Annual report 2013 Institute for Surgical Research 2

Photo: Øystein H. Horgmo, UiO “Odyssé hender i Paris” Jenny Alnæs

Institute for Surgical Research Division of Cardiovascular and Pulmonary Diseases Oslo University Hospital P.O. Box 4950 Nydalen 0424 Oslo, Norway

Visiting address: Oslo University Hospital Rikshospitalet, Building A3, Sognsvannsveien 20, Oslo.

Telephone: (+47) 23073520 Telefax: (+47) 23073530 email: [email protected] www.ous-research.no/isr

Print: Møklegaard Print Shop AS, Fredrikstad - www.mprint.no 3

Table of Contents

Preface...... 5

Organizational charts Division of Cardiovascular and Pulmonary Diseases...... 6 Institute for Surgical Research...... 7

Abbreviations...... 9

Research Groups...... 11 Surgical Intensive Care Medicine...... 13 Molecular Cardiology...... 19 Integrated Cardiovascular Function...... 23 Center for Cardiological Innovation...... 27 Vilhelm Magnus Laboratory for Neurosurgical Research...... 31 Experimental Orthopaedic Research...... 37 Cell Transplantation and Tissue Engineering...... 41 Experimental microsurgery and Transplantation...... 49 Transplantation and Malignancy...... 55 Plastic and reconstructive surgery...... 61

Projects guided by external project leaders...... 67

Training Courses and Seminars (in Norwegian)...... 71 Basalkurs i laparoskopisk kirurgi...... 72 Introduksjon til nyfødtmedisinske teknikker og prosedyrer...... 72 Thorako-/laparoskopisk kirurgi...... 73 Seminars...... 74

Publications and PhD-Theses...... 75 Publications 2008 – 2013...... 76 PhD-Theses...... 85

Egil Amundsen Lecture...... 92 4

Photo: Øystein H. Horgmo, UiO 5

Preface

I am pleased to present this report on the activities at the Marit Kristine Smedsrud, 10 April 2013, Assessment of Institute for Surgical Research in 2013. incipient global myocardial dysfunction by speckle tracking echocardiography. The report provides merely comprehensive overviews of the developments and the results we have obtained during the Jørgen Gravning, 23 May 2013, Cardioprotective past 12 months and I am delighted to report very positive Mechanisms in Ischemia-Reperfusion Injury and Heart results for 2013 at the institute. I hope the report will Failure - Experimental and Clinical Studies Dissecting the illustrate our commitment to scientific work aiming at Functional Effects of Connective Tissue Growth Factor improving medical practice, and thereby contribute to (CTGF/CCN2) in the Heart. provide the best patient care. Thomas Helle-Valle, 24 May 2013, New Non-invasive 2013 was a year in which important strategic decisions in Methods for Quantification of Regional and Global the project on Regenerative Medicine and Tissue Engineering Myocardial Function in the Normal and Ischemic Left were made. Three years ago, three of the Clinics at Oslo Ventricle. University Hospital (OUH) (Division of Cardiovascular and Pulmonary Diseases, Division of Cancer Medicine, Surgery Sebastian Imre Sarvari, 18 September 2013, Detection of and Transplantation, and Division of Surgery and Clinical subtle myocardial alterations by echocardiographic Neuroscience) decided to start a joint project with the aim to techniques for improved prognostic information in patients provide patients with treatment solutions based on tissue with heart disease. engineering techniques. Professor Aksel Foss was chosen as Mrinal Joel, 10 December 2013, Characterization of stem project leader. During 2013, a formal collaboration has been cell-enriched glioblastoma cells in vitro and in xenotypic established with Karolinska Institute in Stockholm, Sweden, tissue environments. and activities on Regenerative Medicine and Tissue Engineering at the hospital and at the University in Oslo were My special thanks go to the enthusiastic and hard work of all joined in Oslo Regenerative Medicine Initiative. This initiative staff at the institute. Our progresses in 2013 would not have has recently been selected as Focused Area of Research at been possible without their tireless efforts, service to all and OUH for 2014 – 2018. support of the institute’s strategy. The following members of the staff deserve to be particularly acknowledged for taking The Egil Amundsen Lecture 2013 was held by Prof. Dr. med. care of important in-house administrative responsibilities: Dr. h.c. mult. Markus W. Büchler and was entitled “Clinical Jorunn Hestenes Larsen (Executive Assistant), Magali Remy- trials in surgery; towards evidence”. It was a strongly analytic Stockinger (Advisor), Signe Flood Kjeldsen (Laboratories), and inspiring presentation which was very well received by all Vivi Bull Stubberud, Sera Sebastian, Aurora Pamplona, attending the conference. Professor Büchler is Managing Roger Ødegård (Operating Theatres), Irene Stensrud Director of the Department of Surgery at Heidelberg University Andersen (Housekeeping), Shakil Ahmed, Biljana Stangeland, Hospital, and Director of the European Pancreas Centre in Espen Remme and Håvard Attramadal (Seminars). Heidelberg, Germany. Under his stewardship The Centre has developed to one of the leading institutions in the world for the The institute is very grateful for very good cooperation with the treatment of pancreatic disease. He has served as President clinical departments of the hospital which form an important of the German Society of Surgery (2011–2012) and is an hon- link between research and clinical practice. orary member of numerous international surgical societies and associations. Since 2009 Professor Büchler has been a The institute sincerely thanks Oslo University Hospital, Univer- member of the German Academy of Sciences Leopoldina. sity of Oslo and external institutions including The Norwegian Research Council, The Norwegian Advisory Committee for 29 articles, with an average impact factor of 4.3, have been Cardiovascular Diseases and the Health Authority of published from the institute in 2013. South-Eastern Norway for important financial support.

The following 7 doctoral theses from the institute were The annual report 2013 was edited by Jorunn Hestenes defended in 2013: Larsen, Magali Remy-Stockinger, Håvard Attramadal and Ansgar O. Aasen. Petter K. Risøe, 31 January 2013, Inflammatory Modulation in Sepsis – Role of Adenylyl Cyclases. Institute for Surgical Research, March 2014

Anders Opdahl, 8 March 2013, Mechanisms of left ventricular early-diastolic untwisting and lengthening. Ansgar O. Aasen, Professor /Head of Institute

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Division of Cardiovascular and Pulmonary Diseases 7

Institute for Surgical Research 8

Photo: OUH 9

Abbreviations

AHUS Akershus University Hospital

CAST Cancer, stem cell innovatin center

CIT Clinical Islet Transplantation Consortium

FP 7, EU Seventh Framework Programme, European Union

LO Lovisenberg Diaconical Hospital

NCCD The Norwegian Council on Cardiovascular Diseases

NRC Norwegian Research Council

OC Orthopaedic Centre, Oslo University Hospital

OUH Oslo University Hospital

SENRHA South-Eastern Norway Regional Health Authority

UiO University of Oslo

UiT University of Tromsø 10

Photo: Øystein H. Horgmo, UiO 11

Research Groups 12

Photo: Øystein H. Horgmo, UiO 13

Surgical Intensive Care Medicine

Leader Ansgar O. Aasen, Professor, MD, PhD (UiO/OUH)

Deputy leader Signe Flood Kjeldsen, Head engineer, MSc (UiO)

Scientific staff Ola Sveen, Prof. Emeritus, MD, PhD Tom Erik Ruud, Senior scientist, MD, PhD (OUH) Yngvar Gundersen, Senior scientist, MD, PhD (OUH) Claus Danckert Krohn, Consultant, MD, PhD (OUH)

Johanna Samulin-Erdem, Senior scientist, PhD (OUH) Professor Ansgar Aasen O. Hamid Shegarfi, Postdoc, PhD (OUH) Kristin Bjørnland, Consultant, MD, PhD (OUH) with organ-protective properties. Except for CCN5, all CCN Yun Yong Wang, MD, PhD (OUH) proteins contain four conserved domains that include Claus Vinter Bødker Hviid, MD, PhD-student (OUH) insulin-like growth factor-binding protein (IGFBP), von Lars Andreas Pedersen, Student of Physics (UiO) Willebrand factor type C repeat (vWC), thrombospondin Runar Strand-Amundsen, Student of Physics (UiO) type I repeat (TSR), and a carboxyl-terminal (CT) domain. Vivi Bull Stubberud, Operating Theater Nurse (OUH) The CCN1/CYR61 protein plays an important role in tissue Sera Sebastian, Operating Theater Nurse (OUH) regeneration and inflammation by regulating a wide range of cellular processes, including immune cell adhesion, cell Research area survival and endothelial proliferation. The vWC and CT domains of the protein have several integrin-binding sites Infections following surgery or trauma continue to be a major that may mediate the CCN1s functions. clinical problem. Due to the immunological consequences of surgery, infections frequently develop into severe septic We have investigated the regulation of CCN1 in the complications and multiple organ injuries. The problem in circulation and intestinal tissue in a porcine model subjected severe sepsis is a paradoxical and self-destructing to long intestinal ischemia-reperfusion (IIR). A jejunal inflammation and disturbances in the plasma protease ischemia-reperfusion model was used in this experiment. Four cascade systems leading to dysfunctional host defense and pigs were fully anesthetized. The abdomen of the animal was injury to vital organ systems. More than one million patients opened and a 30-cm long jejunum was subjected to local are expected to die annually from severe sepsis worldwide. intestinal ischemia by occluding the arteries and veins for six hours. Intestine biopsies were taken from i) ischemic area (IS) Aims ii) randzone area (RZS) -a segment with sub-normal circulation close to the edge of the ischemic area-, and iii) non-ischemic Our aim is to develop novel means to prevent or ameliorate intestine (NIS) -a segment from an area that was not sub- the self-destructive inflammation and the abnormal plasma jected to ischemia as baseline control segment. Portal and proteolysis in patients with infection. A major focus of our work systemic blood samples of the animals were also collected as is research into the cellular mechanisms and proteins involved follows: before induction of ischemia (baseline –BS), 3 hours and to facilitate translation of new knowledge from basic re- after induction of ischemia, 6 hours after induction of ischemia search into clinical practice. or just before reperfusion, and 5, 10 and 30 minutes after reestablishment of intestinal circulation after 6 hours ischemia. Ongoing Projects in 2013 Histological analysis (H&E staining) of these sections Regulation of CCN1 in a Porcine Model showed a little inflammation in NIS while the ischemic, and of Intestinal Ischemia-Reperfusion at lesser level RZS, segments were largely damaged. Messenger RNA of the biopsies measured by real time PCR The extracellular matrix (ECM) has generally received showed a significant upregulation of CCN1 gene in both little attention in research. In contrast to the classical ECM IS and RZS segments compared with NIS as it is shown in proteins, rather being part for the framework for tissue Figure 1. Western blotting corroborated the upregulation of structure, the matricellular CCN family functions to CCN1 at protein level (Figure 2). modulate cellular responses to environmental stimulation 14

elimination of ischemic damage. Our studies now focus on identifying the source of CCN1-protein cells after ischemia as well as identifying a potential role of local increased levels of CCN1 in intestinal ischemic injury.

CYR61 in an orthopaedic wound after major orthopaedic surgery Cyr61/CCN1 is a multifunctional matricellular protein that has recently emerged as a potential player in the regulation of inflammatory responses. Experimental research has uncovered pro-inflammatory effects and chemotactic capacities of this protein. Clinical investigations found it to be elevated in patients with chronic inflammatory conditions. However, its regulation in acute non-infectious Figure 1. CCN1 gene is strongly expressed in the ischemic and inflammation in humans has not been investigated randzone segments in comparison to non-ischemic segment. previously.

Ten otherwise healthy patients (13 - 18 years old) undergoing major orthopaedic surgery for idiopathic thoracic scoliosis were recruited to the study. Fluid from the surgical drain and systemic blood was collected 0, 1, 2, 4, and 6 hours after wound closure and analyzed for CCN1 contents, neutrophil counts, selected cytokines, and mark- ers of primary haemostasis activation. Figure 2. CCN1 protein is strongly upregulated in the ischemic and randzone segments in comparison to non-ischemic segment. CCN1 levels increased rapidly in the drained fluid, reaching significance at 1 hour after wound closure, We also wanted to study the cell types that produce CCN1 whereas its levels remained unaltered in the systemic protein in response to ischemia. Immunofluorescence circulation throughout the observation period. The platelet staining indicated that endothelial and epithelial cells may activation marker soluble (s) P-selectin increased in the have a major role in production and secretion of CCN1 in drained fluid but not in systemic blood during the early response to hypoxia (Figure 3). This suggestion will be post-operative phase, resulting in a strong correlation further studied by double immunofluorescence staining. between CCN1 and sP-selectin in the drained fluid (0.649, Furthermore, animal blood samples taken during ischemia p=0.042). and after reperfusion showed no enhancement of CCN1 and TNF-level, indicating that CCN1 is locally regulated (not shown). CCN1 Platelets 0.591 (p=0.09) sP-selectin 0.649 (p=0.042)* Neutrophils -0.171 (p=0.638) TNF- 0.515 (p=0.232) IL-1 0.519 (p=0.232) NIS IS RZS IL-6 0.401 (p=0.250) Figure 3. Immunofluorescence staining of the intestine biopsies IL-8 0.684 (p=0.02)* shows CCN1-positive cells (green) co-stained with DAPI (blue) in the non-ischemic segment (NIS) ischemic segment (IS) and IL-10 -0.043 (p=0.905) randzone segment (RZS). G-CSF 0.493 (p=0.148) Altogether, these results indicate that CCN1 is upregulated Table 1. Pearson’s correlation of subject means. sP-selectin: in intestine subjected to ischemia. These observations raise soluble P-selectin, TNF-: tumor necrosis factor alpha, IL: the interesting possibility that CCN1 may act as a local interleukin, G-CSF: Granulocyte colony stimulating factor. *p<0.05 danger or survivable signal that may lead to repair and 15

Levels of interleukin (IL)-6 and granulocyte-colony source for CCN1 release to the circulation in sepsis. It stimulating factor (G-CSF) were elevated in the drained fluid demonstrates that the increase in circulating CCN1-protein only from two hours after wound closure and neutrophil is opposed by a profound CCN1 mRNA repression in vital counts, tumor necrosis factor – alpha, IL-8 and IL-10 were organs and suggests that the restoration of pulmonary elevated from 4 hours after wound closure. protein levels is mediated by the sequestering of platelets and innate immune cells in the microvascular network. In conclusion, the present study demonstrates that CCN1 The functional impact of these observations awaits further accumulates in fluid drained from a surgical wound but not investigations but encourages more research into the role in the systemic blood after major surgery. It uncovers that of this protein in lifethreatening infections. CCN1 levels increase before classical cytokine mediators at sites of tissue-injury and suggests a connection between platelet activation and CCN1 accumulation. Altogether, Thrombin Generation is a sensitive tool these data may suggest that CCN1 is an early actor in to predict haemostatic changes in sepsis acute inflammation in humans. in an animal model

Septicemia is almost inevitably associated with changes in The matricellular “cysteine-rich protein 61” the haemostatic equilibrium ranging from a discrete drop is released from activated platelets and in platelet count to full-blown disseminated intravascular increased in the circulation during coagulation (DIC). Routine monitoring of haemostasis in experimentally-induced sepsis septic patients is largely based on evaluation of prothrombin time (PT), International normalized ratio (INR), We have recently provided the first evidence that the CCN- activated partial thromboplastin time (aPTT), platelet count, family members CCN1-CCN6 are regulated in the lung, liver fibrinogen levels, D-Dimer, Thrombo-Elasto-Gram (TEG) and heart of rats subjected to early stage experimentally measurements and, eventually, bleeding time. Abnormal induced sepsis. Interestingly, the magnitude of CCN1 results often indicate poor prognosis and outcome. regulations demonstrated strong correlations with the In septicemia time and early detection of disturbances inflammatory activity in the organs during development of leading to life threatening situations may be of vital dysfunction. Furthermore, exposing hepatocytes to TNF- importance. The tests mentioned are generally insensitive alpha resulted in a dosage-dependent CCN1 mRNA in early septicemia. regulation. There has been increasing interest in so-called “global In the present study we have provided a comprehensive assays” where the final formation of fibrin and fibrinolysis is description of CCN1-regulation in the circulation and vital evaluated to reveal the early changes. The most promising organs during experimentally induced sepsis with of these tests is the fluorogenic Thrombin Generation (TG) developing organ dysfunction. assay, which measures active thrombin in a plasma sample. Female CD-1 mice served as baseline controls or were subjected to coecal ligation and puncture (CLP) for 18 - 96 h and CCN1 regulation was analyzed in selected organs and in the circulation. A 5-, 5-, and 3-fold increase in circulating CCN1-protein were respectively observed at 18, 48, and 96 h post-CLP.

Hepatic and pulmonary CCN1 mRNA expression was down-regulated by 80, 60, and 55% and 85, 80, and 65% at 18, 48, and 96 h post-CLP, and undetectable in circulating white blood cells. To identify a potential source for the circulating protein, mouse and human platelets were explored and revealed to contain CCN1. Human platelets were stimulated in vitro by thrombin and a specific PAR1 agonist (SFLLRN). Both agonists induced an instant CCN1 release and the effect of SFLLRN was blocked by the specific antagonist RWJ56110.Taken together, the current study provides novel evidence that platelets comprise a Figure 4. Thrombin Generation in early-stage experimental sepsis. 16

We have compared this method to TEG, PT, INR, aPTT and evaluated AntiThrombin (AT) and Thrombin/ Antithrombin (TAT) in a rodent model where septicemia was induced by coecal ligation.

Our conclusion is, as expected, that thrombin formation precedes fibrin formation. This measurement is a very sensitive way to detect haemostatic disturbances early in septic animals. The TG assay should be further developed to a “bedside” (easy to use) test for clinicians.

