadi B. 83 Baykal B. 46 Abdulhadi N. H. 45 Baysal K. 46 Abukunna F. E. 45 Bell G. I. 87 Acilan C. 46 Belo J. A. 67 Adams R. H. 20 Beltrami A. P. 49 Adiguzel Z. 46 Beltrami C. A. 49 Ahmad A. 47 Bento M. 67 Akbay E. 48 Benzoni P. 53 Alitalo K. 19 Berns K. I. 24 Ambati J. 23 Bertero A. 37 Amicucci G. 59 Bilio M. 56 Andolfi G. 82 Bisleri G. 53 Angelucci A. 59 Boccardo S. 76 Anversa P. 32 Bock-Marquette I. 28, 70 Apicella I. 23 Boisset G. 41 Aquaro G. D. 73 Bortolotti F. 90 Arcelli D. 65 Brancaccio M. 37 Arese M. 17 Broughton H. C. 87 Arnold H-H 51 Brunelli S. 57 Atsma D. E. 58 Bucci E. 68 Avitabile S. 65 Bussolino F. 17, 54 Avolio E. 49 Buzzi M. 85 Azzoni E. 57 C B Cai W. 38 Bain O. 50, 60 Caldwell J. H. 78 Baker A. H. 26 Campolo F. 69 Balconi G. 88 Cantarella D. 68 Ballerini L. 78 Capogrossi M. 34 Balogh P. 51 Caragnano A. 49 Banfi A. 52, 76, 77, 91 Carmeliet P. 22 Barbagallo F. 69 Carnevale D. 37 Barbuti A. 53 Carrel T. 77
96 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Carrer A. 54 Diena M. 94 Cashman J. 38 Diviani D. 41 Cellot G. 78 Djonov V. 52 Cesselli D. 49 Dolci S. 69 Chien K. R. 33 Drakulic D. 61, 62 Ciccarese F. 55 Duarte A. 92 Cioffi S. 56 Dzobo K. 63 Claesson-Welsh L. 21 Cobellis G. 82 E Collesi C. 66 Condorelli G. 43 Eckstein F. 77 Consolo F. 94 El Naggar A. W. 72 Conti V. 57 Engelhardt S. 42 Contu R. 43 Eulalio A. 64 Cook S. 77 Evtoski Z. 59 Cossu G. 88 Crepaldi T. 68 F Crescini E. 53 Crippa S. 41 Fabregat C. 30 Czömpöly T. 51 Failla C. M. 65 Falcione A. 90 D Fantin A. 86 Faulkner G. 71 D’Aniello C. 82 Felician G. 66 Dal Ferro M. 64, 66, 90 Fibbe W. E. 58 Dawson M. 38 Fico A. L. 82 De Angelis N. 88 Fiore G. B. 94 De Falco S. 23 Fiorenzano A. 82 De la Pompa J. L. 29 Frijia F. 73 De Luca A. 53 Fujikara K. 76 De Oliveira E. M. 47 Furtado J. 67 De Vries A. A. F. 58 Dejana E. 15 G Dell’Era P. 53 Delle Monache S. 59 Gallo S. 68 Denvir M.A. 79 Galvez B. G. 88 Detela G. 50, 60 Gargiulo M. 85 Di Camillo B. 55 Gatti S. 68 Di Siena S. 69 Gesenhues F. 28, 70
97 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Giacca M. 27, 54, 64, 66, 74, 78, 90, 95 Gianfranceschi G. 49 Iaconis S. 82 Gianni-Barrera R. 52, 91 Ibberson M. 41 Gimmelli R. 69 Ibrani D. 47 Giovannetti G. 73 Illingworth E. 56 Giraud M. N. 77 Indraccolo S. 55 Giraudo E. 17, 54 Ippodrino R. 74 Giuliani A. 59 Isidori A. 69 Götz F. 28, 70 Izpisua Belmonte J. C. 30 Goumans M. J. 58 Grassi A. 55 J Groppa E. 91 Grunewald M. 18 Jaconi M. 75 Günaydin S. 48 Jasnic-Savovic J. 71 Gürpinar Ö. A. 48 Jopling C. 30 Gutierrez M. I. 95 K H Kacar O. 46 Hajjar R. J. 25 Katare R. 49 Hannappel E. 28, 70 Kazemi M. 54 Harshman K. 41 Kellermayer Z. 51 Hassan D. A. 45 Keshet E. 18 Hayder A. 83 Kessler O. 16 Heberer M. 52, 77 Klajn A. 61, 62 Helmrich U. 77 Kojic S. 71 Hescheler J. 31 Kolanowski T. 89 Hess D. A. 87 Konofagou E. 76 Hinkel R. 28, 70 Korraa S. 72 Hirano Y. 23 Kovacevic-Grujicic N. 81 Hlushchuk R. 52 Krstic A. 61, 62 Hoffmann P. 75 Kupatt C. 28, 70 Hofmann A. 91 Kurpisz M. 89 Hubbell J. 91 Hussein A. M 45 L
I Lábadi Á. 51 Landini L. 73
98 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Lanier M. 38 Milivojevic M. 80, 81 Latini R. 88 Minchiotti G. 82 Laugwitz K-L 36 Mohamed N. M. 45 Lebherz C. 28, 70 Mohammad F. I. 83 Lembo G. 37 Mohammed S. A. A. 88 Lenzi A. 69 Moimas S. 54 Leo C. 68 Molla F. 88 Licht T. 18 Morales A. 76 Lionetti V. 73 Morton T. 91 Liu K. Y. 87 Mullins J. J. 79 Livi U. 49 Muneretto C. 53 Lombardi M. 73 Myers M. P. 95 Long C. S 78 Lovric J. 74 N Luo J. 76 Lusic M. 66 Naro F. 69 Nemir M. 41 M Nestorovic A. 71 Neufeld G. 16 Madeddu P. 49 Nikcevic G. 80 Maidhof R. 76 Nori S. 69 Maione F. 54 Novelli E. 94 Mano M. 64, 74, 95 Novotová M. 84, 93 Marcu I. C. 75 Marsano A. 76, 77 O Marti M. 30 Martinelli V. 71, 78 Okolotowicz K. 38 Martino M. 91 Olszewska M. 89 Martucciello S. 56 Oltolina F. 94 Mason C. 50 Onur M. A. 48 Mathur A. 50, 60 Orecchia A. 65 Matrone G. 79 Ounzain S. 41 Medico E. 68 Melly L. 77 P Menchetti L. 73 Meneguzzi G. 65 Pantano S. 95 Mercola M. 38 Papadopulos F. 85 Mestroni L. 78 Parker M. I. 63 Mettouchi A. 65 Pasquinelli G. 85
99 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Passier R. 35 Ruvo M. 23 Pattarini L. 95 Pavesi A. 94 S Pawlak P. 89 Pedrazzini T. 41 Sabag A. 16 Pellegrini M. 69 Sabah B. 83 Petrovic I. 80, 81 Sabah M. I. 83 Pfenninger K. 65 Sacchi V. 91 Pfosser A. 28, 70 Sala V. 68 Phillips M. I. 47 Sanità P. 59 Pietronave S. 94 Santarelli F. 73 Pijnappels D. A. 58 Sarre A. 41 Plein A. 86 Sbroggiò M. 37 Poli A. 68 Schalij M. J. 58 Positano V. 73 Schorderet D. 41 Prat M. 94 Schulman I. H. 40 Prato M. 78 Senna M. M. 72 Putman D. M. 87 Serhatlı M. 46 Serini G. 17 R Sevim H. 48 Siatkowski M. 89 Radaelli A. 94 Simon G. 65 Radojkovic D. 71 Sleep E. 30 Ragone G. 65 Smetana J. 43 Rakicevic L. 71 Smolkin T. 16 Ramkisoensing A. A. 58 Soncini M. 94 Raya A. 30, 53 Stachel G. 28, 70 Raya M. 30 Staszewsky L. 88 Recchia F. A. 39, 73 Stevanovic M. 61, 62, 80, 81 Redl H. 91 Sune G. 30 Refaay S. 72 Swildens J. 58 Reginato S. 52 Richaud Y. 53 T Ristagno G. 88 Romano S. L. 73 Tang Y. L. 47 Rozwadowska N. 89 Tarone G. 37 Ruegg C. 80 Tavares A. T. 92 Ruhrberg C. 86 Teixeira V. 92 Ruozi G. 90 Tevaearai H. 77
100 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
Toffolo G. 55 Zecchin A. 95 Toma F. M. 78 Zentilin L. 54, 64, 66, 74, 78, 90 Trani M. 52 Zhang D. H. 43 Trenkwalder T. 28, 70 Tucker C. S. 79 Tudisco L. 23 Tuncer A. 46 Turco A. 78
U
Ullrich N. D. 75 U v a P. 65
V
Vanderheyden M. 93 Vascotto C. 49 Vunjak-Novakovic G. 76
W
Wall I. 50, 60 Weber B. 85 Wiland E. 89 Willems E. 38 Wilson K.S. 79
X
Xenarios I. 41
Z
Zacchigna S. 54, 64, 90 Zahradník I. 84, 93 Zambruno G. 65 Zamperone A. 94 Zaric J. 80
101 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
List of Participants
A
ABUKUNNA Fatima, Department of Biology, Central laboratory, Ministry of Science & Technology, Almamora, Juba street, Khartoum, P.O. Box 7099, SUDAN. Telephone: +249-91-2576093; Fax: +249-155-183855; E-mail: [email protected]