Assessment of the viability of Ischemic Small Intestine using Bioimpedance Measurements In gastrointestinal surgery related to intestinal ischemia, the surgeon has to assess the viability of intestines that have been exposed to ischemic injury. There are at present limited means by which tissue viability can be assessed. The standard clinical method is still visual inspection and palpation. This method is non-specific and unreliable, and requires a high level of clinical experience. Bioimpedance has been utilized to measure changes in electrical parameters during ischemia in tissues like the liver, skeletal muscle, and the heart. The physical changes on the cellular and structural levels after the onset of ischemia result in time-variant changes in the electrical properties of the Figure 5 Left: Control - Right: Ischemic small intestine (Jejunum, tissue. We have investigated the possibility of utilizing porcine model)- The figures show: Tan d (loss tangent) as a bioimpedance measurements to support intraoperative function of frequency. The data show measurements with the two-electrode setup on a porcine model. Each point on the graph assessment of the viability of ischemic small intestinal show the mean from 8 measurements on separate porcine models tissue. at the same frequency, within a +/-5 minute window of ischemic time development. We have accomplished 5 initial pilot studies in a porcine model of small intestinal ischemia, and 7 further studies on modulus, phase, and tan d as a function of ischemic time, porcine models based on the initial findings. After induction compared to the control (Figure 5). Tan d is the loss of anesthesia, a warm ischemic model with full mesenteric tangent, a parameter that compares the loss of occlusion for 6 hrs was implemented in a 30 cm part of the electromagnetic energy to the energy that passes through jejunum. Measurements were conducted placing a medium. electrodes on the serosa of the jejunum, applying a constant voltage, and measuring the resulting admittance. A Matlab program with pilot algorithms has been devel- Several electrode setups were tested. We used the oped showing 78,2% sensitivity, 94,2% specificity, 92,7% recognized and commonly used Solartron 1260/1294 positive predictive value, and 82,0% negative predictive impedance analyzer setup. As a control we used value in assessing if the porcine small intestine is ischemic measurements on parts of the jejunum with normal or not, based upon analysis of the collected data. The perfusion in the same porcine model. program also shows promising results in discerning the time duration of ischemic small intestine within the tested The 2-electrode Silver-Silver chloride setup appeared the 6 hour time frame. Histological samples show ischemic best of the tested setups for measuring small intestinal changes to the small intestinal tissue that correlate with the ischemia interoperably. The collected data from the porcine measured time variable changes (Figure 6). models show significant changes in electrical parameters of

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Figure 6. Cross section of porcine jejunum wall with damaged and affected areas after 6 hours of warm full mesenteric occlusion ischemia.

The time development of the electrical properties measured Based on the results from the porcine models, it is not within a 6 hour period of ischemia is statistically significant, yet possible to assess the viability of the small intestine to and can be correlated with the onset and duration of the point of irreversible ischemic damage, as the 6 hour ischemia (Figure 7). ischemic period of porcine model 2 (PM2) did not create a 100% certain ischemically irreversible damaged porcine small intestine. The animal model developed through these experiments seems suitable for further studies related to the determination of the viability of ischemic small intestine.

Collaborators • Prof. Heinz Redl, Prof. Soheyl Bahrami and Senior Scientist Martin F. Osuchowski, DVM, PhD, Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria • Prof. Michael A. Rogy, University of Vienna, Vienna, Austria • Prof. Christoph Thiemermann, The William Harvey Research Institute, London, UK • Prof. Irshad Chaudry, University of Alabama at Birmingham, Birmingham, Alabama, USA • Prof. Michael Gallimore and Dr DW. Jones, University of Kent, UK Figure 7. Tan d - Control vs. Ischemic small intestine at 10kHz. • Prof. Ståle Petter Lyngstadaas, Dept. of Oral Pathology, Each box plot is based on 8 measurements from 8 separate UiO, Oslo, Norway porcine models, at the same frequency in the same time window Prof. Jan Olav Høgetveit, UiO/OUH, Oslo, Norway (+/- 5 min of the selected hour). They are presented as mean, • 1 and 3 quartile and range. • Prof. Håvard Kalvøy, UiO/OUH, Oslo, Norway 18

Photo: Øystein H. Horgmo, UiO 19

Molecular Cardiology

Leader Håvard Attramadal, Professor, MD, PhD (OUH/UiO)

Scientific staff M. Shakil Ahmed, Senior Scientist, PhD (OUH) Geir Florholmen, PhD, Postdoc (SENRHA) Thomas G. von Lueder, MD, PhD, Consultant (OUH) Jørgen A. Gravning, MD, PhD, Consultant (AHUS) Vladimir N. Martinov, PhD, Research Associate (UiO) Ingvild Tronstad Moe, MD, PhD-Student (NCCD/UiO) Tuyet Anh Pham, MD, PhD-Student (SENRHA) Ole Jørgen Kaasbøll, MD, PhD-Student (NCCD) Liv Kvistad Klemetsaune, MSc, PhD-Student (NCCD) Professor Håvard Attramadal Else Marie Valbjørn Hagelin, MSc, Senior Engineer (SENRHA) Kashif Rasheed, MSc, Research Associate of these investigations is to provide new knowledge of disease mechanisms, enabling development of novel pharmacological Research Area interventions for heart failure. Heart failure, the clinical syndrome of end-stage cardiac disease of diverse etiologies, is a major cause of morbidity Our research group is a multidisciplinary team of experts in and mortality. Indeed, the incidence and prevalence of heart gene technology, molecular and cellular biology, as well as failure in affluent societies are increasing due to demographics experimental and clinical medicine. The research efforts with rising proportion of elderly, as well as increased survival comprise studies of isolated cardiac myocytes and fibroblasts, of myocardial infarction. Despite implementation of several integrated physiology in genetically engineered mice, large new treatment modalities during the last 20 years, heart failure animal studies, as well as clinical investigations. Our research is still a progressive and ominous condition indicating that group is member of the Center for Heart Failure Research, important pathogenic mechanisms remain unmodified by the University of Oslo (www.heartfailure.no ), a thematic research most current treatment modalities. Thus, there is an impetus initiative and focus area of research selected by the Faculty for new and more effective pharmacological interventions. of Medicine. The Center for Heart Failure Research is also a regional research network sponsored by the Regional Health In evolving heart failure, multiple compensatory actions are Authority Helse Sør-Øst. The Institute for Surgical Research triggered in order to maintain cardiac output, among which is provides infrastructure with state-of-the-art equipment for activation of the sympathetic nervous system, the renin- gene technology, as well as for high-resolution angiotensin system, as well as a number of autocrine/ echocardiography and integrated physiologic assessment of paracrine factors synthesized in myocardial tissue. These cardiac function in both small and large animals. compensatory actions also reflect in alterations of cardiac structure, collectively called cardiac remodeling. The most important structural alterations are cardiac myocyte Major Aim Dysfunctional cardiac signaling mechanisms and signals hypertrophy and myocardial fibrosis. Although cardiac astray are considered major causes of pathologic myocardial remodeling may initially balance loss of contractile force, the hypertrophy and predisposition to heart failure. Increasingly, continuum of these structural alterations often feeds into dysfunctional signaling mechanisms are implicated in vicious circles leading to progression of cardiac dysfunction. increased production of free oxygen radicals, mitochondrial Despite substantial new insights into the mechanisms of dysfunction and reduced tolerance to hypoxia. Thus, a major myocardial hypertrophy and fibrosis, many of the nodal points goal of our research group is to dissect the function of that orchestrate these structural alterations still remain to be myocardial autocrine/paracrine factors, their cognate identified. Thus, an important focus of our research group is receptors, and intracellular pathways in cardiac myocytes and to unravel the signal transduction mechanisms that generate fibroblasts. Of particular interest is also the cross-talk and the dysfunctional signals leading to pathologic remodeling intercellular signaling between cardiac fibroblasts and cardiac and progression of heart failure. Another important concep- myocytes. New knowledge on the function and mechanisms tual approach is that of delineating mechanisms that either of signaling pathways in the heart may provide basis for increases or decreases the tolerance of cardiac myocytes to development of new and more effective therapeutic hypoxia or free oxygen radical injury, i.e. potential mediators of intervention in acute coronary syndromes and heart failure. cardiac myocyte damage in evolving heart failure. The purpose 20

Current specific aims of the research group and CCN5 in order to investigate the signaling mechanisms and biologic functions of these proteins in cardiac 1. Elucidate the function of novel G protein-coupled myocytes and cardiac fibroblasts. receptors and their signaling pathways in the heart.

2. Resolve how G protein-coupled receptor kinases (GRK) Report from 2013 control the sensitivity of receptors to their cognate agonists, as well as decipher the role of GRKs in 1) Investigation of substrate specificities and function of controlling G protein-dependent versus G protein- cardiac G protein-coupled receptor kinases (GRKs). independent signaling in the heart in health and disease, with particular focus on heart failure. We have previously investigated distribution of GRK2, GRK3, and GRK5 in myocardial tissue. These studies revealed that 3. Uncover the function of myocardial autocrine/paracrine GRK2 was enriched in endothelial cells, whereas GRK3 was factors or cytokines that are activated or induced in heart confined to cardiac myocytes. GRK5, on the other hand, was failure. Current focus is on delineating the target cells and ubiquitously expressed among the cellular elements of the functions of secreted matricellular CCN proteins, in myocardial tissue. The restricted distribution of GRK3 in particular CCN1, CCN2/CTGF (connective tissue growth cardiac myocytes clearly points to a role for this GRK factor), and CCN5/WISP-2 (Wnt-inducible secreted isoform in regulation of G protein-coupled receptors on protein-2), secreted regulators of Wnt signaling, as well as cardiac myocytes. However, since both GRK2 and GRK3 the TGF-ß superfamily cytokine GDF-15 in heart failure. The could be demonstrated in cardiac myocytes, studies of the CCN proteins (CCN is an acronym for the first three substrate specificity of these kinases were an imminent issue. members of this gene family; Cyr61, CTGF, Nov) are In isolated, fully differentiated, cardiac myocytes we cysteine-rich, modular proteins (Figure 1) considered to investigated the substrate specificities of the GRK’s forms interact with various proteins in the extracellular matrix, GRK2 and GRK3. These studies revealed that GRK2 and including cytokines, growth factors, extracellular matrix GRK3 display striking specificity at G protein-coupled proteins, as well as receptors on the cell surface. Yet, the receptors controlling different aspects of cardiac function. mechanisms of CCN protein action are poorly understood. Overall, our data have uncovered the novel findings that GRK3 We have established eukaryotic expression systems for has substantially higher potency and efficacy than GRK2 at

production and purification of recombinant CCN1, CCN2, endogenous endothelin receptors and alpha1-adrenergic

The Matricellular CCN Gene Family

Figure 1. – Schematic demonstrating the modular structure of the CCN family proteins (CCN1-6). IGFBP; insulin-like growth factor binding protein homology domain, VWC; von Willebrand factor homology domain, TSP1; thrombospondin-1 homology domain; CT; cysteine knot homology domain. 21

receptors. This did not seem to be the case for the contribute to progression of heart failure? Does CCN2 cause

ß1-adrenergic receptor as GRK3 potency at this receptor myocardial fibrosis? In order to elucidate the physiologic appeared to be much weaker than that for the ET-R, and actions of CCN2 in the heart and to investigate how the was found to be equipotent with that of GRK2. Thus, GRK3 actions of CCN2 may contribute in the pathophysiology of emerges as a primary regulator of ET-R and of alpha1- AR- heart failure, we are currently investigating various genetically signaling in cardiac myocytes. The distinct receptor specificity engineered models with constitutive or conditional of GRK3 may have important implications in cardiac function overexpression of CCN2 in the heart. The transgenic mice with as well as in cardiac disease. These functional differences are cardiac-restricted, constitutive overexpression of CCN2/CTGF currently subject of investigations in transgenic and gene- displayed marginal increase of myocardial collagen contents targeted mice. despite 70-fold overexpression of CCN2/CTGF (Ahmed, MS et al. Am J Physiol Heart Circ Physiol. 300: H1291-1302, 2011). In a report from our laboratory published in 2013 (Gravning This finding appears to be consistent with data from J et al. Mol Pharmacol 84:372-383, 2013) we disclosed the transgenic overexpression of CCN2/CTGF in other tissues or novel findings that myocardial GRK5 is upregulated in organs. Thus, the interpretation of the available data both from transgenic mice with cardiac-restricted overexpression of our and other research groups is that additional factors are CCN2/CTGF, as well as in cardiac myocytes pretreated with required for CCN2 to induce fibrosis. A surprising, novel recombinant human CCN2, causing reduced sensitivity of finding in our laboratory was that CCN2 exerts striking cardiac ß-adrenergic receptors to endogenous agonists. cardioprotective actions, increasing tolerance towards Furthermore, increased GRK5 in the heart initiates ischemia-reperfusion injury both ex vivo in G protein-independent signaling by recruitment of ß-arrestin to Langendorff-perfused hearts as well as in vivo in mice the receptor, allowing ß-arrestin to act as a scaffolding protein subjected to transient ligation of the left anterior descending for signaling complexes at the plasma membrane such as the coronary artery in situ. A recent report from our laboratory mitogen-activated protein kinase ERK1/2. These findings have also provides evidence that cardiac myocytes are direct been recapitulated in cardiac myocytes pretreated with targets of recombinant human CCN2 (rec-hCCN2), and that recombinant human CCN2/CTGF. Yet, the signaling rec-hCCN2 also increases the tolerance of cardiac pathway(s) implicated in CTGF-induced induction of GRK5 myocytes to hypoxia/reoxygenation-induced injury and expression in cardiac myocytes is yet to be characterized. oxidative stress (Moe, IT, et al. J Cell Commun Signal. Furthermore, the relative contribution of GRK5 to the 7:31-47,2013). These findings have led to filing of patents for cardioprotective actions afforded by CCN2/CTGF remains to protection of the potential commercial development of CCN2/ be resolved. CTGF as a new pharmacologic treatment in acute coronary syndromes with the objective of minimizing myocardial 2) Role of CCN2 - connective tissue growth factor - in necrosis. Verification of the data in large animal models, regulation of tolerance towards ischemia-reperfusion injury commercial development plans, including plans for early and in resisting maladaptive cardiac remodeling during chronic clinical testing, are currently being pursued in collaboration pressure overload. with Birkeland Innovation/ Inven2 AS, the TTO of University of Oslo and Oslo University Hospital. A recent report from our Myocardial CCN2 is highly expressed in the developing group also demonstrates that CCN2 enhances healing heart in fetal life and apparently plays crucial role in cardiac after myocardial infarction (Gravning, J. et al PlosOne development. However, myocardial expression of CCN2 is 2012;7(12):e52120). However, the mechanisms whereby repressed in the postnatal heart under physiologic conditions. CCN2 enhances scar healing after myocardial infarction is still Interestingly, myocardial expression of CCN2 is reactivated or obscure and a matter of investigation. Interestingly, the study induced during evolving heart failure. Previous findings from also reports data from patients admitted for acute ST-elevation our laboratory demonstrate that induction of myocardial CCN2 myocardial infarction. The patient cohort segregated in two appears to be a general response to evolving heart failure, i.e. groups, one in which plasma CCN2 levels were elevated after induction of myocardial CCN2 occurs in heart failure of diverse myocardial infarction (MI) and the other in which plasma CCN2 etiologies. Induction of tissue expression or increased plasma levels decreased of remained unchanged. Patients that had levels of CCN2 is often associated with diseases in which elevated plasma CCN2 levels after MI displayed improved fibrosis is an important morphologic characteristic. However, healing of the infarction and enhanced myocardial function one to what extent CCN2/CTGF actually elicits fibrosis is yet to be year after the event as assessed by cardiac MRI (Gravning, J. demonstrated. Indeed, the physiologic and/or et al PlosOne 2012;7(12):e52120). Although the patient cohort pathophysiologic functions of CCN2 in myocardial tissue was relatively small, these findings are translational evidence remain to be resolved. Thus, a major focus of our research supporting a cardioprotective function of CCN2. Another effort is to elucidate the function of CCN2 in the heart. Does report from our laboratory published in 2013 provides CCN2 exert salutary actions in heart failure, or does CCN2 evidence that CCN2 attenuates pathologic cardiac 22

hypertrophy associated with chronic pressure overload Our research group is currently member of a national following aortic constriction. The diminished hypertrophic consortium of research groups that in 2012 obtained funding response of cardiac myocytes from transgenic mice from UNIKARD, a research program sponsored by the overexpressing CCN2 was related to inhibition of NFAT Norwegian Research Council and the Regional Health transcriptional activities (Gravning, J. et al. Int J Cardiol. Authorities, on a major effort to investigate the mechanisms 168:2049-2056, 2013). and implications of physical exercise on the progression heart failure. A cognate receptor for CCN2 or for any of the other CCN proteins has not yet been properly characterized. Despite several reported interactions between CCN proteins and Collaborators extracellular matrix-associated proteins, data from our • Prof. Robert J. Lefkowitz, Dept. of Medicine, laboratory indicate that CCN2 may also act directly on cells by Duke University Medical Center, Durham, USA. binding to receptors at the surface of the plasma membrane. • Prof. Walter J. Koch, Dept. of Pharmacology and Center Furthermore, analysis of the phosphoproteome of cardiac for Translational Medicine, Temple University School of myocytes following stimulation in the absence or presence of Medicine, Philadelphia, PA, USA. recombinant CCN2 revealed that the PI3 kinase/AKT/GSK-3ß • Prof. Erhe Gao, Dept. of Pharmacology and Center for pathway is a major intracellular signaling pathway of CCN2 Translational Medicine, Temple University School of (Figure 2). Indeed, our data also demonstrate that this Medicine, Philadelphia, PA, USA. pathway is crucial for CCN2-dependent cytoprotection • Prof. Otto A. Smiseth, Dept. of Cardiology/Inst. for Surgical towards hypoxia/reoxygenation-induced cell injury. The Research, Oslo University Hospital. mechanisms of the cytoprotective actions of CCN2 are • Prof. Thor Edvardsen, Dept. of Cardiology/Inst. for Surgical currently a major endeavor in our research group. However, Research, Oslo University Hospital. the cytoprotective actions of CCN2 apparently entail activation • Professor Jon Storm-Mathisen, Institute of Basic Medical of a transcriptome that includes the gene ontology groups Sciences, Dept of Anatomy, University of Oslo. anti-apoptosis, oxidative stress-associated genes and • Professor Linda Hildegård Bergersen, Institute of Basic hypoxia-induced genes (Moe, IT et al. J Cell Commun Signal. Medical Sciences, Dept of Anatomy, University of Oslo. 7:31-47,2013). To facilitate studies on the mechanisms of • Professor Øivind Ellingsen, Department of Circulation and CCN protein actions in cells, our group has established Medical Imaging Norwegian University of Science and eukaryotic expression systems for large scale production and Technology, Trondheim, Norway, purification of several of these proteins. • Prof. Ivar Sjaastad, Inst. for Experimental Medical Research, Oslo University Hospital • Prof. Guro Valen, Inst. of Basic Medical Sciences, Dept. of Physiology, University of Oslo. • Prof. Geir Christensen, Inst. for Experimental Medical Research, Oslo University Hospital. • Prof. Jan-Bjørn Osnes, Dept. of Pharmacology, University of Oslo. • Prof. Tor Skomedal, Dept. of Pharmacology, University of Oslo. • Prof. Finn Olav Levy, Dept. of Pharmacology, University of Oslo. • Prof. Terje S. Larsen, Dept. of Medical Physiology, University of Tromsø, Norway. • Assoc. prof. Ellen Aasum, Dept. of Medical Physiology, University of Tromsø, Norway. • Prof. Pål Aukrust, MD, PhD, Research Inst. for Internal Medicine, Oslo University Hospital. • Dr. Leif Erik Vinge, M.D., Ph.D., Dept. of Cardiology/ Research Inst. for Internal Medicine, Oslo University Hospital. Figure 2. – Schematic illustrating the intracellular signaling pat­hways • Dr. Per Steinar Halvorsen, M.D., Ph.D., The Intervention of CTGF/CCN2 in cardiac myocytes. A cognate receptor for CTGF Center, Oslo University Hospital. upstream of PI3 kinase has not yet been characterized. 23

Integrated Cardiovascular Function

Leader Otto A. Smiseth, Professor, MD, PhD (OUH/UiO)

Deputy Leader Thor Edvardsen, Professor, MD, PhD (OUH/UiO)

Scientific staff Espen W. Remme, PhD, Senior Scientist (JEBSEN) Helge Skulstad, MD, PhD Senior Researcher (OUH) Kristina H. Haugaa, MD, PhD, Cardiologist, Senior Researcher (OUH)

Stig Urheim, MD, PhD, Cardiologist (OUH) Smiseth A. Professor Otto Morten Eriksen, MD, PhD, Senior Scientist (CCI) Anders Opdahl, MD, PhD (OUH) Marit Kristine Smedsrud, MD, PhD (CCI) 4. To develop better diagnostic methods to identify Thomas Helle-Valle, MD, PhD (OUH) coronary occlusion in acute coronary syndrome. Sebastian Imre Sarvari, MD, PhD (OUH) 5. To investigate LV electro-mechanical interactions and Kristoffer Russell, MD, PhD (OUH) improve risk stratification for ventricular arrhythmias. Espen Bøe, MD, PhD-student (NCCD) 6. To investigate right ventricular function in patients with Nina Eide Hasselberg, MD, PhD-student (CCI) congenital abnormalities which involve the right ventricle. Jørg Saberniak, MD, PhD-student (CCI/OUH) Petter Storsten, MD, PhD-student (OUH) 1. Left ventricular dyssynchrony: Ola Gjesdal, MD, PhD (OUH) A large number of heart failure patients suffer from Aurora Pamplona, Operating Theatre Nurse (OUH) dyssynchronous electrical activation of the heart muscle. Implantation of a pacemaker is a highly efficient treatment for many of these patients (Cleland 2005). In General objectives principle it works by sending electrical activation pulses The Integrated Cardiovascular Function Group studies to electrodes placed in the muscle and by stimulating cardiac mechanics in experimental models and in patients these simultaneously to restore synchronous with the general objective to gain new insights into contraction of the muscle around the ventricles. This is mechanisms of cardiovascular disease and to develop new referred to as Cardiac Resynchronization Therapy (CRT). imaging modalities which quantifies disease processes and In 2012 we published a novel method that non-invasively cardiac function. The idea is to develop better diagnostic estimates the regional work performed by the heart muscle. understanding and solutions and to translate these into Each region of the muscle around the ventricle will normally improved clinical practice. The group participates in the contract through systole when pressure rises and blood is Center for Cardiological Innovation (CCI) which focuses on ejected out to the body and subsequently stretches when improving diagnostic methods for patients with heart failure pressure drops and the ventricle fills. We have refined and patients at risk of sudden cardiac death. The group is this technique further by making separate calculations for also participating in the KG Jebsen Cardiac Research work performed during shortening of the muscle and work Center which focuses mainly on left ventricular performed during elongation of the muscle. The latter is dyssynchrony and diastolic heart failure. negative work, since the elongation of the muscle actually means that mechanical energy is absorbed by the muscle. Negative work is obviously a factor that affects the overall Specific objectives energetic efficacy of the heart. The energy absorbed by 1. To investigate mechanisms of left ventricular (LV) the stretching of muscle tissue is not regenerated, and is dyssynchrony and develop better methods for selecting lost as heat. For each myocardial segment studied, we can patients for cardiac resynchronization therapy (CRT). calculate the ratio between negative and positive work. We 2. To investigate hemodynamic effects of CRT in patients have defined this as wasted work ratio (WWR). A global with heart failure and narrow QRS. WWR can also be calculated as the ratio between summed 3. To investigate mechanisms of LV diastolic dysfunction negative and positive work for all segments. and to develop better diagnostic methods of diastolic heart failure. 24

We have successfully validated our non-invasive method in 2. Narrow QRS: While CRT is designed to correct animal experiments by comparing estimates of positive and electrical dyssynchrony, it has also been reported that negative work with data obtained by invasive means. Using some patients get better with CRT despite having no both the invasive and the non-invasive techniques described indication of electrical dyssynchrony (i.e. the duration of above we have shown that surgically induced left bundle the ventricular activation wave, QRS of the ECG signal is branch block (LBBB) in dogs causes considerable loss of narrow). As more than two thirds of heart failure patients do mechanical energy compared to the baseline situation. not have electrical conduction disturbances, extending the Furthermore, we have also demonstrated similar losses in indications for CRT into this patient group is going to have patients with LBBB. This means that their hearts’ metabolic considerable implications. The mechanisms of possible capacity is partly wasted without contributing to the primary effects of this treatment in heart failure patients with narrow task of the left ventricle of the heart: pumping of blood into QRS have not been studied properly. Two possible the systemic circulation. The global WWR was 0.36, and the mechanisms have been suggested: CRT may correct intraventricular septum was found to be the primary site of electrical dyssynchrony not seen on ECG, or CRT induces energy losses, with values of WWR of about 1.6, meaning changes in the interventricular interaction. The change in that the septum absorbed more energy than it produced. ventricular interaction can be obtained by pacing in the left Implantation of a CRT device in these patients reduced the ventricular lateral wall. The left ventricle (LV) is then WWR in the septum to 0.18, and the global WWR to 0.17. activated earlier than the right ventricle (RV), and the LV will both start contracting and subsequently start filling earlier Our studies have demonstrated that energy loss is an than the RV. A head start of LV filling relative to RV filling important factor in the pathogenesis of cardiac failure with reduces the LV external constraint. External constraint is LBBB. High energy losses indicate substantial room for determined by the RV pressure and the pericardial improvement with CRT since these patients have a pressure and constitutes the external resistance to LV substantial contractile reserve capacity that can be filling. The hemodynamical result of reduced external mobilized by resynchronization. constraint is improved LV filling and increased cardiac output. An experimental dog model was used to explore The paper was published in Am J Physiol Heart Circ these electromechanical and hemodynamic consequences Physiol. 2013. of CRT. The results showed improvement of filling. However, pacing was also shown to introduce electrical and mechanical dyssynchrony and thereby reduce ejection and systolic function. In the animal model the negative effects outweighed the positive effects of pacing. The findings suggest that considerable caution should be taken before applying CRT in patients with narrow QRS. The study has been submitted and is under review at the time of publication of this report.

3. Diastolic dysfunction: About half the patients with heart failure seem to have problems related to filling of the heart as the ejection fraction is normal. Main challenges are therefore to understand what causes diastolic dysfunction and how it can be diagnosed. Early diastolic untwisting of the left ventricle has been proposed as an index of relaxation rate, and hence a marker of diastolic function. We confirmed this hypothesis but we also found that untwisting rate was determined by early diastolic load and restoring forces. In November 2012 this study (Opdahl Estimated LV pressure (top), strain (middle), and calculation of 2012) was published in the cardiology journal with highest wasted work for the septum and lateral wall (bottom) in a impact factor, Circulation. At the end of 2013, the representative patient with left bundle branch block (left) and after Circulation editors picked this study as among the most treatment with cardiac resynchronization therapy (CRT; right). Negative and positive work time intervals during systole are important manuscripts and most read manuscripts marked as red and black, respectively. Wasted work ratios (WWRs) published on the topic of heart failure as well as on the topic were calculated using cumulated work from MVC to MVO. of studies performed in an animal model within the last year From Russell et al 2013 in Circulation and the Circulation subspecialty journals. (Circulation. 2013;127:e845-e858 and 2013;128:e346-e361.) 25

4. Myocardial ischemia and infarct result from occlusion of morbidity in these patients. Right bundle branch block and the coronary arteries which supply the myocardium with dyssynchrony are also frequent clinical problems in these blood. The part of the myocardium that is affected by lack patients. By doing strain analysis and regional work of blood supply may be rescued by reperfusion therapy if calculations, we have, in echocardiographic studies, it is still viable (i.e. the muscle cells are still alive). Patients discovered regional differences in ventricular function, with chest pains and signs of coronary occlusion on ECG especially between the septum and RV free wall. We will (ST-elevation) are directly referred to reperfusion therapy investigate if these differences in regional work are also (PCI). However, some patients without ST-elevation also associated with metabolism (by using PET analysis) in the have a coronary occlusion and may benefit from rapid ventricle. These differences in work may over time be a referral to PCI. Therefore, there is a need for diagnostic cause of ventricular failure. Thus, these findings may methods to identify these patients. We are currently provide a basis for altered treatment, e.g. by using CRT. investigating the ability of a non-invasive echocardiographic The study is ongoing, but some preliminary results have method for estimation of myocardial work (as explained already been presented at international congresses. in point 1 above) to identify infracted myocardium, and therefore the presence of coronary occlusion. This method does not rely on biomarkers and can be performed in most PhD defences in 2013 emergency departments upon admission. The study is • Anders Opdahl: Mechanisms of left ventricular ongoing, but some preliminary results have already been early-diastolic untwisting and lengthening. presented at international congresses. • Marit Kristine Smedsrud: Assessment of incipient global myocardial dysfunction by speckle tracking 5. LV electro-mechanical interactions: Evaluating patients echocardiography. Clinical studies with emphasis on with susceptibility for cardiac arrhythmias and sudden patients with stable coronary artery disease and patients cardiac death is a major challenge in daily cardiology with chronic aortic regurgitation. practice. Electrophysiological studies have demonstrated • Thomas M. Helle-Valle: New non-invasive methods for that damaged myocardium (e.g. infarcted or genetically quantification of egionalr and global myocardial altered) provides the substrate for malignant arrhythmias. function in the normal and ischemicl left ventricle. Echocardiographic techniques can accurately quantify • Sebastian Imre Sarvari: Detection of subtle myocardial regional myocardial function. Over the last few years we alterations by echocardiographic techniques for have studied the interactions between myocardial improved prognostic information in patients with heart mechanical function and risk for ventricular arrhythmias. disease. We have demonstrated that mechanical dispersion can predict ventricular arrhythmias in patients with genetic cardiac diseases and in patients after myocardial infarction. Collaborators The novel measurement principle has been tested on other • Prof. Joao A.C. Lima, Johns Hopkins University, patient populations, including patients with ischemic Baltimore, Maryland, USA dilated cardiomyopathy, mutations in the Lamin A/C gene • Prof. Frits Prinzen, Maastricht University, Maastricht, or arrhythmogenic right ventricular cardiomyopathy, to The Netherlands investigate if it provides clinical diagnostic value. These • Prof. Sherif Nagueh, Methodist DeBakey Heart and studies have resulted in new publications and awarded Vascular Center, Houston, Texas posters on international congresses. In 2013 we published • Dr. Martin Penicka, OLV Hospital Aalst, Belgium a study in JACC Cardiovasc Imaging (Haugaa 2013) • Prof. Jens-Uwe Voigt, Katholieke Universiteit Leuven, showing that this method could predict ventricular Belgium arrhythmias after myocardial infarction in a prospective • Prof. Hans Torp, NTNU, Trondheim multicenter study. Furthermore, we published risk • Prof. Håvard Attramadal, Rikshospitalet, predictors of ventricular arrhythmias in patients with Lamin Research group: Molecular Cardiology A/C mutations (Hasselberg et al 2013). See the Center for • Prof. Ivar Sjaastad, OUS, Institute for Experimental Cardiological Innovation section of this annual report for Medical Research further details. • Consultant Harald Brunvand, MD, PhD, Sørlandet hospital, Arendal 6. Systemic RV: Patients with transposition of the great arteries have ventriculoarterial discordance, which means that the aorta and pulmonary artery have switched places. They were previously operated with an “atrial switch” where the RV is exposed to systemic pressure and increased wall stress. Ventricular failure is therefore a frequent cause of 26

Photo: Øystein H. Horgmo, UiO 27

Center for Cardiological Innovation

Center Leader and Management Thor Edvardsen, Professor, MD, PhD, Center Leader (OUH/UiO) Kristina Hermann Haugaa, MD, PhD, Center Director of cardiology research (OUH) Eigil Samset, PhD, Center Coordinator (GE Vingmed Ultra- sound AS) Samuel Wall, PhD, Deputy Director for Scientific Computing (Simula Research Laboratory) Marianne Weberg, MSc, Administrative Coordinator (OUH)

Scientific Staff, Oslo University Hospital Arne Kristian Andreassen, MD, PhD, Consultant Cardiologist Professor Thor Edvardsen Christian Eek, MD, PhD, Consultant Cardiologist Einar Gude, MD, PhD, Department of Cardiology Erik Kongsgård, MD, PhD, centers that was awarded the status of Centre for Research- Head Section of Electrophysiology Based Innovation (SFI) through the Research Council of Erlend Aune, MD, PhD Norway SFI program. The CCI has a total budget of roughly Espen Wattenberg Remme, PhD, Senior Researcher 210 MNOK, of which 80 MNOK are to be allocated via the Fred Johan Pettersen, Scientist Research Council of Norway. The CCI is hosted by Oslo Hans Henrik Odland, MD, Consultant Cardiologist University Hospital and is a collaborative effort between Oslo Helge Skulstad, MD, PhD, Consultant Cardiologist University Hospital, Simula Research Laboratory, the University Håvard Kalvøy, Scientist of Oslo, and the industry partners GE Vingmed Ultrasound, Jan Olav Høgetveit, Head MTA Research and Development CardioSolv and Kalkulo. In December 2012, Medtronic Jan Otto Beitnes, Acting Consultant Cardiologist Bakken Research Center B.V. joined the CCI as additional Kristoffer Russell, MD, PhD research partner. Medtronic is a leading international Lars Aaberge, MD, PhD, Head Section of Treatment and medical technology company and brings world class expertise Diagnostics in therapy and disease management to the consortium. Each Morten Eriksen, MD, PhD, Senior Researcher research partner represents a unique and required element in Morten Flattum, Chief Engineer Department of Cardiology the research and development chain leading to the industrial Ole-Gunnar Anfinsen, MD, PhD, Consultant Cardiologist innovations targeted by the CCI. Otto Smiseth, Prof, MD, PhD, Head Division of Cardiovascular and Pulmonary Diseases Svend Aakhus, MD, PhD, Head Section of Aim Echocardiography The aim of the Center for Cardiological Innovation (CCI) is to Thorbjørn Holm, Consultant Cardiologist develop the next generation of ultrasound systems for Willian E. Louch, Permanent Scientific Staff cardiology. The proposed tools and technologies will be Margareth Ribe, Research Nurse created through linking currently isolated diagnostic systems with advanced biomedical research, advanced patient-specific computer simulation, and multi-modality visualization PhD- Students techniques. The targeted clinical uses of the proposed Jørg Saberniak, MD, PhD-student innovations are for better triage and treatment of patients at Nina Eide Hasselberg, MD, PhD-student risk of sudden cardiac death or suffering from heart failure, two Sebastian Imre Sarvari, MD, PhD-student of the biggest challenges in cardiology today (Figure 1). Wasim Zahid, MD, PhD-student Ida Skrinde Leren, MD, PhD-student Trine S. Fink Håland, MD, PhD-student Mechanical dispersion Thomas Muri Stokke, MD Student Much of the research at CCI is concentrated around a key number which can be calculated from an ultrasound scan called mechanical dispersion. This term indicates the extent Introduction to which the heart contracts synchronously, or whether The Center for Cardiological Innovation (CCI) was formally different parts of the heart contract at different times. A low established 31st October 2011. It is one out of seven new dispersion (e.g. approximately 30ms) is good, while a high 28

dispersion expresses the risk of sudden cardiac arrest. Researchers working with Integrated Cardiovascular Function Reference values for​​ the mechanical dispersion must be and Prof. Otto Smiseth have developed a new ultrasound established for various cardiac diseases as it may vary from method for assessing regional and global work of the heart in one disease to another. This key figure can be calculated close collaboration with CCI. Before CCI was established, from the curves and figures an ultrasound scanner from GE Oslo University Hospital developed a method to calculate Vingmed Ultrasound generates (Figure 2). Because of the the pressure changes in the heart without inserting a compelling results that CCI’s host institution, Oslo pressure sensor into the heart. The cardiac workload can University Hospital, has conducted and the attention they be calculated by combining the heart’s contraction have received internationally, GE Vingmed Ultrasound has measured by ultrasound with the pressure calculations now included a new index in the product corresponding to (Figure 3). This method can have several different medical mechanical dispersion. The new index is preliminary applications, particularly in the assessment of patients who available in the analysis tool EchoPAC (not the ultrasound have blockages in the coronary arteries. Oslo University scanner). It is not yet able to assess whether the Hospital has applied for a patent for this method, and GE calculation is physiologically correct. Further development Vingmed Ultrasound has signed a license agreement for and cooperation within CCI will make it possible to extend commercial use. GE Vingmed Ultrasound will assess the the concept and promote it in future product releases. utility of the method by involving potential users in hospitals worldwide.

Inefficient cardiac function

Figure 1. Sebastian Sarvari and Nina E. Hasselberg measuring maximal oxygen uptake in a patient performing exercise testing at Oslo University Hospital. 29

mercial product line. An initial version of this research ultrasound sandbox has been made during 2012 including the principles of mechanical dispersion and cardiac workload.

3D TEE probe Ultrasound imaging of the heart is limited by the narrow imag- ing windows between the ribs, as ultrasound cannot penetrate bone. However, more up-close imaging can be achieved by using a trans- esophageal echocardiogram (TEE) probe. Such a probe is inserted down the esophagus while the patient is lightly sedated or intubated. It can give a non-obstructed, high-resolution view of the heart. 2D TEE has been available for a long time, but to make a 3D TEE probe (with many hundreds more transducer elements) is an engineering challenge. 3D imaging can acquire volumes in real-time, which can be rendered semi-transparently in 3D, or as multiple 2D slices. GEVU released such a probe in 2012. Figure 2. Dispersion in measured time to peak strain is a risk factor for life threatening arrhythmias. Results and highlights from 2013 • A multi-center study, initiated by Oslo University Hospital has shown that ultrasound can be used to decrease the risk of life-threatening arrhythmias in patients who have suffered myocardial infarction. This is done by analyzing the pattern of cardiac movements in order to calculate the hearts contractions. • Ultrasound has the potential to predict the patients who are not likely to benefit from a pacemaker implantation after heart failure. Calculations based on the pattern of cardiac movements can reveal which patients might experience adverse effects from a pacemaker implantation. Excluding these patients from pacemaker treatment is of significant importance. • A study of the relationship between physical activity and arrhythmias in patients with the genetic heart disease ARVC was conducted. For these patients, there was a clear correlation indicating that these patients Figure 3. Screenshot of a new tool developed by the CCI to allow should be careful with engaging in too much physical simple analysis cardiac work. The tools has a tight integration with activity. EchoPAC by GE Healthcare. • Patients with a lamin A/C mutation have an increased risk of arrhythmia. A study was conducted to determine which markers should be used in order to assess the Research Ultrasound Sandbox risk. The results revealed that the ECG markers PR The software embedded in the GEVU scanner and workstation interval best define the risk in this patient group. is subject to strict QA procedures for development, testing and • Heart failure caused by a blockage of the heart’s release. In order to allow researchers within the CCI to develop conduction system can often be corrected by a and test methods without the restrictions of corporate proce- pacemaker implantation. The pacemaker will convert dures, the design of research software for sandboxing of new the heart’s wasted effort to useful work. A new method methods has been initiated. This software will communicate for calculation of the heart’s workload shows the percentage closely with the EchoPAC workstation or GEVU scanner, aiming of wasted work. This method is important for assessing the to avoid duplication of functionality already present in the prod- positive effects of the pacemaker implants (Figure 3). uct. The sandbox has been designed in such a way that it will • Patients who have multiple diseases of the aortic valve be possible to port functionality from the sandbox to the com- are in need of a replacement or repaired valve. A new 30

method enables replacement of the valve with minimally • Jussi Koivumäki, Post doctoral fellow, Simula Research invasive technique. Ultrasound can be used to plan and Laboratory monitor parts of the procedure. A method for automatic • Marit Kristine Smedsrud, MD, PhD-student, calculation of the size of the aortic valve implant using University of Oslo 3D ultrasound has been developed and validated. • Martin Reimers, PhD, Computational Mathematics, • The Center for Cardiological Innovation works to acquire University of Oslo new knowledge about the heart by combining ultra • Molly Maleckar, Director of the Simula School of Research sound imaging and mathematical simulation (Figure 4). and Innovation An important milestone is reached by being able to simulate both electrical activity and movements of the heart. This has been done based on one patient’s 3D ultrasound.