ACILAN Ceyda, TUBITAK Marmara Research Center, Genetic Engineering and Biotechnology Institute, 41470, Gebze Campus, Kocaeli, TURKEY. Telephone: +90-262-6773354; Fax: +90-262-6463929; E-mail: [email protected]
ADAMS Ralf H., Max Planck Institute for Molecular Biomedicine, Dept. of Tissue Morphogenesis, Roentgenstrasse 20, 48149 Münster, GERMANY. Telephone: +49-251-70365400; Fax: +49-251-70365499; E-mail: [email protected], [email protected]
AHMAD CHATTHA Aftab, School of Biological Sciences, University of the Punjab, Lahore, New Campus, 54590, PAKISTAN. Telephone: +92-300-7402202; Fax: +92-42-99230980; E-mail: [email protected]
AKBAY Esin, Hacettepe University, Faculty of Science, Department of Biology, 06800 Beytepe, Ankara, TURKEY. Telephone: +90-312-2976440; Fax: +90-312-2992028; E-mail: [email protected]
ALESSANDRINI Federica, Master Degree in Medical Biotechnology, Faculty of Medicine, University of Trieste, ITALY. E-mail: [email protected]
ALI Hashim, ICGEB, Molecular Medicine Group, AREA Science Park, Padriciano 99, I-34149 Trieste, ITALY. Telephone: +39-040-7357325; Fax: +39-040-226555; E-mail: [email protected], [email protected]
ALITALO Kari, Molecular Cancer Biology Research Program, Biomedicum Helsinki 1, University of Helsinki, P.O.Box 63 (Haartmaninkatu 8), 00014 Helsinki, FINLAND. Telephone: +358-9-19125511; Fax: +358-9-19125510; E-mail: [email protected]
ANVERSA Piero, Brigham & Women’s Hospital, Harvard Medical School, 20 Shattuck St., Thorn 1319A, Boston, MA 02115, USA. Telephone: +1-617-5258170; Fax: +1-617-2646320; E-mail: [email protected]
ANZINI Marco, Scuola di Specializzazione in Malattie dell’Apparato Cardiovascolare, Facolta` di Medicina e Chirurgia, Università degli Studi di Trieste, Trieste, Italy. E-mail: [email protected]
102 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
ASTANINA Elena, Laboratory of Vascular Oncology, IRCC, Institute for Cancer Research and Treatment, Strada Provinciale 142, Km 3.95, 10060 Candiolo, Torino, ITALY. Telephone: +39-011-9933506; Fax: +39-011-9933524; E-mail: [email protected]
AVOLIO Elisa, CIME - Centro Interdipartimentale di Medicina Rigenerativa, Università degli Studi di Udine, Piazzale Kolbe 4, 33100 Udine, ITALY. Telephone: +39-340-4684725; Fax: +39-0432-559420; E-mail: [email protected]
B
BACKOVIC Ana, ICGEB, Molecular Medicine Group, AREA Science Park, Padriciano 99, I-34149 Trieste, ITALY. Telephone: +39-040-3757325; Fax: +39-040-226555; E-mail: [email protected]
BAIN Owen William, 115, Roberts Building, Department of Biochemical Engineering, University College London, Torrington Place, London, WC1E 7JE, UNITED KINGDOM. Telephone: +44-7912-608490; Fax: +44-20-72090703; E-mail: [email protected]
BAKER Andrew H., Institute of Cardiovascular and Medical Sciences, BHF Glasgow Cardiovascular Research Centre, University of Glasgow, 126 University Place, Glasgow, G12 8TA, Scotland, UNITED KINGDOM. Telephone: +44-1413301977; Fax: +44-1413303360; E-mail: [email protected]
BALOGH Péter, Department of Immunology and Biotechnology, University of Pécs, Szigeti út 12, H-7624 Pécs, HUNGARY. Telephone: +36-72-536288; Fax: +36-72-536289; E-mail: [email protected]
BANFI Andrea, Cell and Gene Therapy, Basel University Hospital, ICFS 407, Hebelstrasse 20, 4031 Basel SWITZERLAND. Telephone: +41-61-2653507; Fax: +41-61-2653990; E-mail: [email protected]
BARACCHINI Silvia, Master Degree in Medical Biotechnology, Faculty of Medicine, University of Trieste, ITALY. E-mail: [email protected]
BASO Beatrice, University of Trieste, Faculty of Medicine, Italy. E-mail: [email protected]
BELTRAMI Antonio Paolo, Istituto di Anatomia Patologica Universitaria, Azienda Ospedaliero Universitaria di Udine, P.zzle S. Maria della Misericordia, 33100 Udine, ITALY. Telephone: +39-0432-559400; Fax: +39-0432-559420; E-mail: [email protected]
BELTRAMI Carlo Alberto, Istituto di Anatomia Patologica Universitaria, Azienda Ospedaliero Universitaria di Udine, P.zzle S. Maria della Misericordia, 33100 Udine, ITALY. Telephone: +39-0432-559400; Fax: +39-0432-559420; E-mail: [email protected]
103 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
BENZONI Patrizia, Dept. Biomedical Science & Biotechnology, Fibroblast Reprogramming Unit, 11, viale Europa, 25123 Brescia, ITALY. Telephone: +39-030-3717314; Fax: +39-030-3717539; E-mail: [email protected]
BERNS Kenneth I., University of Florida Genetics Institute, Molecular Genetics and Microbiology, College of Medicine, Cancer/Genetics Research Complex, 2033 Mowry Road, P.O. Box 103610, Gainesville, FL 32608, USA. Telephone: +1-352-2738100; Fax: +1-352-2738284; E-mail: [email protected]
BOCCARDO Stefano, Fondazione Istituto Italiano di Tecnologia, Via Morego, 30 16163 Genova, ITALY. Telephone: +39-3290833536; Fax: +39-010-7170817; E-mail: [email protected]
BON Carlotta, Master Degree in Medical Biotechnology, Faculty of Medicine, University of Trieste, ITALY. E-mail: [email protected]
BORTOLOTTI Francesca, ICGEB, Molecular Medicine Group, AREA Science Park, Padriciano 99, I-34149 Trieste, ITALY. Telephone: +39-040-3757325; Fax: +39-040-226555; E-mail: [email protected]
BOSSI Fleur, University of Trieste, Via Valerio 28, 34127 Trieste, ITALY. Telephone: +39-040-5584035; Fax: +39-040-5584023; E-mail: [email protected]
BRAGA Luca, ICGEB, Molecular Medicine Group, AREA Science Park, Padriciano 99, I-34149 Trieste, ITALY. Telephone: +39-040-3757325; Fax: +39-040-226555; E-mail: [email protected]
BREGAR Dejan, University in Ljubljana, Medical Faculty, Institute for Histology and Embriology, Korytkova2, 1000 Ljubljana, SLOVENIA. E-mail: [email protected]
BUSSOLINO Federico, Department of Oncological Sciences, University of Turin, Sp 142, Km 3.95, 10060 Candiolo, Turin, ITALY. Telephone: +39-011-9933347; Fax: +39-011-9933524; E-mail: [email protected], [email protected]
C CAMERINI Fulvio, Azienda Ospedaliero - Universitaria “Ospedali Riuniti” Trieste, ITALY. Telephone: +39-040-3992327; Fax: +39-040-3992533; E-mail: [email protected]
CAMPAGNOL Davide, Master Degree in Medical Biotechnology, Faculty of Medicine, University of Trieste, ITALY. E-mail: [email protected]