Figure 4. Creation of high quality 3D geometries for numerical simulations from readily obtainable ultrasound images.

Scientific staff from collaborating partners • Aleksandar Babic, PhD-student, GE Vingmed Ultrasound AS • Andreas Heimdal, Senior Staff, GE Vingmed Ultrasound AS • Aslak Tveito, Professor, Managing Director, Simula Research Laboratory • Bastien Mariyathas, Scientific programmer, GE Vingmed Ultrasound AS • Bjørn Fredrik Nielsen, PhD, Adjunct Research Scientist, Simula Research Laboratory • Brock M. Tice, Vice President Operations, Cardiosolv • Christian H. Tarrou, PhD, Director, Kalkulo • Edward Joseph Vigmond, President, CardioSolv • Fredrik Orderud, Permanent Scientific Staff, GE Vingmed Ultrasound AS • Glenn Terje Lines, PhD, Senior Research Scientist, Simula Research Laboratory • Gunnar Hansen, Senior Staff, GE Vingmed Ultrasound AS • Joakim Berdal Haga, Kalkulo • Lars Ove Gammelsrud, Medtronic • Joakim Sundnes, PhD, Senior Research Scientist, Simula Research Laboratory • Jørn Bersvedsen, PhD-student, GE Vingmed Ultrasound AS 31

Vilhelm Magnus Laboratory for Neurosurgical Research

Leader Iver A. Langmoen, Professor of Neurosurgery MD, PhD (UiO/OUH)

Scientific staff Einar Vik-Mo, Associate Scientist, MD, PhD Wayne Murrell, Senior Scientist, PhD Biljana Stangeland, Posdoc, PhD (CAST/Norwegian Research Council) Awais Mughal Ahmad, MD, PhD Student Jinan Behnan, PhD Student (Lytix Biopharma) Artem Fayzullin, MD, PhD Student (Norwegian Cancer Society) Mrinal Joel, MSc, PhD Student (HSØ) Professor Iver A. Langmoen A. Professor Iver Cecilie Sandberg, MSc, PhD Student Håvard K Skjellegrind, MD, PhD Student (VIRUUS) Kirsten Strømme, PhD Student of the 20th century, studies in birds and rodents came to Zanina Grieg, Research Technician, MSc question this doctrine as new markers for labeling neurons, (Norwegian Research Council) combined with techniques for identifying cells that had been Birthe Mikkelsen, Research Technician, MSc born in adult life, suggested that new neurons sometimes Emily Palmero, Research Technician, BSc may develop later in life in some species. Inger Marit Stark Valstad, Assistant & Study Nurse At the turn of the century, these findings were to some degree extended to the human brain, as a few research groups Network partnerships had been able to culture immature cells from the human ventricular wall and hippocampus. It was still not known, however, whether it would be possible to differentiate these cells into functional neurons, i.e. cells with typical neuronal action potentials and the ability to communicate via synapses.

The putative existence of an adult human brain SC with the Research area ability to proliferate and differentiate into mature neurons Vilhelm Magnus Lab is the translational research group for created huge interest as one could now envisage treatment neurosurgery at Oslo University Hospital. The goal of the labo- of neurological diseases with either transplantation of SCs ratory is to build a bridge between the basic biological scienc- that have been expanded in vitro or by mobilization of es and clinical neurosurgery, to explore the biology endogenous progenitor cells. underlying neurosurgical conditions, and to facilitate translation of new knowledge from basic research disciplines The work on developing functional neurons from cells from into the clinic. Research efforts therefore encompass both the human ventricular wall was started in Professor normal brain cell development and disease states such as Langmoen’s laboratory at Karolinska Institutet (KI) in Stockholm tumours. Investigations aim to understand these processes in 2002. Instrumental in this work was Morten Moe, who came and develop methods to treat disease as well as promote cell to KI as a postdoc, and the PhD students Mercy Varghese and replacement to heal damaged brain tissue. Håvard Ølstørn. Human tissue was harvested from the wall of the lateral ventricle in temporal lobe specimens resected due to epilepsy. Aims • To characterize human brain stem cells and develop cell Following dissociation and exposure to differentiation cues types for neurodegenerative disorders (mainly withdrawal of growth factors and addition of serum) • To characterize brain tumor stem cells these cells went through characteristic steps of morphological • To identify therapeutic targets in brain tumors and electro-physiological changes (Moe et al, Brain, 2005) and • To initiate clinical protocols based on translational research developed into the three principal building blocks of the brain: 1. Astrocytes Stem cells (SC) from the adult human brain 2. Oligodendrocytes A central dogma in neuroscience has been that the mature 3. Neurons brain is unable to produce new neurons. Towards the end 32

Functional neurons from adult human SCs first prize at the Scandinavian Neurosurgical Society’s Annual More importantly, the group became the first to demonstrate Congress in 2005. See Olstorn H et al Neurosurgery, 2011. that it is possible to transform immature progenitor cells from the adult human ventricular zone into functional neurons, i.e. cells with typical neuronal action potentials and the ability to Dopaminergic cells for Parkinson’s disease communicate through synapses (Figure 1). Essentially, we An avenue where adult human neural SCs offer significant were able to develop a small nervous system from a single promise is in the treatment of degenerative diseases such as human SC in vitro. Parkinson’s disease. Parkinson’s disease is characterized by loss of pigmented dopamine-secreting cells in the substantia The neurons in this “nervous system” communicated via nigra. Though transplantation of fetal brain tissue has shown synapses. These results were selected from more than 15.000 promising results, the results have not been consistent and the abstracts for presentation at the press conference of the method is controversial due to the use of tissue from Society for Neuroscience eight years ago. See Westerlund U abortions. et al Exp Cell Res 2003, Moe MC et al Brain, 2005 and Neurosurgery 2005. Dopaminergic cells derived from adult human brain SCs have obvious benefits: firstly, they pose no ethical challenges, and secondly, they make autologous transplantation possible. We are investigating the potential of adult human brain SCs to develop into dopaminergic neurons. Mercy Varghese, a previous PhD student in our lab, obtained preliminary results that adult human brain SCs can develop into dopaminergic neurons in vitro. This has been confirmed and extended by Wayne Murrell and Emily Palmero.

SCs from different CNS regions Mercy Varghese and coworkers isolated neural progenitors from the adult human filum terminale. This terminal end of the spinal cord has been referred to as a fibrovascular tag without neurogenic potential and of no clinical significance. Similar to brain SCs mentioned earlier, these cells from the filum Figure 1 – Dual patch-clamp recordings from neighboring neuron-like terminale generated functional neurons capable of firing cells demonstrates functional synaptic communication (Moe et al, Neurosurgery, 2005). action potentials. When transplanted into the adult central nervous system, the filum terminale-derived neural progenitors Transplantation to another adult brain survived, differentiated and showed targeted migration to a site of injury (Varghese M et al Stem Cells and Development, For SCs to be useful in the clinical situation they must be able 2008). More recently we have also shown that SCs from other to survive and integrate following transplantation into another brain regions than the subventricular zone may have a mature brain. Using rats with a selective lesion of the potential in clinical situations (Murrell et al, PLOS One, 2013). hippocampal CA1-region (a small part of cerebral gray matter), Håvard Ølstørn and Morten Moe demonstrated that SCs from the adult human brain are not only able to survive in the adult Optimizing cell culturing rat brain, but also selectively target and migrate to the area One problem with regard to the utilization of adult human with the lesion (Olstorn H et al Neurosurgery, 2007). Use of neural SCs both in research and in clinical trials has been that specific antibodies against human nuclei (HuN) demonstrated it has been difficult to expand these cells into sufficient survival of the transplanted cells and showed that the grafted numbers. Emily Palmero and Wayne Murrell have therefore cells frequently express the immature marker human nestin. tested a number of different culture conditions systematically. Less frequently, the cells expressed the neuronal marker They arrived at a robust technique for cell culture that doublecortin and the glial marker GFAP. produces billions of cells (Murrell et al, PLOS One, 2013). At present, studies are being performed to assess the fate of Ølstørn and Moe further showed that by using these cells and their differentiation potential. Directed ‘predifferentiation’, i.e. “pushing” the cells in a neuronal differentiation of these cells towards a specific fate such as direction prior to transplantation, it was possible to significantly down the dopaminergic pathway to obtain dopaminergic enhance the development of neurons following neurons is being examined. transplantation. The first part of the study secured Ølstørn the 33

Transplantation of adult human neural SCs in the rat model of normal SCs should define both the quintessence of the cancer Parkinson’s disease is an ongoing aim of the lab. The specific and possible targets for treatment. Consequently, we have aims of this study are: 1.To investigate the ability of systematically compared these two cell types. transplanted neural SCs to reduce the signs of disease in In a leading study, Mercy Varghese and Morten Moe showed Parkinsonian rats. 2. To estimate and compare the that these cells share a number of the properties with normal therapeutical efficacy of neural SCs transplants from different neural SCs of the adult human brain. Håvard Ølstørn sources (olfactory mucosa, subventricular zone and filum demonstrated that SCs isolated from tumours could terminale). 3. To test the hypothesis that neural SC transplants reproduce those tumours in immunodeficient rodents, will produce dopaminergic neurons in the striatum and/or will whereas SCs from normal human brain did not result in promote increased innervation of grafted striatum by surviving tumours. (Varghese M et al Neurosurgery, 2008). dopaminergic afferents. Both normal SCs and GSCs showed a high proliferation rate. Interestingly, GSCs could differentiate and then lost most of SCs and brain cancer their proliferative capacity. Normal and tumour SCs otherwise Glioblastoma multiforme (GBM) is both the most common and showed a similar pattern of differentiation, i.e. in neuronal and lethal type of brain cancer. GBMs always recur despite glial directions, although differentiated cells from tumours were apparent complete removal under the operating microscope clearly abnormal morphologically and differentiation in itself and subsequent adjuvant therapy. Median survival in progressed much faster. Einar Vik-Mo performed a number of unselected groups is ~ 10 months. experiments studying the effect in vitro-culturing had on GSCs ability to form tumours, differentiate, and undergo genotypic In parallel with other research institutions, our group showed and expressional changes (Vik-Mo et al, Neuro Oncol. 2010). that GBMs contain a subpopulation of cells that share central He also explored the cellular organization of neuro- and properties with SCs from the adult human brain, and further tumourspheres, looking at the cellular heterogeneity of such that only this cell population has the ability to proliferate and spheres (Vik-Mo et al, Exp Cell Res. 2011). By sorting tumour initiate new tumours following transplantation to cells based on surface antigens, we hope to establish immunodeficient mice. These cells therefore became known methods for better identification of the progenitor population. as glioblastoma SCs (GSCs). GSCs infiltrate surrounding brain tissue, appear resistant to both irradiation and chemotherapy, In a second leading study, Cecilie Sandberg compared gene and are the likely explanation for recurrence (Figure 2). expression in GSCs to expression in normal SCs in order to identify new targets for treatment. This was done by microarray technology / global gene expression. This investigation revealed a significant up-regulation in GSCs of genes connected to regulation of focal adhesion, actin cytoskeleton, axon guidance, as well as the Wnt signalling pathway. Putative target genes were confirmed at the protein level using immunohistochemistry and Western blot (Sandberg et al, Exp Cell Res, 2013). Currently, the genes’ roles in GBM are investigated using shRNA-knockdown based technology and its effect on proliferation, apoptosis and sphere-forming capacity.

Figure 2. Transplantation of normal human brain SCs (left) and human GSCs (right) to rodent brains. Normal brain SCs partially integrate in brain circuitry but make no tumor. GSCs produce a vopy of the mother tumor in the rodent brain (B and D).

Comparing cancer SCs to normal SCs A key concept, conceived when we started the investigation on GSCs more than 10 years ago, was that a spectrum of qualities differentially expressed in GSCs as compared to 34

Fig. 3 – Pathway significance in GSCs (from Sandberg et al 2013).

Confirmation of potential pathways not least whether they also are up-regulated at the protein and target genes level. She performed further analyzes by qPCR, Western blot, Having identified pathways and individual genes through and immunohistochemistry on tissue sections and cell global gene expression as mentioned above we decided to cultures. In addition, the genes were evaluated in relation to confirm the relevance of these pathways and genes using public data bases (Rembrant, TCGA). The study confirmed other methods. that nine of the 20 candidate genes from Sandberg’s investigation may be potential targets for treatment In Sandberg’s study, Wnt came up for the first time as a (Stangeland et al, in prep). central pathway in GBM. Kirsten Strømme has since then further characterized the dysfunction of this pathway by PCR To explore a putative functional importance of these target of almost one hundred pathway members and other methods. genes we have established lentiviral-based shRNA delivery One important finding in her project has been that and started to test the potential of gene knock-downs (KDs) downregulation of certain pathway members may be just as to inhibit growth of tumour cells. Using these and other significant as up-regulation of others. techniques, Mrinal Joel and Awais Ahmad have explored individual genes (Joel el al, submitted; Ahmad et al, in prep). Biljana Stangeland has investigated a set of 20 genes that were up-regulated in all GBM tumour cultures in Sandberg’s micro-array data. The identified genes are involved in Studying GSCs following transplantation cell-cycle/division, epigenetic regulation, signaling and Studying GSCs following transplantation to other nervous down-regulation of tumour-suppressors. systems may serve to throw light on several aspects of GBM biology. Stangeland’s main task has been to further test to which extent the 20 candidate genes are of significance in GBMs, Håvard Ølstørn started to transplant GSCs to rodents. 35

Following this, Einar Vik-Mo has shown how human GSCs Clinical protocols consistently produce tumors in the brain of SKID mice Based on our preclinical studies, we developed a clinical (Vik-Mo et al, Neuro Oncol. 2010; Exp Cell Res. 2011). protocol and have completed inclusion into a phase I/II study where we immunized patients against their own brain cancer Mrinal Joel transplanted these cells to chicken embryos. SCs. In this study, which made the front page of the journal Interestingly, it turned out that within proliferative embryonic Cancer Immunology and Immunotherapy last September tissue, GSCs exhibited reduced proliferation and survival, and (Figure 4), Einar Vik-Mo and Birthe Mikkelsen developed altered gene expression and formed no tumors at all (Joel et methods for cultivating SCs and extracting/amplifying mRNA al, Dev Dyn 2013). The chicken embryo thus contains factors from GSCs under GMP conditions. that control these very malignant cells. Professor Kvalheim, in the Department of Cell Therapy, Awais Ahmad has, in a separate study, followed the isolated monocytes from the same patients and cultivated development of tumors in mice brain following transplantation dendritic cells (DCs). The DCs, which are antigen presenting of GSCs by serial high-tesla MRI imaging. cells, were then transduced with mRNA from GSCs and used as “vaccine”. This clinical trial is backed up from the In parallel with this, Håvard Skjellegrind and Artem Fayzullin collaboration through the Cancer SC Innovation Center have started to film SCs over several days. This is done both (SFI CAST) and is acollaboration between the departments in cell culture and following transplantation to brain slices. of Neurosurgery, Cell Therapy, Clinical Cancer Research and They have been able to film both synapse formation and the Oncology (Figure 4). The study, which was the first clinical dynamics of GSC infiltration of the surrounding brain. They trial targeting cancer SCs in a solid tumor, was published last are at the present finalizing a study where they have identified summer. The vaccine led to a decrease in tumor associated various GSC subtypes based on kinetic and structural contrast enhancement on MRI scans starting at the time of a properties. confirmed immune response (Figure 5 A,B) and three times longer recurrence free survival (Vik-Mo et al, Cancer Immunol Skjellegrind and Fayzullin are also visualizing the time course Immunother, 2013). Other clinical trials based on our work on of important cellular functions, like intracellular calcium GSCs are currently being planned. concentration, in response to cellular stress.

Figure 4. Inset: Front page of Cancer Immunology and Immunotherapy Sept 2013. Main figure: Schematic overview Figure 5. Changes in size of contrast-enhancing tumor over time. of the production of DCs targeting glioblastoma stem cells. A Brain MRI images after contrast. Days before (negative) and after Left circle: GSCs were cultivated from tumor biopsies and mRNA surgery are noted on scans. B Maximal area of contrast was purified from these cells. enhancement plotted against days since surgery (abscissa). Right circle: Monocytes were harvested by leukapheresis of the The lower part of the figure indicates the timing of concomitant same patients and differentiated into immature dendritic cells. chemo-radiotherapy (blue box), DC vaccinations (blue arrows), Center: The dendritic cells were transfected with autologous GSC and immune response (red arrow). mRNA by electroporation. 36

Photo: Øystein H. Horgmo, UiO 37

Experimental Orthopaedic Research

Leader Lars Nordsletten, Professor, MD, PhD (OC/UiO)

Scientific staff Lars Engebretsen Prof, MD, PhD (UiO/OC) Olav Reikerås Prof, MD, PhD (UiO/OUH) Jan Erik Madsen Prof, MD, PhD (UiO/OC) Harald Steen Prof, MD, PhD (OUH) Stig Heir MD, PhD (Martina Hansens Hospital) Sanyalak Niratisairak, PhD, Head Engineer (UiO/OUH) Sverre Løken MD, PhD (OC) Ulf Sigurdsen MD, PhD (UiO/AHUS) Sigbjørn Dimmen, MD, PhD (LO) Professor Lars Norrdsletten Finn Reinholt Prof, MD, PhD (UiO/OUH) Jan Brinchmann Prof, MD, PhD (UiO/OUH) Stein Erik Utvåg, MD, PhD (UiO/AHUS) wounded, portions of articular cartilage with newly formed Geir Hjorthaug, MD, PhD-student (OUH) fully functional cartilage is possible remains one of the Jan-Egil Brattgjerd, MD, PhD-student (OUH) unsolved problems in orthopaedic practice. Knee patients have more problems than patients with rupture of the anterior cruciate ligament and experience severe Research areas limitations in their daily life. Despite this, knowledge about Musculoskeletal injuries are main causes of disability in the the best treatment, and if surgical treatment do offer a community and are often subjected in both younger and better outcome than the natural history, is still not older age groups. It induces large socioeconomic costs, documented. A better understanding of articular cartilage and improved health care in this area is important for both biology, pathophysiology, and biomechanics are definitely the individual quality of life and how the society handles warranted. High priority research projects of the group have increasing health expenditures. The Experimental been better understanding of the cartilage repair process Orthopaedic Research (EOR) group applies experimental and assessment of the role of the mesenchymal stem cells methods on different aspects of orthopaedics. This versus chondrocytes in studies of cartilage repair. includes research on human substances (biopsies, joint The ongoing work of the cartilage research group can be fluid, and retrievals), animal experiments and cell culture divided into three main areas. studies. Mechanical testing of structures, including live anaesthetized animals, and materials has been one of the main research methods. The experimental work in the Experimental cell-culturing cartilage research laboratory is closely connected to ongoing or planned The group has worked intensively to provide the best cell clinical studies, aiming to improve orthopaedic care of source for cartilage repair. Mesenchymal stem cells, these patients in the community. Involvement of the harvested from the bone marrow, implanted in hyaluronic- clinicians is one of the strengths of the group. based scaffold have been studied in the laboratory for production of articular hyaline cartilage specific markers. Aims Theoretically, these cells have a promising potential for To: repair of normal hyaline cartilage. The collaboration with the • develop a novel treatment of focal cartilage defects Ex Vivo Laboratory (Rikshospitalet) headed by Jan Brinchman • reduce the numbers of deficient fracture healing has enhanced the supply of improved cells for cell based • improve healing of tendon grafts in orthopaedic surgery cartilage repair and the ability to test and develop new • delineate the best biomaterial surface for prosthesis scaffolds. surgery • reveal biomechanical factors in internal fixation of hip Experimental cartilage research in an animal model fractures An important factor in cartilage treatment is the location of the defect in the knee and the consequences for the treatment of the defect and the risk of degenerative Cartilage research changes. An experimental study has been undertaken to Malfunction of the knee joint is often associated with specifically investigate this factor. The location of the defect cartilage injury. Whether healing, or restoration, of lost, or is essential, as it is demonstrated in the current study. The 38

difference in treatment outcomes between microfracture potential positive effects of bone formation stimulators and and mosaicplasty has been difficult to assess clinically and bone resorption inhibitors. another study has been conducted to evaluate some clinical assessment techniques. We observed changes in the subchondral bone and their relation to the long-term risk of degenerative changes in the joint. Mesenchymal stem cells implanted under a commercially available scaffold for repair of articular defect have been assessed experimentally to look at feasibility of this technique and at the amount of cartilage obtained in the defect.

Clinical cartilage projects The experimental work is closely connected to the clinical studies of the group and, currently, there is an ongoing study on stem cell treatment versus chondrocyte implantation. Patients with cartilage defects of the knees treated with chondrocyte implantation have been evaluated with electron microscopy biopsies to study ultra- Figure 1. - In most of our fracture and tendon projects we use organization of the tissue and deaths of chondrocytes. biomechanical tests as the primary endpoint to evaluate healing. Samples are loaded until fracture, rupture or pull-out in a highly Results have recently been published. sensitive materials testing machine (MTS). We obtain data on load, time and deformation. This figure illustrates a typical load– deformation curve from a cantilever bending test of an adult rat Fracture and tendon-to-bone healing tibia tested to failure. It shows the biomechanical structural properties of strength, stiffness and energy to fracture. Delayed fracture healing or non-union may occur in high energy fractures, in patients with systemic diseases, with hormonal or nutritional deficiencies, or in patients taking Previous experiments on intramedullary nailing and external specific medications like non-steroidal anti-inflammatory fixation in lower leg fractures assessed bone healing by drugs (NSAIDs and COXIBS). Healing of tendon material quantitative microcomputer tomography (qmCT), in to bony surfaces is of major importance in both shoulder addition to mechanical testing and densitometric and knee surgery where understanding of the biology and measurements (Figure 1). A collaboration program between biomechanics in this process is a key element. Rupture of EOR and the Robotics and Intelligent Systems (ROBIN) the reconstructed tendon/ligament and increased laxity by research group at the Department of Informatics is under repeated loading constitute the major problems. planning. The scope is to utilize modern robot technology to standardize bone-implant and fracture-implant systems We have previously studied fracture healing in rats on a for improved experimental biomechanical evaluation of short time medication with NSADIs and COX-2-inhibitors, fracture fixation techniques in both composite and human and in rats made severely osteoporotic by estrogen and cadaver bones. A pilot study included one group of vitamin-D depletion. We have demonstrated the potential standard mechanical testing of 4th generation (Figure 2) negative effects of these drugs on bone metabolism and composite bones (radius) with manual osteotomy and bone, tendon and tendon-to-bone repair. fixation with different devices. A similar group of composite bone is now in the process of being tested in the robotic Manipulation of bone resorption by osteoclast inhibitors has setup. become possible with several drugs including monoclonal antibodies. Bone formation stimulators have been more difficult to develop, but several candidates are now Biomechanical studies of hip fractures available. In ligament reconstructions, stability is achieved Most hip fractures are operated with reduction and internal only by solid tendon to bone healing. This may pose a fixation. An improvement in this procedure is thought to clinical challenge considering the necessity of early active reduce the complications related to fracture healing. movement and loading to facilitate the best possible However, there is a lack of documentation of the concept functional outcome. For both fracture and tendon to bone and the effect of optimal reduction and fixation. On the healing, it would be desirable to be able to speed up the choice of implant for hip fractures, no clear conclusions healing processes. There is growing evidence that can be made. pharmacological adjuvants may have an important role to play and may be pivotal for the clinical outcome. We are Our hypothesis is that a new implant design provides currently developing our rat models and analyses to test higher stiffness in fixation of intracapsular hip fractures, and 39

Figure 2. Photograph of a 4th generation radius composite bone with a standardised osteotomy fixated with the Orthomedic DVR volar plate. that for extracapsular hip fractures, a new constellation of a was also awarded the yearly SE Health Authority Region well-known implant will increase stiffness and reduce Innovation award. Novel orthopaedic devices and implants fracture healing complications. We want to perform have been mechanically tested and characterized in our biomechanical studies on synthetic and cadaveric femurs laboratory as a part of the global patent seeking process. to analyze the stiffness of different fixations in different Data cannot be published yet due to patenting procedural types of hip fractures. To correct for osteoporosis we use issues. bone mineral content measured by quantified CT.

International Collaborators Innovation • Rob LaPrade, Steadman Philippon Research Institute The EOR group has also collaborated with AHUS in an Vail, Colorado, USA innovation project funded by OUH and Inven2. The inventor 40

Photo: Per Marius Didriksen, DNR 41

Cell Transplantation and Tissue Engineering

Leader: Aksel Foss, Prof., MD, PhD, MHA, FEBS (OUH/UiO)

Cell Transplantation Scientific Staff Cell Transplantation Hanne Scholz, Senior Scientist, Msc, PhD (OUH) Simen W. Schive, PhD student , MD (OUH) Shadab Abadpour, PhD student, Msc (OUH) Kristine Kloster-Jensen, PhD student , MD, (OUH) Afaf Sahraoui PhD student , MD (OUH) Tormod Lund, MD, PhD (OUH) Merete Høyem, Research technician (OUH) Ragnhild Fjukstad, Bioengineering (OUH) Professor Aksel Foss Tim Scholz, MD, PhD, FEBS (OUH) Trond Jenssen, Prof, MD, PhD (OUH/UiT) Karsten Midtvedt, MD, PhD (OUH) Background Geir Hafsahl, MD, PhD (OUH) Type 1 diabetes (T1D) is an autoimmune disease caused Stein Bergan, Prof., MSc, PhD (OUS/UiO) by destruction of the alpha- and beta cells in the pancreas. Prevention of T1D is hampered by the fact that the actual mechanism of cell destruction is largely unknown. Standard Research Area; Cell Transplantation of care in T1D is life-long therapy with exogenous insulin. The rapidly emerging achievements in cell In spite of optimal insulin therapy (including digital insulin transplantation and stem cell-based treatments have the pump devices and subcutaneous glucose sensors), potential of providing new therapeutic options for several periods of hyperglycemia induce serious vascular- and illnesses. As such, Islet transplantation (see below) neurological complications, such as accelerated represents a research model for other cellular treatments. arteriosclerosis, kidney failure and impaired vision. There The Research Group for Cell transplantation is part of the is currently no cure for diabetes, and exogenous insulin Nordic Network for Clinical Islet Transplantation (NNCIT) administration, either as injection or via a subcutaneous and works in close collaboration with Uppsala University. pump, is the selected treatment for most patients suffering During the last years, extensive work has been carried out from the disease. However, it is apparent from numerous to refine established methods for islet isolation and to clinical studies that insulin does not offer the effective develop new strategies for cell-based engineering glucose homeostasis required to prevent the deadly long- technology to improve the long-term outcome of islet term complications of the disease, and better treatment transplantation as treatment for type 1 diabetes. The group options are highly warranted. As many as 25% of these has leaded the work with the aim to create a “Center for patients have impaired awareness for hypoglycemia and Cell Replacement in Diabetes” at Oslo University Hospital. some develop life threatening brittle diabetes, with The intention is to develop a multidisciplinary center complete unawareness (lack of symptoms) for severe composed of experts within basic research and clinicians hypoglycemia. The estimated prevalence of unawareness with a unique and comprehensive knowledge and expertise among T1D patients is 2-3%, i.e. 500-1000 patients in in diabetes, cells and stem cells biology, pharmacokinetics Norway suffer from this condition. These patients have and transplantation. The strength in this organization is that constant concerns regarding their blood glucose levels the people involved are a mix of full time researchers and with frequent day and night measurements, working active clinicians who quickly can transfer new knowledge disability and severe quality of life impairment. from the laboratory into the clinic, which is mandatory in Hypoglycemia is a significant cause of cardiovascular modern medical science. events and death among persons with diabetes, and the mortality rate of brittle diabetes patients (dead-in-bed syndrome) is markedly increased compared to the normal Aims population. The group’s overall aim is develop and improve islet isolation, engraftment and clinical outcome after islet It is well documented that beta cell replacement therapy, transplantation for treatment of patients with diabetes. either as whole organ pancreas transplantation or as islet transplantation may cure or stabilize T1D. More than 1000 patients worldwide have received islet transplantation since 42

Figure 1 (A) Illustration of clinical islet transplantation (organ procurement, islet isolation and intraportal transplantation. (B) Schematic presentation of loss of islets from the time of organ retrieval (1), islet isolation (2), pre-transplant culture (3), transplantation (4), and post-transplant period (5). Green area represents insulin-independence, red area insulin dependence. From Olle Korsgren the breakthrough in 2000. Islet transplantation is highly Experimental Cell Transplantation successful in improving glycemic control and reducing life-threatening episodes of hypoglycemia,. Up to 80% of 1. Investigate the intracellular immunosuppressive the patients are temporarily free from exogenous insulin concentrations in human islets and the regulation after transplantation (most often with islets from multiple thereof. donors), but the long-term efficacy is too low and further Following islet transplantation the recipients are in need refinement of the treatment is highly needed. of lifelong immunosuppression. Known diabetogenic side effects of immunosuppressive therapy are particularly

Current Research activity deleterious in the situation of a reduced beta cell mass (like in islet transplantation), possibly contributing to the

historically poor success rate of human islet allografts. Clinical islet transplantation The islets engrafted in the liver are exposed to relatively Islet transplantation is currently standard treatment of care high concentrations of immunosuppressive drugs, even in selected patients with T1D, refunded through the DRG when systemic drug levels are carefully controlled for. system. The high liver concentrations may be toxic to the islets and could impair revascularization and proliferation, and are This year we established a GMP human cell processing most likely not required to prevent acute transplant facility for large-scale production of human islets to be used rejection. Studies to determine the right dosages and in clinical islet transplantation at the Department for Cellular optimal combinations of available immunosuppressants for Therapy (Radiumhospitalet) in close collaboration with optimal islet survival are needed. Professor Gunnar Kvalheim (Figure 2).

Figure 2. Human islet isolation at ex vivo laboratory, Department for Cellular Therapy, Radiumhospitalet. Photo: Per Marius Didriksen, DNR 43

Together with the i2mc research group, headed by Prof. Stein Bergan, we are investigating the pharmacokinetics, pharmacodynamics and pharmacogenomics of the immunosuppressive drugs in human islets in vitro. The potential direct toxic (e.g. diabetogenic) effects of immunosuppressants on islets during engraftment may influence upon their survival and function. Protective mechanisms like efflux transporters (e.g. p-glycoprotein) and drug-metabolizing enzymes may counteract the damaging effects of immunosuppressants on the transplanted islets. Preliminary data from our group (in preparation for publication) indicate that there is significant expression of some of these transporters and enzymes in the islets, and also that the intra-islet concentrations of immunosuppressants are significantly influenced by concomitant drugs.

2. Adult stem cells promote islet survival Mesenchymal stem cells (MSCs) are multipotent stromal cells found in many different types of tissue. The MSCs show immunomodulatory and regenerative properties. Figure 3. Bioluminescence in B6 Albino mice transplanted with MSCs from bone marrow have been documented to synergistically MIP-luc islets under kidney capsule. Representative reduce inflammation and promote islet survival both in vitro image of transplanted MIP-luc islets before (baseline) and day 3 and 7 after induction of diabetes with alloxan treatment and in vivo. The use of intravenous administration of MSCs (correspondent blood glucose levels shown below). in animal experiments has also proven to have an anti- diabetic effect in T1D. It has been suggested that MSCs 3. Develop a transplantation model to evaluate the transdifferentiate into pancreatic islets. But recently it was potency of human islet grafts in vivo. reported that the most likely effect of MSCs in diabetes is In collaboration with AstraZeneca AB research team and due to their immune-modulatory effect, restoring Th1/Th2 Uppsala University, we have established a double islet balance and modifying the pancreatic microenvironment. transplantation model to be able to investigate the impact Most of the work has been done on bone marrow derived of new compounds on the expansion of beta cell mass and MSCs. However, the frequency of MSCs in bone marrow is improvement of function (Fig.4). Briefly, two islet reported as low. Adipose tissue yield 100-500 times more transplantations are performed in the same animal. First, cells than bone marrow, and generates less pain and mice islets are transplanted under the left kidney capsule to morbidity for the donor. The stromal vascular fraction restore hyperglycemia, the second transplantation is (SVF) can be cultured and expanded in vitro to adipose performed after a recovery time of 2-4 weeks. stem cells (mesenchymal cells). In a pre-clinical setting A suboptimal dose of human islets are transplanted under we will explore whether ex vivo expanded MSCs from the right kidney capsule and the grafts are allowed for a fat and bone marrow and fresh ADRCs can improve islet recovery period for up to 3 weeks, the first graft is removed transplantation. In addition, in vitro and in vivo by left sided nephrectomy which allows for followed islet co-culturing experiments will be established. function and possible proliferation in the second graft in re- sponse to different interventions in vivo. Exendin-4 has the Experimental imaging of beta cells and MSCs potential to increase beta- cell mass either by stimulating Non-invasively monitoring of changes in islet mass proliferation or inhibiting apoptosis. We study the effects of following islet transplantation is important to provide new Exendin-4 on metabolic parameters of aged human islets in insight into factors regulating post transplant function. vivo and report that Exendin-4 could play an important role For in vivo surveillance cells can be labeled with protein. in the first period after islet transplantation where In house, we have mouse strains that express a beta normal levels of blood glucose are desirable to reduce cell-specific reporter (luciferase) which allows for glucose toxicity to the islets (manuscript in preparation). bioluminescence imaging of the beta cell mass (Figure 3). In a set of experiments we will combine GFP labeling of MSCs with luciferase positive beta cells for non-invasive imaging of the fate of the co-culture using IVIS Spectrum (already in use in our labs). 44

Fig. 4. Transplantation of human islets under the kidney capsule into diabetic nude mice. (A) Human islets (white arrow) at the sub-capsular site. (B) Immunofluorescence section of human islets transplanted under mouse kidney capsule showing insulin-producing cells (red). DAPI staining shows cells nucleus (blue).

4. Beta cell regeneration and stem cells by assembling the protein during activation promotes the Expanding the beta cell mass would greatly improve maturation of the pro-inflammatory cytokines IL-1beta and opportunities for cell-based therapy in diabetes. The fact IL-18. This project seeks to unravel the role of the NLRP3 that the beta cell mass in a person is capable of expanding inflammasome in islet transplantation. Pancreatic islets are in response to insulin resistance, obesity, pregnancy and susceptible to hypoxia-induced dysfunction. Central to the trauma is the background for our research focus on injurious effects of hypoxia in many cell types, is the conversion of non-beta cells within the pancreas to involvement of inflammasomes like NLRP3. The NL- beta-like cells. The adult pancreas consists of both RP3-ASC inflammasome complex is involved in the exocrine and endocrine parts with different functions. activation of casapse-1, interleukin- (IL)-1 and IL-18 The acinar, ductal and pancreatic progenitor cells of the secretion, and has been implicated in type 2 diabetes. exocrine pancreas have been suggested as source for Whether hypoxia could activate NLRP3 in islets is not conversion into beta-like cells or to prevent hyperglycemia established. In this study, we aimed to investigate whether in diabetic animals. In addition, it is believed that other cells ablation of the NLRP3 complex (NLRP3-/- or ASC-/-) could belonging to the endocrine part of the pancreas can be alleviate the negative impact of hypoxia on islets. Our transformed into beta-like cells, especially when the preliminary results showed that ablation of the NLRP3 transformation occurs in the native microenvironment. inflammasome in islets significantly improves hypoxia- Among the endocrine cells, the glucagon-producing alpha induced cell death, and reduces the mRNA expression of cells seem to be the potential source. In human T1D, HIF-1 and VEGF. This suggests that the NLRP3 random beta cells are found scattered around the inflammasome conveys the deleterious effect of hypoxia in pancreas. Whether this is a result of regenerating new beta islets. cells in the adult pancreas or persistence of existing cells that have escaped from autoimmune reaction, is unknown. Scientific Collaborators Cell transplantation In this study we will investigate different approaches to • Professor Olle Korsgren, Uppsala University Hospital, induce human islets proliferation and survival, and explore Sweden the mechanisms involved by in vitro models of whole islets • Professor Peter Stock and Ass. Professor Qizhi Tang, or dispersed islets (Fig.5). University of California, San Francisco, USA • Professor Gunnar Tufveson Uppsala University Hospital, 5. Role of NLRP3 inflammasome in type 1 diabetes. Sweden This project is performed in collaboration with Dr. Arne • Professor Peetra Magnusson, Uppsala University Yndestad and Dr. Trine Ranheim, Institute for Internal Hospital, Sweden Medicine OUH. Recently publications indicate a role of • Advanced Center for Translational REgenerative the NLPR3 inflammasome in type 2 diabetes. The Medicine (ACTREM), Karolinska Institutet, Sweden inflammasome is part of the innate immune system and • Professor Tomas Totterman, Uppsala University 45

Figure 5. (A) Immunofluorescence section of pancreas showing insulin-producing cells (green) in a proliferative state, BrdU positive cells (red) as a marker for proliferation pointed by arrows. (B) Dispersed human islets showing insulin producing cells (green) and glucagon producing cells (red). DAPI staining shows cells nucleus (blue).