104 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
CAMPANER Elena, Master Degree in Medical Biotechnology, Faculty of Medicine, University of Trieste, ITALY. E-mail: [email protected]
CAPOGROSSI Maurizio, Laboratory of Vascular Pathology, Istituto Dermopatico dell’Immacolata, IRCCS, Via dei Monti di Creta, 104, 00167 - Rome, ITALY. Telephone: +39-06-66462429; Fax: +39-06-66462430; E-mail: [email protected], [email protected]
CARMELIET Peter, Center for Transgene Technology & Gene Therapy, Flanders Interuniversity Institute for Biotechnology, University of Leuven, Herestraat 49, box 912, 3000 Leuven, BELGIUM. Telephone: +3216373204; Fax: +3216372585; E-mail: [email protected], [email protected]
CARPI Andrea, Università degli Studi di Padova, Dip. di Scienze Biomediche, Viale G. Colombo 3, 35121 Padova, ITALY. Telephone: +39-049-8276414; Fax: +39-049-8276040; E-mail: [email protected]
CARRER Alessandro, ICGEB, Molecular Medicine Group, AREA Science Park, Padriciano 99, I-34149 Trieste, ITALY. Telephone: +39-040-3757325; Fax: +39-040-226555; E-mail: [email protected]
CEH Katerina, Veterinary faculty, Cesta v Mestni Log 47, 1000 Ljubljana, SLOVENIA. Telephone: +386-1-4779382; Fax: +386-1-4779340; E-mail: [email protected]
CHENG Shuk Han, Dept of Biology & Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, HONG KONG. Telephone: +852-34429027; Fax: +852-34420522; E-mail: [email protected]
CHIARUTTINI Giulia, ICGEB, Cellular Immunology Laboratory, AREA Science Park, Padriciano 99, 34149 Trieste, ITALY. Telephone: +39-040-3757371; Fax: +39-040-226555; E-mail: [email protected]
CHIEN Kenneth, Director, MGH Cardiovascular Research Center, Charles Addison and Elizabeth Ann Sanders Professor, Harvard University, Department of Stem Cell and Regenerative Biology, 159 Fairchild Labs, 7 Divinity Avenue, Cambridge, MA 02138, USA. Telephone: +1-617-6433440; Fax: +1-617-6433451; E-mail: [email protected]
CICCARESE Francesco, Dipartimento di Scienze Chirurgiche, Oncologiche e Gastroenterologiche, Sezione di Immunologia e Oncologia, Via Gattamelata 64, 35128, Padova, ITALY. Telephone: +39-049-8215851; Fax: +39-049-8072854; E-mail: [email protected]
CIOFFI Sara, CNR-IGB Adriano Buzzati Traverso, Via Pietro Castellino 111, 80131 Naples - ITALY. Telephone: +39-081-6132230; Fax: +39-081-5609877; E-mail: [email protected]
105 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
CLAESSON-WELSH Lena, Uppsala University, Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Dag Hammarsköldsv. 20, 75185 Uppsala, SWEDEN. Telephone: +46-18-4714363; Fax: +46-18-4714808; E-mail: [email protected]
COAN Michela, Master Degree in Medical Biotechnology, Faculty of Medicine, University of Trieste, ITALY. E-mail: [email protected]
COBELLIS Gilda, Dipartimento di Patologia Generale, Seconda Università di Napoli, Via L. De Crecchio, 7, 80138 Napoli, ITALY. Telephone: +39-081-292399; Fax: +39-081-292399; E-mail: [email protected]
COLLESI Chiara, ICGEB, Molecular Medicine Group, AREA Science Park, Padriciano 99, I-34149 Trieste, ITALY. Telephone: +39-040-3757375; Fax: +39-040-226555; E-mail: [email protected]
CONDORELLI Gianluigi, Department of Medicine, National Research Council CNR, P.le Aldo Moro 7, 00185 Rome, ITALY. Telephone: +39-06-49932458; Fax: +39-06-49932498; E-mail: [email protected], [email protected]
CONTI Valentina, Division of Regenerative Medicine, Stem Cells and Gene Therapy, DIBIT, San Raffaele Scientific Institute, Via Olgettina, 58, 20132 Milan, ITALY. Telephone: +39-02-26436196; Fax: +39-02-26434621; E-mail: [email protected]
COSENTINO Simona, Centro Cardiologico Monzino, via Carlo Parea, 4, 20138 Milano, ITALY. Telephone: +39-02-58002018; Fax: +39-02-58002342; E-mail: [email protected]
COZZI Francesca, Master Degree in Medical Biotechnology, Faculty of Medicine, University of Trieste, ITALY. E-mail: [email protected]
D DAL FERRO Matteo, ICGEB, Molecular Medicine Group, AREA Science Park, Padriciano 99, I-34149 Trieste, ITALY and Scuola di Specializzazione in Malattie dell’Apparato Cardiovascolare, Facoltà di Medicina e Chirurgia, Universita` degli Studi di Trieste, Trieste, ITALY. Telephone: +39-040-3757325; Fax: +39-040-226555; E-mail: [email protected]
DAL MAS Andrea, ICGEB, Human Molecular Genetics Laboratory, AREA Science Park, Padriciano 99, 34149 Trieste, ITALY. Telephone: +39-040-3757388; Fax: +39-040-226555; E-mail: [email protected]
106 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
DAPAS Barbara, Lab. Research (Molecular Biology Section), Division of Internal Medicine, University Hospital of Cattinara, Strada di Fiume 447, Trieste, ITALY. Telephone: +39-040-3996228; Fax: +39-040-3994593; E-mail: [email protected]
DAPAS Marina, ICGEB, Molecular Medicine Group, AREA Science Park, Padriciano 99, I-34149 Trieste, ITALY. Telephone: +39-040-3757325; Fax: +39-040-226555; E-mail: [email protected]
DE FABRIS Ilaria Maria, Master Degree in Medical Biotechnology, Faculty of Medicine, University of Trieste, ITALY. E-mail: [email protected]
DE FALCO Sandro, Institute of Genetics and Biophysics – CNR, Via Pietro Castellino 111, 80131 Naples, ITALY. Telephone: +39-081-6132354; Fax: +39-081-6132706; E-mail: [email protected]
DE IACO Doriano, Master Degree in Medical Biotechnology, Faculty of Medicine, University of Trieste, ITALY. E-mail: [email protected]
DE LA POMPA MÍNGUEZ José Luis, Fundación CNIC Carlos III, Centro Nacional de Investigaciones Cardiovasculares, Cardiovascular Developmental Biology, Cardiovascular Development and Repair, Melchor Fernández Almagro 3, P.O. Box 28029, Madrid, SPAIN. Telephone: +34-620-936633; Fax: +34-91-4531304; E-mail: [email protected]
DE SABBATA Giulia, ICGEB, Protein Networks Laboratory, AREA Science Park, Padriciano 99, 34149 Trieste, ITALY. Telephone: +39-040-3757327; Fax: +39-040-226555; E-mail: [email protected]
DE VRIES Antonius Adrianus Franciscus, Associate Professor, Department of Cardiology, Leiden University Medical Center, Postal Zone: C5-P, P.O. Box 9600, 2300 RC Leiden, THE NETHERLANDS. Telephone: +31-71-5265989; Fax: +31-71-5266809; E-mail: [email protected]
DEBELJAK Nataša, Institute of Biochemistry, Faculty of Medicine University of Ljubljana, Vrazov trg 2, SI-1000 Ljubljana, SLOVENIA. Telephone: +386-1-5437663; Fax: +386-1-5437641; E-mail: [email protected]
DEJANA Elisabetta, IFOM Foundation, Via Adamello 16, 20139 Milan, ITALY. Telephone: +39-02-574303234; Fax: +39-02-574303088; E-mail: [email protected]
DEL TORTO Alberico, c/o Scuola Superiore Sant’Anna, P.zza Martiri della Libertà, 33, 56127 Pisa, ITALY. Telephone: +39-050-883614; Fax: +39-050-883250; E-mail: [email protected]
107 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
DELL’ERA Patrizia, Dept. Biomedical Science & Biotechnology, Fibroblast Reprogramming Unit, 11, Viale Europa, 25123 Brescia, ITALY. Telephone: +39-030-3717539; Fax: +39-030-3717539; E-mail: [email protected]
DELLE MONACHE Simona, Department of Biomedical Science and Technologies Coppito II, Via Vetoio, 67100, L’Aquila, ITALY. Telephone: +39-349-5891154; Fax: +39-086-2433433; E-mail: [email protected]
DELPIN Anna, Reparto di Chirurgia Plastica, Ospedale di Cattinara, XII piano torre Medica, Strada di Fiume 447, 34149 Trieste, ITALY. Telephone: +39-328-3013204; Fax: +39-040-3994388; E-mail: [email protected]
DENDORFER Andreas Markus, Walter Brendel Center for Experimental Medicine, Ludwig-Maximilians-University Munich, Clinic of the University Munich, Marchioninistr. 15, 81377 Munich, GERMANY. Telephone: +49-89-218076535; Fax: +49-89-218076532; E-mail: [email protected]
DETELA Giulia, Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UNITED KINGDOM. Telephone: +44-20-76793819; Fax: +44-(0)20-72090703; E-mail: [email protected]
DRAKULIĆ Danijela, Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, P.O. Box 23, 11010 Belgrade, SERBIA. Telephone: +381-11-3976212; Fax: +381-11-3975808; E-mail: [email protected]
E EDOSEGHE Isoken Faith, University of Nigeria, Nsukka, Faculty of Medical Sciences, UNN, Nsukka, Enugu State, NIGERIA. Telephone: +234-8033431056; Fax: +234-8033431056; E-mail: [email protected]
EGBADON Benjamin, Benue State University, College of Health Science, Km.1 Gboko Road, Makurdi, Benue State, NIGERIA. Telephone: +234-703-3326160; Fax: +234-44-3265432; E-mail: [email protected]
EGUN Frankie, Nnamdi Azikiwe University, Faculty of Medical Science, Awka (Main Campus), Along Enugu - Onitsha Express Way, Awka Anambra State, NIGERIA. Telephone: +234-8183865954; Fax: +234-15880803; E-mail: [email protected]
ENGELHARDT Stefan, Technische Universität Muenchen, TUM, Faculty of Medicine, Institute of Pharmacology and Toxicology, Biedersteiner Str. 29, 80802 Munich, GERMANY. Telephone: +49-89-41403260; Fax: +49-89-41403261; E-mail: [email protected], [email protected]
108 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
EULALIO Ana, ICGEB, Molecular Medicine Group, AREA Science Park, Padriciano 99, I-34149 Trieste, ITALY. Telephone: +39-040-3757323; Fax: +39-040-226555; E-mail: [email protected]
F FAILLA Cristina Maria, Molecular and Cell Biology Laboratory, IDI-IRCCS, Via Monti di Creta 104, 00167 Roma, ITALY. Telephone: +39-06-66464734; Fax: +39-06-66464705; E-mail: [email protected]
FALCIONE Antonella, ICGEB, Molecular Medicine Group, AREA Science Park, Padriciano 99, I-34149 Trieste, ITALY. Telephone: +39-040-3757325; Fax: +39-040-226555; E-mail: [email protected]
FELICIAN Giulia, ICGEB, Molecular Medicine Group, AREA Science Park, Padriciano 99, I-34149 Trieste, ITALY. Telephone: +39-040-3757325; Fax: +39-040-226555; E-mail: [email protected]
FRANFORTE Elisa, Master Degree in Medical Biotechnology, Faculty of Medicine, University of Trieste, ITALY. E-mail: [email protected]
FRUNCILLO Eleonora, Master Degree in Medical Biotechnology, Faculty of Medicine, University of Trieste, ITALY. E-mail: [email protected]
FURTADO João, Departamento de Ciências Biomédicas e Medicina. Universidade do Algarve, Campus Gambelas, C8, 8005-139 Faro, PORTUGAL. Telephone: +351-916048142; Fax: +351-289-818419; E-mail: [email protected]
FUSCO Laura, Master Degree in Medical Biotechnology, Faculty of Medicine, University of Trieste, ITALY. E-mail: [email protected]
G GALLO Simona, Dipartimento di Anatomia, Farmacologia e Medicina Legale, Corso Massimo D’Azeglio 52, 10126 Torino, ITALY. Telephone: +39-011-6707747; Fax: +39-011-6705931; E-mail: [email protected]
GALLO Stefania, Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche, Via Abbiategrasso 207, 27100 Pavia, ITALY. Telephone: +39-0382-546324; Fax: +39-0382-422286; E-mail: [email protected]
109 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
GAMBINI Elisa, Centro Cardiologico Monzino Spa, Via Carlo Parea 4, 20138 Milano, ITALY. Telephone: +39-02-58002023; Fax: +39-02-58002425; E-mail: [email protected]
GATTI Stefano, Dipartimento di Anatomia, Farmacologia e Medicina Legale, Corso Massimo d’Azeglio 52, 10126 Torino, ITALY. Telephone: +39-011-6707747; Fax: +39-011-6705931; E-mail: [email protected]
GIACCA Mauro, ICGEB Trieste, Molecular Medicine Laboratory, Area Science Park, Padriciano 99, I-34149 Trieste, ITALY. Telephone: +39-040-3757324; Fax: +39-040-3757380; E-mail: [email protected]
GIANNOTTA Monica, IFOM Fondazione Istituto Firc di Oncologia Molecolare, Via Adamello 16, 20139 Milano, ITALY. Telephone: +39-02-574303200; Fax: +39-02-574303088; E-mail: [email protected]
GIMMELLI Roberto, Sapienza university of Rome, via Antonio Scarpa 16, 00161 Rome, ITALY. Telephone: +39-06-49766580; Fax: +39-06-4462854; E-mail: [email protected]
GRASSI Gabriele, Dep of Life Sciences, Via L. Giorgieri 1, I-34127 Trieste, c/o UCO di Clinica Medica Generale e di Terapia Medica, Ospedale di Cattinara, Edificio Anatomia Patologica, piano o, laboratori di Medicina Interna‚ Strada di Fiume 447, Trieste, ITALY. Telephone: +39-040-3996227; Fax: +39-040-3994593; E-mail: [email protected]