Hospital, Sweden CMO Per Gjorstrup, Resolvyx types also including endocrine and nerves. Pharmaceuticals, Inc. Bedford, MA, USA • AstraZeneca R&D Mølndal, Sweden Tissue engineered tracheal replacement • Professor Bo Gøran Ericzon, Karolinska Institutet, For the first time, a patient, in 2011, was given a new Huddinge, Sweden trachea made by TE technique using a synthetic scaffold • Professor Pål Aukrust and Professor Bente Halvorsen, seeded with his own stem cells at Karolinska University Institute for Internal Medicine, Oslo University Hospital Hospital in Huddinge, Stockholm, by Professor Paolo • Senior scientist Thor Ueland and Arne Yndestad, Macchiarini. Professor Macchiarini led an international team Institute for Internal Medicine, Oslo University Hospital including colleagues from UK and USA who provided the • Professor Gunnar Kvalheim, Department of Cellular tracheal scaffold and a specifically designed bioreactor Therapy, Oslo University Hospital used to seed the scaffold with the patient’s own stem cells. The TE-project group has an ongoing collaboration with Advanced Center for Translational Regenerative Medicine Tissue engineering at the Karolinska Institute (ACTREM).

Background Organ and tissue bioengineering Tissue Engineering combines research within cellular Proof-of-principle studies of tissue engineered bioartificial biology and cellular transplantation with material- and heart, liver, pancreas, including kidney have been engineering science to develop biologic substitutes. The established. goal is to restore and maintain normal organ function that has been damaged due to disease, trauma, and cancer Organization and research plan therapy or by other causes. At Oslo University Hospital the Division of Cancer Medicine, Tissue engineering is a multi disciplinary field of research Surgery and Transplantation and Division of Cardiovascular that has seen intense developments in recent years. and Pulmonary Diseases decided, in 2011, to initiate a joint It involves the in vitro seeding and attachment of cells onto project in tissue engineering with the aim to provide a scaffold. After proliferation and migration, reinforced by patients with treatment solutions based on regenerative growth factors, cells differentiate into the specific tissue. medicine and bioengineering techniques. Professor Aksel Scaffold technology is also developing fast and is made Foss, Transplantation Surgery, accepted to lead the up of synthetic and/or degradable biomaterials. Tissue project. Initial phase of the project was estimated to last engineering utilizes living cells as engineering materials. for 2-3 years and included establishment of the research Recently, there has been a great trend towards the use of group on tissue engineering and international network adult mesenchymal stem cells from bone marrow or fat and cooperation. Recently, all activities on regenerative iPSCs. These cells can differentiate into a variety of tissue medicine and tissue engineering at OUH and UiO were 46

joined in Oslo Regenerative Medicine Initiative ORMI, Transplantation (Research area: Adipose tissue stem (Figure 6) and the initiative has been selected as Focused cells in tissue repair) Area of Research at OUH 2014-2018. A formal collabora- • Dr. Kim A. Tønseth, Department of Plastic and tion with Karolinska Institute has been established. A joint Reconstructive Surgery, Division of Surgery and Clinical initiative is preparing an EU Horizon 2020 proposal (Pro- Neuroscience (Research area: Regenerative medicine in gram PHC 16 – 2015: Tools and technologies for advanced plastic surgery) therapies on Bioengineered Human Kidney Transplantation • Prof. Stefan Krauss, SFI-CAST Biomedical Innovation (NEOrgan consortium) to advance cell- and organ-based Center, University of Oslo (Research area: Chemical technologies into human clinical bioengineered kidney biology in regenerative medicine) transplantation.) The consortium partners consist of ORMI, • Dr. Hanne Scholz, Department of Transplantation Karolinska Institute, The Ottlab, Harvard Medical School Medicine, Division of Cancer Medicine, Surgery and and the industrial partners AstraZeneca - Center for Transplantation (Research area: Cell replacement and CardioMetabolic Research and Regenerative Medicine -, tissue engineering) Organ Recovery Systems and Corline Systems AB • Dr. Jan E. Brinchman, Norwegian Center for Stem Cell (participating SME). ORMI will be the Principle Investigator Research and Department of Immunology (Ex vivo), of the project. Division of Diagnostics and Intervention (Research area: Cellular therapy) Scientific Staff Tissue Engineering • Prof. Jørgen J. Jørgensen Department of Vascular (Oslo Regenerative Medicine Initiative (ORMI): Diseases (Oslo Vascular Centre), Division of Cardiovascular and Pulmonary Diseases (Research area: • Prof. Aksel Foss (leader) Department of Transplantation Tissue-engineered allogenic vein valves in treatment of Medicine, Division of Cancer Medicine, Surgery and chronic venous insufficiency) Transplantation • Prof. S. Petter Lyngstadaas, Faculty of Dentistry, • Prof. Lars Engebretsen, Department of Orthopaedic University of Oslo (Research area: Scaffold technology Surgery, Division of Surgery and Clinical Neuroscience and biomaterials) (Research area: Cartilage tissue engineering) • Dr. Einar Martin Andahl, Department of Transplantation • Prof. Morten C. Moe, Center for Eye Research, Medicine, Division of Cancer Medicine, Surgery and Department of Ophthalmology, Division of Surgery and Transplantation and the Biotechnology Center of Oslo Clinical Neuroscience (Research area: Stem cells and (Research area: Transplantation surgery, experimental tissue engineering in the treatment of ocular disorders) immunology and tissue engineering) • Prof. Joel C. Glover, Norwegian Center for Stem Cell Research, OUH/University of Oslo (Research area: Work Packages (WP) (short version) timeframe Human somatic and pluripotent stem cell differentiation, 2014-2020 characterization and genetic manipulation) • Prof. Iver A. Langmoen, Department for Neurosurgery, WP1: Kidney decellularization/recellularization Division of Surgery and Clinical Neuroscience.Stem cells WP1a: Decellularization of kidney scaffold in the adult human brain (Research area: Stem cells in WP1b: Repopulation with endothelial and epithelial cells brain cancer. Vilhelm Magnus Laboratory for matured to functional kidney constructs in vitro for Neurosurgical Research) subsequent transplantation • Prof. Gunnar Kvalheim, Department of Cellular Therapy, • Small and large animal models Division of Cancer Medicine, Surgery and • Semi-xeno models (pig kidney architecture – human recellularization)

Figure 6 47

WP2: Stem cell technologies WP2a: Improving culture of stem cells for directed differentiation and up-scaling WP2b: Integrating novel mRNA technologies into regenerative medicine WP2c: Cell culture (differentiation, expansion and up-scaling) technologies (nephrospheres, kidney organoids)

WP3: In situ clinical stem cell kidney repair WP4: Transplantation of human bioengineered kidney WP5: Regulatory compliance, administration of the consortium, training, exchange of personnel.

ORMI Steering Committee

CEO, Dr. Bjørn Erikstein, OUH Prof. Ansgar Aasen, OUH/UiO Prof. Otto Smiseth, OUH/UiO Prof. Frode Vartdal, OUH/UiO Prof. Erlend Smeland, OUH/UiO Prof. Sigbjørn Smeland, OUH/UiO

Scientific Collaborators Tissue Engineering • ORMI partners, Oslo University Hospital and University of Oslo • Prof. Urban Lendahl, Karolinska Institute, Stockholm, Sweden • Prof. Stephen Strom, Karolinska Institute, Stockholm, Sweden • Prof. Paolo Macchiarini, Karolinska Institute, Stockholm, Sweden • Prof. Harald Ott, The Ottlab Massachusetts General Hospital, Harvard Medical School, Boston, USA • AstraZeneca (Center for Cardio Metabolic Research and Regenerative Medicine) • Organ Recovery Systems • Corline Systems AB (participating SME) 48

Photo: Øystein H. Horgmo, UiO 49

Experimental microsurgery and Transplantation Group

Leader Pål-Dag Line, MD, PhD, Head of department of transplan- tation medicine, Division of Cancer Medicine, Surgery and Transplantation (OUH)

Cooperation Partners Jihua Shi, MD, PhD Student (OUH) Bjarte Fosby, MD, PhD Student (OUH) Helge Scott, Prof, MD, PhD (OUH) Henrik Huitfeldt, MD, PhD (OUH) Vivi Bull Stubberud, RN (OUH) Aksel Foss, Prof, MD, PhD (OUH) Svein Dueland, MD, PhD, Consultant (OUH) Head of Department Pål-Dag Line Introduction The research group consists of members from the Trans- transplantation of various size is achieved by performing lobar plant Unit at the Department of transplantation medicine and resections on the graft. The model can also be combined with other research groups at Rikshospitalet, in particular from the porto-caval shunting, utilizing a carotid artery graft as a shunt Institute of Pathology. After the foundation of the Division of (Figure 2). Cancer medicine, Surgery and Transplantation and new research-group organization, we have joined the research group for Transplantation and malignancy headed by Svein Ongoing research topics Dueland MD PhD. Our main research topic for 2013 has been related to growth Our group has for many years established various and differentiation of hepatocellular carcinoma, finalizing the experimental transplantation models and other surgical thesis of PhD-student Jihua Shi. Hepatocellular carcinoma models in order to form a solid platform that allows us to (HCC) ranks fifth in frequency worldwide among all explore our main fields of interest: transplantation immunology malignancies and is the third most common cause of cancer and hepatic regeneration and cancer. mortality, with about 600,000 cancer related deaths annually. Liver resection or transplantation represents the only potential The transplantation models curative treatment modalities for most HCC patients. Despite successful surgery achieving R0 resection, tumor recurrence is In the cuff-based cervical heterotopic transplantation a major problem and might be as high as 75 % at five years of technique we transplant the donor heart to the neck of the observation. recipient. Anastomoses between the graft aorta and the carotid artery, and between the pulmonary artery and the external jugular vein are cuffed as reported by Heron.

Compared to conventional techniques of anastomosis the cuff method reduces graft cold ischemia and bleeding from the anastomoses. Another advantage with the method is the superficial localisation of the graft in the recipient’s neck which simplifies registration of graft function and enables exact judgment of endpoint for the rejection process. Graft ischemia is typically 5-10 min, and the technical success rate above 90%.

We have also established orthotopic liver transplantation models in the rat (Figure 1). This is a non arterialized, model, where the portal vein and the infrahepatic cava is anastomosed by cuff technique whereas the proximal, suprahepatic cava is sutured. The model is performed partly as a whole graft trans- plant but, when indicated, partial liver 50

(a microvessel density marker), vascular endothelial growth factor (VEGF), and vascular endothelial growth factor receptor 2 (VEGFR2) were evaluated and compared between the groups. The tumor’s volume and number increased significantly with the size of the partial hepatectomy (P < 0.05). The largest resections were also associated with increased hepatoma cell infiltration in the lungs and significant up-regulation of cyclin D1, AFP, CAPNS1, CD34, VEGF, and VEGFR2. The results suggest that liver regeneration after partial hepatectomy facilitates the growth and malignant transformation of microscopic HCC. This could be significant for liver resection and partial liver transplantation strategies for HCC. Results presented in Liver Transpl 17:866–874, 2011 and 2011 AASLD.

RAF targeted therapy for hepatocellular carcinoma in the regenerating liver

Figure 1. – Transplanted rat liver. Background Post-operative liver regeneration may contribute to tumor recurrence. There is a theoretical need for an adjuvant therapy that can suppress tumor growth without adversely affecting post-operative liver regeneration.

Objective To evaluate the effect of RAF inhibitor Sorafenib on cell viability and proliferation of hepatoma cells and hepatocytes in vitro and in an in vivo rat model.

Methods Cell viability, DNA synthesis, and RAF/MAPK kinase activity in the primary hepatocyte and hepatoma cell lines were Figure 2. – Portocaval shunt where a carotid artery graft is sutured end- to-side to the vena porta and cava respectively. investigated after Sorafenib exposure. Sequence analysis of the B-RAF gene in hepatic cells was determined. Tumor Growth of Hepatocellular Carcinoma in the markers were compared within the rats after 70% regenerating liver hepatectomy with or without daily oral gavages of Sorafenib. Liver resection and liver transplantation are the treatment Liver regeneration was assessed by liver function tests and modalities with the greatest potential for curing hepatocellular proliferation markers. carcinoma (HCC). Tumor recurrence after resection for HCC is, however, a major problem, and an increased rate of Results recurrence after living donor transplantation versus Primary hepatocytes showed higher cell viability, proliferation cadaveric whole liver transplantation has been suggested. rate, and stronger RAF/MAPK kinase activity compared with Factors involved in liver regeneration may stimulate the growth hepatoma cell lines. The in vivo tumor volumes, size, and of occult tumors. The aim of this project was to test the metastases were significantly decreased (P < 0.05) whereas following hypothesis: a microscopic HCC tumor in the setting no significant change in liver regeneration related to Sorafenib of partial hepatectomy would show enhanced growth and exposure was found (P > 0.05). B-RAF V600E mutation was signs of increased invasiveness corresponding to the size of not detected neither in the hepatic cells nor in untransformed the liver resection. Hepatectomy was performed to various hepatocytes (Figure 3). degrees in groups of Buffalo rats with the concomitant implantation of a fixed number of hepatoma cells in the Conclusions remnant liver; a control group underwent only resection. The RAF targeted inhibitor can reduce tumor growth without After 21 days, the sizes and numbers of the tumors and the retarding liver regeneration in this experiment. Paper published expression of alpha-fetoprotein in J. Surg. Oncol. ß 2012 Wiley Periodicals, Inc. (AFP), cyclin D1, calpain small subunit 1 (CAPNS1), CD34 51

C B

Figure 3. – Sorafenib selectively inhibited the proliferation and angiogenesis of HCC in vitro and in vivo.

A: Expression of tumor specific marker (AFP), proliferation Marker (Cyclin D1) , and angiogenesis marker (CD34) in tumors assessed by Western blotting. Note: Sorafenib treatment in Group TS1 and TS2 vs. vehicle control Group T.

B: Quantification of AFP, Cyclin D1, and CD34 (/ß-tubulin) by Western blotting. Mean value of aFP, Cyclin D1, and CD34 expression from the different groups were determined by densitometry of Western blotting (P < 0.05). Mean value and standard error was expressed as the bar and vertical error bars.

C: Cell viability of hepatoma cell lines and primary hepatocytes incubated with Sorafenib (0–12.5 mM) and EGF (10 mM) for 48 h (n = 3).

The effect of cells with stem-like properties on experimental hepatocellular carcinoma in the regenerating liver

Liver regeneration following surgery may stimulate the growth of occult HCC tumors. Large liver resections can, in itself, mobilize peripheral hematopoietic stem cells into the liver that differentiate into hepatic progenitor cells (HPC). It has been speculated whether progenitor cells might participate in hepatic tumor genesis or contribute to tumor growth and metastasis after surgery. The aim of this project was to explore how activation of HPCs affects tumor growth and malignant development in vivo following partial hepatectomy and to study the effect of HPCs on hepatoma cells in vitro. Varying grades of hepatectomy (0%, 30% and 70%) with concomitant implantation of hepatoma cells in the liver remnant were performed in Buffalo rats. Progenitor cell like activation was found by double immunofluorescence staining for cytokeratin Figure 4. – Double immunofluorescence staining with AFP (red), CK19 19 and AFP/CD133 (Figure 4). (green) and Hoechst 33258 (blue) (400).

A McA-RH7777 cells. The effects of HPCs (WB-F344) on wild type hepatoma cells B Tumor tissue. (JM1/WT) in vitro were investigated by co-culturing technique C Regenerating liver after 70% hepatectomy. D Positive expression of HPC in liver tissue after 70% hepatectomy whereby the HCC cells (JM1/WT) were cultured in the with tumor supernatant of the HPC cells (WB-F344) for 6 passages, thus 52

Figure 5 . – mRNA expression of ABCG2 and SOX2 in JM/WT and Figure 6 . Expression of AFP, CD133, MMP9 and SOX2 in JM/WT and JM/+6 cells by RT-PCR. JM/+6 cells by WB. creating a distinct subgroup of hepatoma cells (JM1/6+). The results from the current study indicate that progenitor cells Proliferation rate, sensitivity to Adriamycin and expression of could play a critical role in growth and biological behaviour invasive factors and factors linked to «stemness» of the of HCC development after surgery. This could also give new tumor (AFP, CD133, MMP9 and SOX2) were assessed. JM1 insights to the role of liver cancer stem cells. Results from this cell co-cultured with conditioned medium from WB-F344 study will be sent for publication shortly. (JM1/6+) showed more aggressive characters marked with the increased drug resistance (IC50) to Adriamycin and enhanced expression of AFP, ß-catenin, MMP9, CD133 and ABCG2 in The effect of LTX-315 peptide on vitro with RT-PCR (Figure 5) and WB (Figure 7). The in vivo experimental HCC tumors effect of HPCs on tumor growth was tested in 3 groups of LTX-315 is a cationic peptide derived from bovine lactoferricin Fischer rats undergoing 70% hepatectomy and implantation (LfcinB) that has been shown to have potent direct anti tumor of JM1 hepatoma cells: wild type (group TJ70), wild type effects in experimental sarcoma and lymphoma in rodents. co-cultured with HPC (Group TJ/+70) and wild type + HPC transplantation (group TJW70). Vigorous tumor development after WB-F344 injection in rats was evidenced as the increased morphological tumor size, metastasis and an enhanced expression of tumor biological markers including Cyclin D1 and MMP9.

Figure 7. Treatment and protective immune responses of LTX-315 in a Figure 8: Hallmarks of immunogenic celldeath adapted from rat hepatocellular carcinoma model Kroemer and Zitvogel 2012 53

The effect is attributable both to direct lysis of tumor cell opposed to controls, and that this is a quick process membranes and to an activation of T-cell. peaking at about one hour after exposure (figure 9). During 2012 we have cooperated with Gunnar Kvalheim and Meng Yu Wang at the Institute for Cellular therapy, Our group has been given a 3 year post-doctoral research Radium- hospitalet, Janne Nestvold at the Institute of grant from Health region South East for the project: Novel wAnatomy, and Prof. Øystein Rekdal and Baldur Immunotherapeutic principle for primary and secondary Sveinbjørnsson at the Uni-versity of Tromsø/Lytix biopharma. liver tumours – From bench to bedside. The intention of The experiments performed have been focused on: this project is, as the name implies, to bring our 1. The effect of intratumoral LTX-315 on subcutaneous HCC experiences and findings in treating experimental cancers tumors in the liver in rodents closer to clinical practice and a phase 2. The effect of transfer of specific immunity on growth of 1 trial. experimental tumor 3. The effect of preemptive vaccination with LTX-315+ tumor cell lysate before tumor implantation 4. The effect of intratumoral LTX-315 on intrahepatic HCC tumors

We have been able to show that treatment with LTX-315 kills experimental tumor in the subcutaneous tissue as well as in the liver (figure 8). Interestingly, this invokes a strong immune response, since rechallenge of the animals with tumors does not result in any tumor growth (Figure 7). The immune response appears to be t-cell dependent and invokes memory. Adoptive transfer of plenocytes from cured animals to total body irradiated naive animals resulted in protection from tumor when we tried to establish tumors in these animals s.c. and in the liver. Our focus now has been to investigate whether LTX-315 treatment causes an immunogenic cell death of cancer cells. This is an important theoretical concept that opens up a range of possibilities Immunogenic cell death. is according to Guido Kroemer and Laurence Zitvogel at Institut Gustav Roussy in Paris characterized by externalization of calreticuling, release of ATP and high mobility box group 1 (HMGB1) as illustrated in figure 9 (adapted from : Kroemer G, Galluzzi L, Kepp

JM1 Cells

HMGB-1

Figure 9: Western blot of HMGB1 release from HCC cells (JM1) exposed to LTX-315 for 60 min

O, Zitvogel L. Immunogenic Cell Death in Cancer Therapy. Annu Rev Immunol. 2013;31(1):51–72.doi:10.1146/an- nurev-immunol-032712-100008.)

We have now started experiments to test the hypothesis that LTX-315 invokes immunogenic cell death in the described manner. So far, we have looked at HMGB1 release and confirmed that HCC cells exposed to LTX-315 release HMGB1 to the supernatant of the cell culture as 54

Photo: OUH 55

Transplantation and Malignancy

Leader Svein Dueland, Consultant, MD, PhD (OUH)

Main members Aksel Foss, MD, Professor, PhD, MHA, FEBS (OUH/UiO) Marit Andersen, MSc, PhD (OUH) Pål-Dag Line, MD, PhD (OUH) Jihua Shi, MD (OUH) Rune Horneland, MD (OUH) Morten Hagness, MD, PhD-fellow (OUH) Jon M. Solheim, MD, PhD-fellow (OUH) Consultant Svein Dueland Associate members Einar Martin Aandahl, MD, PhD (OUH) Tormod Lund, MD, PhD (OUH) and stabilization of disease in different types of cancer, such as advanced breast and renal cancer that has previously progressed on other treatments. mTOR inhibitor has also Introduction shown increased survival in high risk metastatic renal cancer Organ transplantation requires lifelong immunosuppression. patients compared to interferon treatment. Accordingly, A side effect is increased post-transplant de novo malignancy. Rapamycin is an effective anticancer drug in addition to its During the last decades, the effectiveness of standard immunosuppressive effects. This supports the use of the drug immunosuppressants in allograft transplantation has improved in patients transplanted for cancer and in patients with de and so has the incidence of de novo cancer. A recent study novo post transplant malignancy. of 905 recipients of transplanted hearts, lungs, or both, has shown a 7.1 times increase in de novo cancers compared to the general population. Cancer-related death following The SECA study transplantation is increasing and accounts for 13% of post In 2006 we acquired an ethical approval (S-05409 Regional transplant mortality. Regulatory T cells (T regs) maintain self- Ethics Committee, Helse Sør-Øst) for a clinical pilot study tolerance to autoantigens and are involved in the pathogenesis (SECA-study) to investigate liver transplantation as treatment of various clinical conditions such as autoimmune diseases, option for selected patients with non-resectable liver metastases chronicviral infections and cancer. T regs appear more after colorectal carcinoma (CRC), using the mTOR inhibitor frequently in peripheral blood lymphocytes of cancer patients Rapamycin as standard immunosuppression from postoperative than healthy controls and, interestingly, it seems that high day 1. By March 2012, 23 patients have been transplanted levels of T regs are a prerequisite for allograft tolerance in the study. Based on data from the first 21 patients with following transplantation. By manipulation of the interaction observation time to November 2013, Kaplan-Meier estimates of between CD4+ CD25+ T regs and dendritic cells, it may 5 years overall survival (OS) post Lt was 56%. (Figure 1). become possible to influence host offence and defense in cancer and organ transplantation. In these aspects it is of Non-resectable colorectal liver metastases is a devastating particular interest that immunosuppressive drugs used in disease with a 5-year overall survival of about 10% from transplantation have both an anti-rejection and anti-neoplastic start of first line chemotherapy. Sixteen of the 21 patients activity. in the SECA study had progressed on first or later lines of chemotherapy at the time of inclusion in the study. Such Rapamycin (Sirolimus, RapamuneR) is an established drug patients have a median expected OS of 12 months or for preventing allograft rejection by blocking the intracellular less. Furthermore, 6 patients had progressed on all lines of pathway complex mTOR. It also appears that a Sirolimus- standard chemotherapy at time of liver transplantation. These based immunosuppression protocol has beneficial effects patients have a median overall survival of 5 months. on tumor recurrence and survival with an acceptable rate of rejection and toxicity in liver transplanted HCC patients. The results are also better than most studies for liver resection Rapamycin is a potent VEGF antagonist showing significant for colorectal metastases. Furthermore, it should be kept in anti-angiogeneic effects in addition to a direct inhibitory mind that all SECA patients were not eligible for liver resection. effect on tumor growth and proliferation. mTOR inhibitor has The overall 5-year survival in liver resection studies is generally shown clinical effect and objective radiological responses between 30-40%. 56

each patient were scored according to Fong clinical risk score for resectable colorectal liver metastases.

Metastatic recurrence of disease appeared in 20 of 21 patients. Median progression free survival (PFS) was only 10 months. However, most of the relapses were pulmonary and these progressed slowly untreated. Also, a significant proportion of the recurrences were accessible for surgery and, at follow up, seven patients (33%) had no evidence of disease after surgical treatment. Patients who developed lung metastases only had long overall survival after relapse. In contrast, patients with liver metastases after liver transplantation had a short overall survival from time of detection of liver metastases (Ref. 2). Five year overall survival Figure 1- Kaplan-Meier overall survival for patients included in the liver after detection of relapse was 53%. Hepatic tumor load prior transplantation group (SECA-study: red line), and the chemotherapy to liver transplantation, time from primary surgery to Ltx, group (NORDIC-VII-study: blue line). In A: all SECA patients (n=21) vs. and progressive disease on chemotherapy at time of liver all NORDIC VII patients (n=47). In B: all SECA patients (n=21) vs. the NORDIC VII patients with the longest overall survival (n=21). transplantation were identified as significant prognostic factors for survival (Figure 2). Based on these factors, a 5 year overall survival of 75% was obtained. A combination of these factors For larger hepatic tumor load, the results are even lower. The can likely be used as prognostic factors to select patients that patients in the SECA study had generally a large tumor burden have a high probability of 5 years as well as 10 years survival. (the median number of liver tumors was 8 (Range 4-40) and These strategies could further improve the outcome after Ltx. the median size of largest metastatic lesion was 4.5 cm (range 2.8- 13cm) (Ref. 1). All patients were deemed non-resectable even when available treatment options such as liver expansion The SECA-II study techniques, repeat resections and ex situ liver resections The SECA II trial was initiated in January 2011 as a after chemotherapy were taken into account. If resectable, randomized trial where Ltx was compared to best available expected 5 year OS of the study population based on the oncological treatment. Because of the large difference in clinical risk score (CRS) would have been only 25% when survival in the SECA-I study compared to all previously

Figure 2 – Preoperative factors affecting survival (A) Tumor diameter, (B) plasma CEA levels, (C) Time from surgery of primary tumor, (D) Response to chemotherapy at time of Ltx, partial response + stable disease vs. progressive disease. (E) Each negative factors in A-D were given a value of 1. Overall survival in relation to number of negative factors. 0-1 vs 2-3 vs 4. 57

reported chemotherapy studies, an amendment in the patients have a 5 years survival of only 0-30%. Patients protocol was approved by the Regional Ethics Committee in randomized to Ltx will continue chemotherapy until Ltx. November 2011. This changed the objective of the SECA-II Patients randomized to Ltx who have not received a Ltx within study to a randomized controlled trial between Ltx and hepatic 2 months after randomization due to donor deficiency, will be resection for patients with large but resectable colorectal taken off the transplantation list and included in the resection metastases (arm A of the study), and a non-randomized Ltx arm of the study. study for patients with non-resectable liver only metastases and favorable prognostic factors arm B (metachronous In part B, CRC patients with metachronous non-resectable disease), and arm C (synchronous disease) of the study. liver only metastases with at least 10% response according to RECIST criteria on first line chemotherapy will receive liver Study Objectives of SECA-II transplantation. Further inclusion criteria in part B is having a • In a randomized controlled trial, to explore whether liver primary pN0 disease and CEA<100 ng/ml at time of primary transplantation in selected patients with liver metastases diagnosis as well as at time of metastatic disease. This from CRC can obtain significant life extension and better selected group of patients is expected to obtain a 5 year health related quality of life compared to patients receiving survival of 60-80%. surgical resection. • To explore if patient selection according to risk factors from In part C, patients that may be included are patients with the SECA-I study and nomograms for outcome of relapse of liver metastases after second liver resection or colorectal cancer can define a subgroup of patients with a patients with liver metastases not eligible for curative liver 5 year survival of at least 60% or even cure from the resection, who have received first line treatment with at disease least 10% response (RECIST-criteria) on chemotherapy. The patients must be accepted for transplantation before Study endpoints progressive disease on ongoing chemotherapy and before Primary endpoint start of chemotherapy, no lesion should be larger than 10 cm, • Overall survival and the total number of lesions should be 30 or less. Patients may also be included if they have less than 10% response Secondary endpoints on chemotherapy, if they obtain at least 20% response after • Survival related to SECA-I risk factors and Memorial treatment with TACE (DEB-IRI) or by 90Y-spheres. Sloan-Kettering nomograms for recurrence after liver resection of metastatic CRC. At time of progressive disease all patients will be considered • Disease free survival (RECIST criteria and time to lung for the best treatment options, including further chemotherapy lesions of 10 mm) as well as surgical resections/radiation therapy/radiofrequency • Time to start of new treatment (change in strategy) ablation (RFA). • Quality of life (EORTC QLQ-C30), time to decrease in physical function and global health score At the end of 2013 two patients have been randomized in part • Registration of symptoms (NCI-CTCAE version 4.0) A of the SECA-II study and 7 patients with non-resectable • Diagnosis of other malignancies disease transplanted in part C of the study. • Determine nomogram scores of patients who survive for 5 years or more Characterization of the primary tumor, liver • Survival in relation to biological markers (plasma metastases and temporal T-cell immunity in the TIMP- 1 and TPA, circulating tumour cells and SECA-patients micro-metastatic disease in bone marrow biopsies at 1.1 Recent studies have addressed the importance of time of Ltx, different micro-arrays of liver biopsies, regulatory T-cells (T regs) in suppressing T-cell immunity to regulatory T-cells, infiltrating T-lymphocytes in liver tumor-associated antigens. There is evidence that T regs may biopsies and KRAS and BRAF mutations. be a main obstacle to successful tumor immunotherapy and • Correlation between recurrence/metastases and other that patients who exhibit allograft tolerance have increased biological parameters (proteomics, micro array, micro levels of T regs. Temporal levels of T regs are measured both metastases in bone marrow and lymph nodes and preoperatively and at fixed intervals postoperatively. Lymphoid circulating tumour cells) infiltration in tumors will be assessed, as this has shown to be an independent prognostic factor in CRC. Study population In part A of the study, patients with 6 or more resectable 1.2 Cytokine profiles will be assessed at Ltx and at liver lesions will be randomized to liver resection or liver defined time points at follow up. The cytokine profiles will be transplantation since surgical resection in this group of evaluated in regard to immunosuppressive treatment, to the 58

cancer disease, and in recognition of the liver as an active is collected for DTCs at Ltx, both at induction of anaesthesia immunological organ participating in local and systemic host and after manipulation of the liver in order to assess the defense. potential effect of manipulation of theliver. Samples from BM, portal- and systemic blood will be processed at the 1.3 Samples of tissue from the metastases as well as Department of Tumor Biology immediately after sampling, normal liver tissue are being collected and frozen (-700C) using standard procedures for mononuclear cell isolation for later investigations. The properties of this tissue and the i.e. immunobead rosetting and preparation of cytospins possible relation to the clinical course of the patients will be for immunocythochemical analysis. Tumor cells isolated explored. The exact modalities of analysis are not yet fully by immunobead rosetting will be further enriched using the established s de-freezing is a non reversible event that needs CellPick system followed by RNA/DNA isolation and molecular meticulous planning. The analyses will be performed after profiling. all patients have been included in the trial. There are several possible paths to follow: Mechanisms of regulatory T cell immune suppression and characterization of the regulatory T cell – effector T cell 1.4 Tumor gene expression profiles will be determined interaction in vitro, with special emphasis on the potential of in metastases and in normal liver tissue (oligonucleotide micro cytostatic drugs as immunosuppressants in SECA-patients array analysis) and in transplanted patients with de novo malignancy.

1.5 The role of disseminated tumor cells (DTC, The concept of transplantation of cancer patients raises micrometastases). The prognostic value of disseminated several immunologic considerations. 1) Immunosuppressive tumor cells in bone marrow (BM) has been investigated in treatment to prevent rejection may promote cancer several studies comprising patients with epithelial cancers recurrence. 2) High tumor load in the liver may lead to a such as breast-, colorectal- and gastric cancer. The relative immune suppression that possibly involves regulatory presence of DTC in BM seems to be of predictive value for T cells (T regs), thus the patients may need less intense the development of metastases not only in bones but also immunosuppressive treatment than liver transplant recipients in other distant organs, even in tumors that rarely develop treated on other indications. 3) Cytostatic drugs with skeletal metastases such as CRC. In breast cancer it has immunosuppressive effects may be an alternative to standard been shown that the detection of disseminated tumors immunosuppressive treatment for the SECA patients. cells in bone marrow by immunocythochemical methods is a strong and independent prognostic factor. Although not 1.1 The role of regulatory T cells in cancer completely unequivocal, possibly because of a certain degree immunity in SECA patients. Regulatory T cells have been of methodological concern and lack of standardization, shown to inhibit anti- tumor immune responses in various identification of DTC in BM also seems to predict disease studies in patients with malignant diseases such as colorectal free survival for patients with CRC. The incidence of DTC in cancer, ovarian cancer, breast cancer and melanoma. In this BM in patients with isolated liver metastases from CRC is low project we aim to investigate the suppressive role of regulatory when compared to patients with primary CRC or disseminated T cells in anti- tumor immune function in the SECA patients. CRC disease, but their presence is associated with a poor This cohort of patients will be compared to patients with lower prognosis. There is strong evidence, at least from breast tumor burden and who are eligible for local liver resection (the cancer, that DTC in the peripheral blood from patients with Ullevål patient cohort). Anti-tumor immune function will be N0 disease is correlated with a high incidence of systemic evaluated i) prior to transplantation; ii) four months after; iii) one relapse and mortality. In CRC the inconsistency of detection year after transplantation and; iv) at the time of recurrence. is larger and a more standardized methodology and sets of markers are called for. Nevertheless, it is probable that such a 1.2 Identification of down-stream cell-signaling correlation will be found using a combination of markers. pathways involved in T regs function. As a part of a broader strategy to test known anti-cancer drugs with Little is known about the molecular characteristics of immunosuppressive effects to prevent rejection and reduce isolated DTC cells. Recent technological advances involving risk of recurrence in the SECA-patients, we wish to assess enrichment of the relevant cell population, amplification the effects of known anti-cancer drugs, immunosuppressive procedures for DNA and RNA as well as high-throughput drugs and small molecular compounds (Src-, PI3-, MEK and molecular screening tools now render such characterization p38-kinase inhibitors) on T regs function and effector T cell feasible. Samples containing DTCs in BM, systemic- and function. portal blood opens the possibility to compare molecular characteristics of cells from these compartments with each Compounds with a preferential, different or selective effect other and with the hepatic metastatic tissue. Peripheral blood on either T cell subset, may point to important physiological 59

differences in the intracellular signal transduction machinery transplant recipients and other conditions. Further, the model with potential clinical value. Cell proliferation and intracellular does provide a tool for studying T regs suppression and close cytokine production will be assessed by flow cytometry in in on the core-mechanism of T regs suppression. CD4+ and CD8+ T cells.