GUTIERREZ Maria Ines, ICGEB, Molecular Medicine Group, AREA Science Park, Padriciano 99, I-34149 Trieste, ITALY. Telephone: +39-040-3757325; Fax: +39-040-226555; E-mail: [email protected]
H HAJJAR Roger, The Mount Sinai Medical Center, Weiner Family Cardiovascular Research Laboratories, One Gustave L. Levy Place, New York, NY 10029-6574, USA. Telephone: +1-212-2414082; Fax: +1-212-2414080; E-mail: [email protected]
HESCHELER Jürgen, Institute for Neurophysiology, Faculty of Medicine, University of Cologne, Robert-Koch-Str. 39, D-50931 Cologne, GERMANY. Telephone: +49-0221-4786960; Fax: +49-0221-4783834; E-mail: [email protected]
HINKEL Rabea, Internal Medicine I, Klinikum Großhadern - LMU, Marchioninistr. 15, 81377 Munich, GERMANY. Telephone: +49-89-70953092; Fax: +49-89-70956075; E-mail: [email protected]
110 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
I
IPPODRINO Rudy, ICGEB, Molecular Medicine Group, AREA Science Park, Padriciano 99, I-34149 Trieste, ITALY. Telephone: +39-040-3757325; Fax: +39-040-226555; E-mail: [email protected]
IZPISUA BELMONTE Juan Carlos, The Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd., La Jolla, CA 92037, USA. Telephone: +1-858-4534100; Fax: +1-858-4532573; E-mail: [email protected]
K KALAJA Odeta, Master Degree in Medical Biotechnology, Faculty of Medicine, University of Trieste, ITALY. E-mail: [email protected]
KAZEMI Maryam, ICGEB, Molecular Medicine Group, AREA Science Park, Padriciano 99, I-34149 Trieste, ITALY. Telephone: +39-040-3757325; Fax: +39-040-226555; E-mail: [email protected]
KESHET Eli, Dept of Developmental Biology & Cancer Research, The Hebrew University-Hadassah Medical School, Jerusalem 91120, ISRAEL. Telephone: +972-2-6758496; Fax: +972-2-67575195; E-mail: [email protected], [email protected]
KOJIC Snezana, IMGGE, Vojvode Stepe 444a, PO Box 23, 11010 Belgrade, SERBIA. Telephone: +381-11-3976658; Fax: +381-11-3975808; E-mail: [email protected]
KORRAA Soheir Saad Mohamed, National Center for Radiation Research and Technology, Egyptian Atomic Energy Authority, NCRRT - 3 Ahmed El Zomour St. 8th Sector Nasr City, P.O. Box 29, Cairo, EGYPT. Telephone: +20-2-22727679; Fax: +20-2-22754403; E-mail: [email protected]
KUPATT-JEREMIAS Christian, Ludwig Maximilians-University of Munich, Klinikum Großhadern, Medizinische Klinik und Poliklinik I, Marchionini str. 15, 81377 Munich, GERMANY. Telephone: +49-89-70953092; Fax: +49-89-70956075; E-mail: [email protected]
L LAMPRECHT Gabriella, Direzione Sanitaria – AOUTS, Trieste, ITALY. Telephone: +39-040-3994915, +39-333-2616552; Fax: +39-040-910690; E-mail: [email protected]
111 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
LAUGWITZ Karl-Ludwig, Klinikum rechts der Isar - Technische Universität München, I. Medizinische Klinik, Ismaninger Strasse 22, 81675 München, GERMANY. Telephone: +49-89-41402947/2350; Fax: +49-89-41404901; E-mail: [email protected]
LIONETTI Vincenzo, Via G. Moruzzi, 1, 56124 - Pisa, ITALY. Telephone: +39-0503153572; Fax: +39-0503152166; E-mail: [email protected]
LONG Carlin, 777 Bannock, Cardiology Box 0960, Denver, CO 80204, USA. Telephone: +1-303-4365498; Fax: +1-303-4367739; E-mail: [email protected]
LUCIC Bojana, ICGEB, Molecular Medicine Group, AREA Science Park, Padriciano 99, I-34149 Trieste, ITALY. Telephone: +39-040-3757325; Fax: +39-040-226555; E-mail: [email protected]
LUSIC Marina, ICGEB, Molecular Medicine Group, AREA Science Park, Padriciano 99, I-34149 Trieste, ITALY. Telephone: +39-040-3757375; Fax: +39-040-226555; E-mail: [email protected]
M
MANO Miguel, ICGEB, Molecular Medicine Group, AREA Science Park, Padriciano 99, I-34149 Trieste, ITALY. Telephone: +39-040-3757323; Fax: +39-040-226555; E-mail: [email protected]
MARCU Irene Cristina, Department of Physiology, University of Bern, Bühlplatz 5, CH-3012 Bern, SWITZERLAND. Telephone: +41-31-6313503; Fax: +41-31-6314611; E-mail: [email protected]
MARINI Bruna, ICGEB, Molecular Medicine Group, AREA Science Park, Padriciano 99, I-34149 Trieste, ITALY. Telephone: +39-040-3757325; Fax: +39-040-226555; E-mail: [email protected]
MARINI Elisa, Master Degree in Medical Biotechnology, Faculty of Medicine, University of Trieste, ITALY. E-mail: [email protected]
MARSANO Anna, University Hospital Basel, ICFS, lab 405, Hebelstrasse 20, 4031, Basel, SWITZERLAND. Telephone: +41-61-2652387; Fax: +41-61-2653990; E-mail: [email protected]
MARTELLETTI Elisa, ICGEB, Molecular Medicine Group, AREA Science Park, Padriciano 99, I-34149 Trieste, ITALY. Telephone: +39-040-3757325; Fax: +39-040-226555; E-mail: [email protected]
112 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
MARTINELLI Valentina, ICGEB, Molecular Medicine Group, AREA Science Park, Padriciano 99, I-34149 Trieste, ITALY. Telephone: +39-040-3757322; Fax: +39-040-226555; E-mail: [email protected]
MATRONE Gianfranco, University/BHF Centre for Cardiovascular Science - Room E3.24, The Queen’s Medical Research Institute, University of Edinburgh, 47 Little France Crescent, Edinburgh EH16 4TJ, UNITED KINGDOM. Telephone: +44-(0)753-0158675; Fax: +44-(0)131-2429101; E-mail: [email protected]
MEDIC Nevenka, ICGEB, Molecular Medicine Group, AREA Science Park, Padriciano 99, I-34149 Trieste, ITALY. Telephone: +39-040-3757325; Fax: +39-040-226555; E-mail: [email protected]
MERCOLA Mark, Muscle Development and Regeneration Program, Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA. Telephone: +1-858-7955242; Fax: +1-858-7955221; E-mail: [email protected], [email protected]
MESTRONI Luisa, University of Colorado Denver Anschutz Medical Campus, 12700 E 19th Ave # F442, Aurora, Colorado 80045-2507, USA. Telephone: +1-303-7240577; Fax: +1-303-7240858; E-mail: [email protected]
MEZZINA Nicolò, University of Trieste, Faculty of Medicine, ITALY. E-mail: [email protected]
MIHAJLOVIC Milos, Master Degree in Medical Biotechnology, Faculty of Medicine, University of Trieste, ITALY. E-mail: [email protected]
MILAN Marta, Master Degree in Medical Biotechnology, Faculty of Medicine, University of Trieste, ITALY. E-mail: [email protected]
MILIVOJEVIC Milena, Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, P.O. Box 23, 11010 Belgrade, SERBIA. Telephone: +381-11-3976212; Fax: +381-11-3975808; E-mail: [email protected]
MINCHIOTTI Gabriella, Institute of Genetics and Biophysics “A. Buzzati-Traverso”, CNR, Via Pietro Castellino 111, 80131 Naples, ITALY. Telephone: +39-081-6132354; Fax: +39-081-6132595; E-mail: [email protected]
MOHAMMAD Farooq Ibrahem, Biotechnology Research Center, Al Nahrain University, Al Jaderia, Baghdad, IRAQ. Telephone: +964-7702522348; E-mail: [email protected]
113 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
MOIMAS Silvia, ICGEB, Molecular Medicine Group, AREA Science Park, Padriciano 99, I-34149 Trieste, ITALY. Telephone: +39-040-3757325; Fax: +39-040-226555; E-mail: [email protected]
MOSCA Sarah, Master Degree in Medical Biotechnology, Faculty of Medicine, University of Trieste, ITALY. E-mail: [email protected]
MY Ilaria, Università Vita-Salute San Raffaele, via olgettina 58, 20132 Milano, ITALY. Telephone: +39-3493351380; E-mail: [email protected]
N NEUFELD Gera, Cancer Research Center, Faculty of Medicine, Technion, Israel Institute of Technology, 1 Efron Street, Haifa, 31096, P.O. Box 9679, ISRAEL. Telephone: +972-4-8295430; Fax: +972-4-8523672; E-mail: [email protected]
NORDIO Andrea, ICGEB, Molecular Medicine Group, AREA Science Park, Padriciano 99, I-34149 Trieste, ITALY. Telephone: +39-040-3757325; Fax: +39-040-226555; E-mail: [email protected]
NOVOTOVÁ Marta, UMFG SAV, Vlárska 5, 83334 Bratislava, SLOVAKIA. Telephone: +421-903225227; Fax: +421-254773666; E-mail: [email protected]
P PACE Alice, Interdisciplinary Laboratory for the Advanced Studies, SISSA, via Bonomea 265, 34136 Trieste TS, ITALY. Telephone: +39-3495541477; Fax: +39-040-3787528; E-mail: [email protected]
PAPA Giovanni, UCO Plastic Surgery Cattinara Hospital, Strada di Fiume 447, Trieste, ITALY. Telephone: +39-349-7543197; Fax: +39-040-3994388; E-mail: [email protected]
PAPADOPULOS Francesca, Dep.of Specialistic Surgery and Anesthesiological Sciences, University of Bologna, S. Orsola-Malpighi Hospital, via massarenti 9, 40138 Bologna, ITALY. Telephone: +39-3292242290; Fax: +39-051-306861; E-mail: [email protected]
PARON Greta, Master Degree in Medical Biotechnology, Faculty of Medicine, University of Trieste, ITALY. E-mail: [email protected]
PASSIER Robert, Leiden University Medical Center LUMC, Einthovenweg 20, P.O. Box 9600, Postal zone S1-P, 2300 RC Leiden, THE NETHERLANDS. Telephone: +31-715269359; Fax: +31-715268289; E-mail: [email protected], [email protected]
114 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
PEDRAZZINI Thierry, Experimental Cardiology Unit, Department of Medicine, University of Lausanne Medical School, 1011 Lausanne, SWITZERLAND. Telephone: +41-21-3140765; Fax: +41-21-3145978; E-mail: [email protected]
PERESSIN Elisa, ICGEB, Molecular Medicine Group, AREA Science Park, Padriciano 99, I-34149 Trieste, ITALY. Telephone: +39-040-3757325; Fax: +39-040-226555; E-mail: [email protected]
PETERLUNGER Isabel, University of Trieste, Faculty of Medicine, Italy. E-mail: [email protected]
PETRIS Gianluca, ICGEB, Molecular Immunology Laboratory, AREA Science Park, Padriciano 99, 34149 Trieste, ITALY. Telephone: +39-040-3757310; Fax: +39-040-226555; E-mail: [email protected]
PIROZZI Fabrizio, ICGEB, Molecular Medicine Group, AREA Science Park, Padriciano 99, I-34149 Trieste, ITALY and Scuola di Specializzazione in Malattie dell’Apparato Cardiovascolare, Facolta` di Medicina e Chirurgia, Universita` degli Studi di Trieste, Trieste, ITALY. Telephone: +39-040-3757325; Fax: +39-040-226555; E-mail: [email protected]
PLEIN Alice, UCL Institute of Ophthalmology, 11-43 Bath Street, London, EC1V 9EL, UNITED KINGDOM. Telephone: +44-(0)20-76084018; Fax: +44-(0)20-76086810; E-mail: [email protected]
PRAT Maria, Dept. Health Sciences, Università del Piemonte Orientale, Via Solaroli 17, 28100 Novara, ITALY. Telephone: +39-0321-660662; Fax: +39-0321-620421; E-mail: [email protected]
PRATI Giulio, Scuola di Specializzazione in Malattie dell’Apparato Cardiovascolare, Facolta` di Medicina e Chirurgia, Universita` degli Studi di Trieste, Trieste, ITALY. E-mail: [email protected]
PRETE Francesca, ICGEB, Cellular Immunology Laboratory, AREA Science Park, Padriciano 99, 34149 Trieste, ITALY. Telephone: +39-040-3757371; Fax: +39-040-226555; E-mail: [email protected]
PROSDOCIMO Giulia, Master Degree in Medical Biotechnology, Faculty of Medicine, University of Trieste, ITALY. E-mail: [email protected]
PUTMAN David, Hess Lab, Department of Physiology and Pharmacology, The University of Western Ontario, Vascular Biology Research Group, Krembil Centre for Stem Cell Biology, Robarts Research Institute, P.O. Box 5015, 100 Perth Drive, London, ON, N6A 5K8, CANADA. Telephone: +1-519-6635777 ext. 24190; Fax: +1-519-663-3789; E-mail: [email protected]
115 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
PUZZI Luca, ICGEB, Molecular Medicine Group, AREA Science Park, Padriciano 99, I-34149 Trieste, ITALY. Telephone: +39-040-3757325; Fax: +39-040-226555; E-mail: [email protected]
R RASO Andrea, Master Degree in Medical Biotechnology, Faculty of Medicine, University of Trieste, ITALY. E-mail: [email protected]
RECCHIA Fabio, Scuola Superiore Sant’Anna, Piazza Martiri della Liberta` n. 33, 56127 Pisa, ITALY; Department of Physiology, Temple University School of Medicine, 3500 North Broad Street, Philadelphia, PA 19140, USA. Telephone: +1-215-7074482; Fax: +1-215-7075737; E-mail: [email protected]
REGINATO Silvia, Master in Giornalismo Scientifico Digitale, SISSA, via Bonomea 265, 34100 Trieste, ITALY. Telephone: +39-333-3670771; Fax: +39-333-3670771; E-mail: [email protected]
REŽEN Tadeja, Institute of Biochemistry, Faculty of Medicine, Vrazov trg 2, SI-1000 Ljubljana, SLOVENIA. Telephone: +386-1-5437595; Fax: +386-1-5437588; E-mail: [email protected]
RING Nadja, ICGEB, Molecular Medicine Group, AREA Science Park, Padriciano 99, I-34149 Trieste, ITALY. Telephone: +39-040-7357325; Fax: +39-040-226555; E-mail: [email protected]
RISTAGNO Giuseppe, Istituto di Ricerche Farmacologiche Mario Negri, via La Masa 19, Milano, 20156, ITALY. Telephone: +39-02-39014613; Fax: +39-02-33200049; E-mail: [email protected]
RIU Federica, Level 7, Bristol Royal Infirmary, Marlborough Street, Bristol, BS2 8HW, UNITED KINGDOM. Telephone: +44-117-3422365; Fax: +44-117-9299737; E-mail: [email protected]
RIVA Silvano, Institute of Molecular Genetics/CNR, Via Abbiategrasso 207, 27100 Pavia, ITALY. Telephone: +39-0382-546320; Fax: +39-0382-546213; E-mail: [email protected]
ROGALSKA Małgorzata, ICGEB, Human Molecular Genetics Laboratory, AREA Science Park, Padriciano 99, 34149 Trieste, ITALY. Telephone: +39-040-3757388; Fax: +39-040-226555; E-mail: [email protected]
ROZWADOWSKA Natalia, Institute of Human Genetics Polish Academy of Sciences, Ul. Strzeszynska 32, 60-479 Poznan, POLAND. Telephone: +48616579229; Fax: +48618233235; E-mail: [email protected]
116 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
RUOZI Giulia, ICGEB, Molecular Medicine Group, AREA Science Park, Padriciano 99, I-34149 Trieste, ITALY. Telephone: +39-040-3757325; Fax: +39-040-226555; E-mail: [email protected]
RUSSO Marco, Scuola di Specializzazione in Malattie dell’Apparato Cardiovascolare, Facolta` di Medicina e Chirurgia, Universita` degli Studi di Trieste, Trieste, ITALY. E-mail: [email protected]
RUSSO Matteo Antonio, Dept. Experimental Medicine, Sapieza University of Rome, Viale Regina Elena 324, 00161 Rome, ITALY. Telephone: +39-06-49970806; Fax: +39-06-499770019; E-mail: [email protected]
S SACCHI Veronica, Cell and Gene Therapy Group-Lab 406, ZLF, Hebelstrasse 20, 4031 Basel, SWITZERLAND. Telephone: +41-61-2653423; Fax: +41-61-2653990; E-mail: [email protected]
SALA Valentina, Dipartimento di Anatomia, Farmacologia e Medicina Legale, Corso Massimo d’Azeglio 52, 10126 Torino, ITALY. Telephone: +39-011-6707734; Fax: +39-011-6705931; E-mail: [email protected]
SALAZAR NASSAR Johanna, Hospital Calderón Guardia, Neuroscience Department, Neurophisiology Service, San José, COSTA RICA. Telephone: +506-87150855; E-mail: [email protected]
SASSET Linda, ICGEB, Molecular Immunology Laboratory, AREA Science Park, Padriciano 99, 34149 Trieste, ITALY. Telephone: +39-040-3757311; Fax: +39-040-226555; E-mail: [email protected]
SCHULMAN Ivonne Hernandez, Clinical Medicine, Division of Nephrology and Hypertension and Interdisciplinary Stem Cell Institute, University of Miami Miller School of Medicine, Biomedical Research Building / Room 824, 1501 N.W. 10th Avenue, Miami, FL 33136, USA. Telephone: +1-305-2438879; Fax: +1-305-2433906; E-mail: [email protected]
SECHI Andrea, Scuola Superiore Sant’ Anna, Piazza Martiri delle Libertà 33, 56127 Pisa, ITALY. Telephone: +39-3346407222; Fax: +39-050-883225; E-mail: [email protected]
SILVESTRIC Ana, Master Degree in Medical Biotechnology, Faculty of Medicine, University of Trieste, ITALY. E-mail: [email protected]
117 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
SINAGRA Gianfranco, Polo Cardiologico, Ospedale di Cattinara, Strada di Fiume 447, 34100 Trieste, ITALY. Telephone: +39-040-3994477; Fax: +39-040-3994153; E-mail: [email protected]
SMOLKIN Tatyana, Cancer Research and vascular Biology Center, The Bruce Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, P.O. Box 9679, 1 Efron St., Haifa, 31096, ISRAEL. Telephone: +972-4-8295425; Fax: +972-4-8523672; E-mail: [email protected]
SOCIN Martina, University of Trieste, Faculty of Medicine, Italy. E-mail: [email protected]
SPAZZAPAN Luca, UCO Chirurgia Plastica, Strada di Fiume 447, Ospedale di Cattinara, Trieste, ITALY. Telephone: +39-340-1945044; Fax: +39-040-3994388; E-mail: [email protected]
STELLA Matteo, University of Trieste, Faculty of Medicine, Italy. E-mail: [email protected]
T TARONE Guido, Department of Genetics, Biology and Biochemistry, Molecular Biotechnology Center, University of Turin, Via Nizza 52, 10126 Turin, ITALY. Telephone: +39-011-6706433; Fax: +39-011-6706432; E-mail: [email protected]
TAVARES, FERNANDES NUNES Ana Teresa, Faculdade de Medicina Veterinaria, Avenida da Universidade Tecnica, 1300-477 Lisboa, PORTUGAL. Telephone: +351-21-3652800 ext. 1021; Fax: +351-21-3652889; E-mail: [email protected]
TRIOLO Lelio, S.C. 1a Medica, Ospedale di Cattinara, Azienda Ospedaliera Ospedali Riuniti di Trieste, Strada di Fiume 447, 34100 Trieste, ITALY. Telephone: +39-040-3994530/4827; Fax: +39-040-3994442; E-mail: [email protected]
U
UKOVICH Laura, Resident in Vascular Surgery, Cattinara Hospital, Trieste, ITALY. Telephone: +39-349-5811336; E-mail: [email protected]
UMA Obed Brown, Osun State University, College of Health Sciences Osogbo, Osun State, NIGERIA. Telephone: +234-8033431056; Fax: +234-35206440; E-mail: [email protected]
118 Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
V VALDIVIA Hector Horacio, Hector H Valdivia, MD, PhD, North Campus Research Complex, University of Michigan, 2800 Plymouth Rd, 28-235N, Ann Arbor, MI 48109, USA. Telephone: +1-734-647-4001; Fax: +1-734-9987711; E-mail: [email protected]
VALENTE Matteo, UCO III Medica Ospedale di Cattinara, Trieste, ITALY. Telephone: +39-335-8210845, +39-040-3994528; Fax: +39-040-3994586; E-mail: [email protected]
VALENTINI Chiara, Master Degree in Medical Biotechnology, Faculty of Medicine, University of Trieste, ITALY. E-mail: [email protected]
W WALL Ivan, Department of Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UNITED KINGDOM. Telephone: +44-20-76793918; Fax: +44-20-72090703; E-mail: [email protected]
Z ZACCHIGNA Serena, ICGEB, Molecular Medicine Group, AREA Science Park, Padriciano 99, I-34149 Trieste, ITALY. Telephone: +39-040-3757325; Fax: +39-040-226555; E-mail: [email protected]
ZAHRADNÍK Ivan, UMFG SAV, Vlarska 5, 83334 Bratislava, SLOVAKIA. Telephone: +421-915422808; Fax: +421-254773666; E-mail: [email protected]
ZECCHIN Annalisa, ICGEB, Molecular Medicine Group, AREA Science Park, Padriciano 99, I-34149 Trieste, ITALY. Telephone: +39-040-3757325; Fax: +39-040-226555; E-mail: [email protected]
ZENTILIN Lorena, ICGEB, Molecular Medicine Group, AREA Science Park, Padriciano 99, I-34149 Trieste, ITALY. Telephone: +39-040-3757325; Fax: +39-040-226555; E-mail: [email protected]
119 ICGEB TRIESTE
More than 200 people representing some Scientific activity at the Trieste Labs focuses 30 nationalities work in the 16 laboratories on advanced projects in current biomedical that operate out of Trieste. The Trieste research, include basic science projects such Component provides a dynamic, scientific as control of gene expression, DNA replication, and educational environment of the DNA repair and RNA processing; studies on highest standard and is fully equipped to human viruses such as HIV, HPV and rotavirus, undertake modern molecular and cellular molecular immunology, cardiovascular and biology research. Almost 60 graduates neurodegenerative disorders, molecular are enrolled in the international PhD genetics, experimental haematology and course, undertaken in collaboration with human gene therapy. Other Groups focus on the high-level academic institutions of projects in bacteriology and yeast genetics, the Open University, UK and the “Scuola protein structure and bioinformatics. Over the Normale Superiore” in Pisa, Italy. Over past 5 years, over 400 scientific papers have 125 PhD students have completed their been published in high impact, international PhD studies since 1992, with an average scientific journals. The research programme of over 3 publications in peer-reviewed is evaluated through an international panel international journals per student. of scientists with specific expertise in the The PhD course includes an intense, respective fields. The research activities are international seminar programme. Movies also supported through a large number of of seminar, and course presentations are grants awarded by numerous, international freely available for download on iTunes U, funding agencies. and have gained huge popularity. ICGEB on iTunes U ICGEB on Twitter
ICGEB on Facebook ICGEB NEW DELHI The Delhi Component is located within resistance and biopesticidals, abiotic and the ICGEB Campus in South Delhi,. biotic plant stresses and crop improvement situated alongside the Jawaharlal Nehru through biotransformation. University (JNU). The Laboratories comprise ICGEB New Delhi has 36 Principal Investigators a main building, a Guest House facility, distributed across 9 Research Groups. The Bioexperimentation Unit, BSL-3 Facility and Component has a flourishing PhD programme a number of greenhouses for agriculture in association with the JNU, with up to 100 related research. PhD students at any given time.There are The main research areas focus on mammalian almost 100 junior scientists working in and plant biology. Biomedical projects are various, extra-mural funded research projects. pursued in virology (hepatitis B and E viruses, ICGEB’s PIs have obtained grants from various human immunodeficiency virus and SARS national funding agencies as well as extra- virus), immunology (biology of the immune mural funds from international agencies, response and tuberculosis), development such as The Wellcome Trust, European Malaria of diagnostics and vaccine candidates Vaccine Initiative, European Commission, for dengue, structural biology (synthetic International Aids Vacine Initiative, National antibiotics, crystal structure determination Institute of Health, Bill and Melinda Gates of proteins) and both in basic research and Foundation, Dupont and Pepsico. An vaccine and drug development in malaria, innovative collaboration programme has as well as development of technologies also been established with the Emory Global for biopharmaceuticals and diagnosis Vaccine Centre to develop vaccines against of infectious diseases. In plant biology, diseases such as tuberculosis and malaria. research projects address the study of insect ICGEB CAPE TOWN There is increasing international recognition African scientists on topics of direct relevance that science- and technology-intensive to the major health and agricultural problems solutions can significantly improve quality of the continent is the ultimate mission of the of life. This is particularly true for Africa, a ICGEB Cape Town Component. Inaugurated in vast continent with variegated geography, 2007, and located within the campus of the cultures, resources, strengths and University of Cape Town, it aims to develop weaknesses. Africa still pays an outrageously research and training activities on subjects high death toll to infectious diseases, such as of major impact for the African continent. malaria (700,000 children under the age of 5 Research focuses on infectious diseases, die every year), or HIV/ AIDS (where every including HIV-AIDS, malaria, tuberculosis 12 hours, 3,000 people die of this disease). and neglected infectious diseases. Plant In Africa, hunger or malnourishment affect biotechnology research aimed at improving one third of the population. In addition, non- the staple crops essential for the African communicable diseases are also increasing population will also be pursued. at an alarming rate. All of these are issues There are approximately 70 people currently that can be effectively tackled through operating at ICGEB Cape Town, of whom a science-based approach focusing on more than 60 are scientists in the laboratories. research and its biomedical and agricultural Two thirds of these originate from African applications. Capacity building of young countries. Frontiers in Cardiac May 30 - June 2 and Vascular Regeneration Trieste, Italy
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The ICGEB acknowledges the financial support to the meeting under the CARDIOCELL project -New technologies of the utilization of bone marrow stem cells for cardiac regeneration of the Friuli Venezia Giulia Region, Italy - LR 26/2005.
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Info: ICGEB Padriciano, 99 34149 Trieste, ITALY - Tel. +39-040-37571, Fax: +39-040-226555 [email protected]
www.icgeb.org