1.3 The physiological role of regulatory T cells and MECA-study the mechanisms of action in interaction with effector Ocular malignant melanoma is a rare disease. Patients who have T cells. In this in vitro project, we will aim to elucidate the a relapse of the disease often have metastases to the liver. In physiologic role of regulatory T cells and the mechanisms of many patients liver is the only site of metastases. There are no action in interaction with effector T cells. Regulatory T cells treatment options that have shown increased survival in patients have been extensively studied for the last ten years. However, with liver metastases from ocular malignant melanoma. These several fundamental questions remain unanswered. The patients have a dismal prognosis with median overall survival of molecular interactions underlying the immunosuppressive 6-10 months in most studies. Two patients with ocular malignant activity of regulatory T cells are not known. Furthermore, the melanoma have been included in the study. One patient with temporal and cellular requirements of the immunosuppressive extensive disease at time of liver transplantation had a relapse activity of the regulatory T cells during the cell-cell interaction after a few months and died 6 months post transplant with with the effector T cells during an ongoing immune response relapse at multiple sites. The other patient is without a relapse have not been thoroughly dissected. and has no treatment related symptoms 12 months after liver transplantation. By selecting patients with limited liver tumor load and long disease free interval from diagnosis to development of Naturally Occurring Regulatory T cells liver metastases, a group of patients with long overall survival We have been addressing several basal properties of T regs post transplant may be defined. function by series of experiments studying the activation requirements and kinetic properties of human. References • 1. Hagness M, et al. Liver transplantation for non Regulatory T cells resectable liver metastases from colorectal cancer. Naturally occurring regulatory T cells are crucial in maintaining Ann. Surg. 2013; 257:800-806. self tolerance by dominant suppression of potentially • 2. Hagness M, et al. Patterns of recurrence after liver self-reactive T cells in peripheral tissues. The activation transplantation for non resectable liver metastases from requirements, the temporal aspects of the suppressive activity colorectal cancer. Ann.Surg. Oncol. 2013. Dec 27. and mode of action of human naturally occurring T regs are subjects of great controversy. Ex vivo, T regs display great Main Collaborators and variability in the suppressive activity dependent on donor Participating Institutions and experimental conditions. Here we show that by ex-vivo • Kjetil Tasken, Prof, MD, PhD, The Biotechnology anti-CD3/anti-CD28 stimulation and subsequent fixation in Centre of Oslo paraformaldehyde, T regs acquire full suppressive activity after • Bjørn Naume Prof, MD, PhD, Dep. of Oncology 6 hours of stimulation. However, the activation requirements • Kirsten Muri Boberg, MD, PhD, Dep. of Internal Medicine are heterogenous as 60% of healthy blood donors have fully • Ivar P. Gladhaug Prof, MD, PhD, Division of Surgery suppressive T regs without the need of ex-vivo stimulation. • Øystein Mathisen MD, PhD, Division of Surgery The suppressive activity of T regs operates in a contact- • Stein Bergan, Prof, MSc, PhD, Dep. of Clinical dependent manner that is not dependent on dendrittic cells Pharmacology or other antigenpresenting cells. The suppressive activity of • Gunhild M. Malandsmo, Prof MD, PhD, paraformaldehyde fixated T regs can be fully obliterated by Dep. of Tumor Biology trypsin treatment indicating that cytokine secretion is not • Gunnar Kvalheim, Prof, MD, PhD, Clinical Stem Cell required for suppressive activity, but that a cell-surface protein Laboratory is directly involved. By using inhibitors for down- stream signaling pathways involved in T cell activation, we were not International Collaborators: able to inhibit the upregulation of T regs mediated suppressive • Prof Olle Korsgren, MD, PhD, Department of Immunology, activity, indicating that this mechanism may be dependent Uppsala University Hospital, Sweden on several pathways that operate in concert. We recently • Prof Bo-Goran Ericzon, MD, PhD, Head of division published the results from these studies (1). An unexpected Transplantation Surgery, Center for Surgical Sciences, finding was the presence that 60% of healthy blood donor had Karolinska Institutet, Stockholm University pre-stimulated T regs. The heterogeneity discovered might • Prof Julia S. Johansen, MD, Ph.D. Herlev University have impact both on the immunosuppressive treatment of Hospital, Copenhagen University 60

Photo: Øystein H. Horgmo, UiO 61

Plastic and reconstructive surgery

Leader Kim Alexander Tønseth, MD, PhD, Head of department of Plastic and Reconstructive Surgery (OUH/UiO)

Scientific staff Hans Erik Høgevold, MD, PhD (OUH) Thomas Moe Berg, MD, PhD (OUH) Charles Filip, MD, PhD (OUH) Tyge Tindholdt, MD, PhD (OUH) Michael Matzen, MD (OUH) Torjus Wester, MD, PhD-student (OUH) Haris Mesic, MD (OUH) Michael Schneider, MD (OUH) Head of Department Kim Alexander Tønseth Alexander Head of Department Kim Therese Halvorsen Bjark, MD (OUH) Christian Sneistrup, MD (OUH) Tor Utheim, MD, PhD (OUH) reanimation with free tissue transfer with 3-dimentional Alexander Vigen, MD, PhD (Sykehuset Telemark) measurements. In addition our group is involved in a large Milorad Knezevic, MD (Bærum sykehus) randomized, double-blind, placebo-controlled, multicenter Cathrine Wold Knudsen, MD, PhD (Bærum Sykehus) trial looking at the effect of drug treatment on patients with Bell`s palsy.

Introduction Another new area in almost all surgical fields is the Plastic and reconstructive surgery is performed to restore introduction of regenerative medicine. With this method normal anatomy and function in patients with congenital new cells and tissue structures can be cultured to and acquired disorders, and in patients with tissue defects reconstruct various kinds of defects. after trauma or cancer surgery. During the last decades research in plastic and reconstructive surgery has led to Our research group has focused on the following areas: development of a large number of treatment options for patients with different kinds of disorders and defects. These 1. Microcirculation, wound healing and methods are often based on experimental research which microsurgery has been refined through clinical procedures. The main outcome is improved quality of life and patient satisfaction a. Microcirculation in random flaps on rats and the effect of prostaglandin E1 based on restoration of anomalies and dysfunction. In order to investigate the distribution of blood and

microcirculation in random flaps we have designed a rat

Research areas model that enables us to perform multiple measurements with laser Doppler perfusion imaging (LDPI)*. A random Free tissue transfer is a technique which has revolutionized flap is raised with width-length proportions of 1:5. The flap the field of reconstructive surgery over the past four is monitored in 5 equally sized squares on which a LDPI decades. Tissueflaps, based on small vascular vessels measurement is performed. The circulation is evaluated for (±1mm), can be transposed from a distant part of the body every square with regards to the blood distribution within (donor site) to a location where reconstruction is needed the flap. and the vessels can be anastomosed to the recipient artery and vein. A new area of free flap surgery was initiated with Several studies suggest a positive effect of prostaglandin the introduction of flaps based on perforator vessels. This E1 (PGE1) on the circulation of flaps. In the same rat model technique improved reconstruction by reducing donor site as described above we compare the circulation of random morbidity and by allowing new alternative flap designs. flaps with i.v. infusion of PGE1 alternative saline and There is a constant need for optimising the reconstruction perform LDPI measurement. The circulation is evaluated techniques to give the best possible result with minimal and comparison between the control and intervention disadvantages at the donor site. groups is performed and verified statistically.

Reconstructive surgery in patients with facial palsy and b. Microcirculation and wound healing related clinical problems is a very challenging task. We To resemble a clinical situation, we are using animals with are evaluating our new surgical techniques such as facial 62

skin structure and function similar to the human skin. may be valuable in diagnosing sepsis at an earlier phase, Pig skin has many similarities to human skin, including and may also have a potential in treatment guiding. Still histological appearance and wound healing ability. We microcirculatory monitoring is not used as a routine in any are using Norwegian pigs (Norsk landsvin) with weight clinical field to examine patients with sepsis or cardiogenic ± 25 kg in our studies. Microcirculation and histological shock. Our hypothesis is that systemic diseases will induce measurements are performed to evaluate the effect of microcirculatory changes everywhere in the organism. We different reconstructive procedures or other interventions also believe that we will see changes with our microscope on wound healing. To investigate microcirculation and before we see them in central hemodynamics and blood wound healing in an isolated setting, we use rat models as tests. In our last study we have induced fecal sepsis in described below. eight pigs and three controls. In the model they have been measured regularly from before sepsis induction to immediately before death by four different non-invasive c. Experimental perforator flaps and other rat models techniques in four areas of interest (two skin sites, eye and Dissection of the perforator flaps preserves the muscle tongue). The techniques are Laser Doppler techniques and minimizes the donor site morbidity. Nevertheless, (LDPM, LDPI*), spectroscope (for microvascular tissue the method may have undesirable effects on the muscle oxygenation) and an “in vivo” microscope. The analysis of because of damage of its innervation, blood supply or microcirculatory data are done by two blinded observers by direct injury when dissecting the perforator. We are and microcirculatory data will be compared to central performing studies to evaluate the surgical technique hemodynamics and bloodchemistry and immunology. to reduce this damage to a minimum using Wistar rats where two symmetrical abdominal lipocutaneous flaps are e. Microcirculation in human perforator flaps. raised around the midline. One side is used for intervention The deep inferior epigastric artery perforator (DIEAP) flap which is compared to the other side. After dissection, from the abdomen is one of the most suitable perforator the flaps are fixed to the original position by a continuous flaps used for breast reconstruction. This procedure suture. Microcirculation, flap viability, wound strength and has had a significant impact on the field of plastic and histological changes are measured preoperatively and reconstructive surgery, because of the high number during the first week after the operation. of women requiring breast reconstruction after cancer surgery. Based on the experimental research and clinical To continue improvements in both a clinical and scientific experience, our group is performing investigations to setting research using animal models is important. The optimize the reconstruction technique and to minimize the groin flap based on the superficial inferior epigastric artery donor morbidity. (SIEA) is well described. We have established a new model where the SIEA flap is transposed to the back of the rat Through better understanding of flap anatomy, physiology with good conditions for flap monitoring, without danger and better surgical technique the complication rate has of flap autocannibalisation. This model is used when decreased and the cosmetic outcome has improved. performing studies on microcirculation and histological However, partial flap necrosis is still a recurrent changes where we want to compare different interventions complication that can affect the final cosmetic result and on the flap or the animal over a longer period of time. the patient satisfaction. In most cases this can be avoided by discarding parts with unreliable capillary refilling after transferring the flap to the recipient site. We are performing d. Changes in microcirculation of the skin during quantitative evaluation of the perfusion zones and skin sepsis and cardiogenic shock areas with LDPI* in order to get a more exact picture of the Sepsis and cardiogenic shock are diseases with high microcirculatory differences in the DIEAP flap (fig. 1) and mortality. New equipment for monitoring central other skin flaps. hemodynamics has not improved survival as expected. In 1922 Freedlander studied skin microcirculation with a Laser induced fluorescence of (ICG) is a new sensitive microscope in patients with sepsis and found decreased method for evaluation of tissue perfusion. In another project capillary density and increased heterogeneity of capillary ICG videoangiografy is used to evaluate tissue perfusion of density. New studies with advanced microscopes the DIEAP flap during conventional abdominoplasty. The on patients with sepsis and cardiogenic shock show perforators are isolated to investigate their effect on the microcirculatory alterations in tongue mucosa in the microcirculation of the flap. way Freedlander described. In addition, alterations in erythrocyte flow velocity appear without changes in These studies will have major clinical impact on all surgical central hemodynamics. These characteristic changes procedures involving flap surgery in order to improve 63

surgical outcome of the reconstructed part and to reduce the donor morbidity.

*Measurements of microcirculation with laser Doppler perfusion imaging (LDPI)

Measurements of microcirculation are a central part of all our animal and human experiments. It is performed with a PIM 3.0 LDPI from Perimed, Stocholm, Sweden. The LDPI generates processes and displays colour-coded images of tissue perfusion. An optical scanner guides a low power laser beam stepwise to the tissue surface. The LDPI measures microcirculation to a depth of a few hundred micrometers. When the laser beam hit moving erytrocytes in the subepidermal plexus the light is backscattered and detected by a photodetector, this converts the light intensity to electrical signals and colour-coded images. Figure 1. Colour map of blood flow for the DIEAP flap processed by laser Doppler perfusion imaging. The flap has been divided into perfusion zones. The colour scale red-yellow-green-blue-black 2. Treatment of facial palsy represents perfusion values where red is the highest and black the a. 3-dimentional evaluation of outcome after surgical lowest value. reanimation of facial palsy.

Patients with persistent facial palsy are evaluated for 3. Regenerative medicine surgical treatment. One of the treatment options is Regenerative medicine is of great interest in plastic surgery dynamic reconstruction with cross-facial nerve grafting due to the possibility to reconstruct defects that have been and subsequent gracilis muscle transfer to the face. difficult or impossible to handle with traditional surgery. In cooperation with the department of plastic surgery Still, there are many aspects of the techniques which have in Vienna, Austria, we are analysing the 3-dimentional to be improved before they can replace the methods used outcome of these surgical procedures. to day. Our group has focused on the following areas: b. Medical treatment of Bell’s palsy a. Fat transplantation Bell’s palsy is an acute, idiopathic, unilateral peripheral Transplantation of autologous fat has been performed for facial palsy of unknown cause with an incidence of 30 many decades. Improvements in harvesting techniques per 100,000 inhabitants per year. Treatment of Bell’s and advantages such as availability and biocompatibility palsy has been a matter of debate for decades, and have led to its widespread application. In addition, potential treatment with corticosteroids and antivirals have been positive effects of regeneration on the surrounding cells the most commonly described treatments. To evaluate have been described. We are using fat transplantation in a the treatment effect of these two drugs, the Scandinavian number of different clinical conditions. However, there are Bell’s palsy study has been performed at 17 different clinics still areas where the use of fat transplantation has not been in Scandinavia. This randomized, double-blind, placebo- sufficiently described, and where the long term outcome controlled, multicenter trial included 839 patients with Bell’s of the procedure is unknown. We have investigated the palsy with a 12-month follow-up. Patients were randomized use of fat transplantation to the velum and pharynx (fig 2) to treatment with prednisolone plus placebo, valacyclovir in patients with velopharyngeal insufficiency (VPI). In these plus placebo, and placebo plus placebo. The primary patients there is an incomplete velopharyngeal closure endpoint and several secondary endpoints have already during speech, which may give rise to hypernasality. been published showing significantly higher recovery Fat transplantation to the velopharynx has proven to among prednisolone treated patients. We are still analyzing reduce the velopharyngeal gap during phonation when some secondary endpoints to evaluate which patient measured by MRI. In addition, blinded perceptual speech groups benefit the most of prednisolone. Furthermore, assessments have shown a significant reduction in we will evaluate the long term result of patients who were hypernasality. excluded from the study and did not received treatment. We will also analyze the outcome of pregnant patients with The survival of autologous fat transplantation cells is Bell’s palsy. described to be about 50%. In cooperation with the Norwegian Centre of Stem Cell Research, studies are 64

performed to investigate how this survival can be improved and to evaluate the effect of the transplanted fat on tumorigenesis in the recipient tissue. b. Cultured urothelial cells In reconstructive surgery within the genitourinary tract, autologous urothelial cells cultured in vitro could be of considerable value. To acquire urothelial cells for in vitro engineering of urothelium, bladder washings from adult patients as well as children can be performed. These samples will contain enough proliferative and colony- forming uroepithelial cells to regenerate urethral mucosa in vitro. The cultures could be expanded to confluent, stratified sheets, which can be used for reconstruction of the urethra in urogenital anomalies or in patients with other needs (transsexuals, reconstruction after trauma or cancer Figure 2 . Axial image preoperatively during phonation. surgery). The laboratory work will give a large improvement The velopharyngeal gap area is measured. in the clinical treatment of these patients. c. Cultured epidermal cells This project focus on how cultured epidermal epithelial cells can be (1) successfully cultured on electrospun scaffolds, (2) optimally stored within a small temperature interval, (3) successfully stored in a tailor-made medium, and (4) reliably transported under specific conditions. These methods might ultimately be applicable in the treatment of a number of diseases as for example burns, chronic wounds, stem call deficiency in the cornea, and more.

International collaborators • Prof. G. Kratz, Dep. of plastic surgery, Lindköping University Hospital, Sweden • Prof. S. Monstray, Dep. of plastic surgery, Ghent University Hospital, Belgium • Prof. M. Frey, Dep. of plastic surgery, Vienna University Hospital, Austria • Prof. W. Shaw, School of Dentistry, University of Manchester, United Kingdom • Prof. G. Semb, School of Dentistry, University of Manchester, United Kingdom • FRCS B.C. Sommerlad, Great Ormond Street Children Hospital, London, United Kingdom • Doc. L. Jonsson, Uppsala Univerity Hospital, Sweden • Prof. M. Hultcrantz, Karolinska University Hospital, Sweden • Prof. A. Pitkäranta, Helsinki University Hospital, Finland 65

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Photo: Øystein H. Horgmo, UiO

Photo: OUH 67

Projects guided by external project leaders 68

Neuroprotective effects of cannabidiol after hypoxic-ischemia in newborn pigs

Department of Pediatric Research, Oslo University Hospital – Rikshospitalet

Investigators: Håvard Tetlie Garberg, MD, PhD student (OUH, Vestre Viken HF) Marianne Ullestad Huun, MD, PhD student (OUH) Javier Escobar, PhD (OUH) Rønnaug Solberg, MD, PhD, (OUH, SivHF)

Professor Ola D. Saugstad, MD, PhD, FRCPE, (OUH) PhD student MD, Garberg, Tetlie Håvard

randomly assigned to the different study groups. 9,5 hours Background after end hypoxia the piglets are euthanised. Perinatal asphyxia is defined as severe lack of oxygen and perfusion to the fetus/newborn during/after delivery. It is a We aim to elucidate the hypothesised neuroprotective major cause of death and disability in newborns worldwide. effects of CBD by a wide range of analyses in blood (during One of the severe consequences of perinatal asphyxia is and at end of study), cerebrospinal fluid, urine, brain, hypoxic-ischemic encephalopathy (HIE). Currently, the only heart, lungs, liver and kidneys. Half the brain will be used effective treatment of newborns after perinatal asphyxia is for histological/immunohistological analysis as well as hypothermia (cooling) and there is a great need to further possible radiological studies. From the other half, samples investigate other possible neuroprotective strategies. from regions of specific interest will be snap-frozen in liquid nitrogen before further analysis (e.g MR-spectroscopy, Cannabidiol (CBD) is, second to tetrahydrocannabinol oxidative stress, inflammation, apoptosis). (THC), the major constituent of the cannabis plant. CBD is non psychogenic and believed to mitigate the Results negative effects of THC. CBD exerts its effect through an At present the experimental part of the study is about to be endogenous system called the endocannabinoid system. finished and we do not yet have any results. In the setting of neonatal hypoxic-ischemic encephalopathy (HIE) it is believed that cannabidiol, by modulation of the endocannabinoid system, mitigates several of the pathogenetic processes involved. Several studies have explored the possible neuroprotective effects of CBD with very promising results.

Aim To explore the neuroprotective effects of cannabidiol (CBD) in newborn pigs after hypoxic ischemic injury, both alone and in conjugation with hypothermia, to look for possible augmentation.

Experimental set up Newborn piglets (age 12-36h) are anesthetized, ventilated, and receive invasive vascular catheters for monitoring and blood sampling. They are then randomized to either hypoxia: breathing 8% oxygen until B.E -20 mmol/L or mean arterial blood pressure <20mmHg, or to sham (control group n=6). After reoxygenation they are then 69

Can docosahexaenoic acid (omega 3) augment the effect of hypothermia treatment following birth asphyxia? An experimental study in newborn piglets.

Department of Pediatric Research, Oslo University Hospital – Rikshospitalet

Investigators Marianne Ullestad Huun, MD, PhD student (UiO) Håvard Tetlie Garberg, MD, PhD student (UiO) Javier Escobar, PhD (OUH)

Rønnaug Solberg, MD, PhD (UiO, OUH) PhD Student MD, Huun, Marianne U. Ola Didrik Saugstad, Professor, MD, PhD (UiO, OUH)

Background Birth asphyxia is a major killer of newborns worldwide. There is little offered treatment to this group of patients amounting to almost 1 000 000 newborns/year globally. Advanced medical facilities have the option of performing hypothermia treatment. This significantly improves outcome for a substantial amount of newborns. Yet, far from all benefit from this treatment and the search is on for additional treatments. During asphyxia there is inadequate supply of glucose and oxygen to the tissues leading to intracellular devastating alterations with increased reactive oxygen species (ROS). This then leads to lipid peroxidation which is a contributor to apoptosis and increased inflammation in the asphyxiated cell. Docosahexaenoic acid (DHA) is a major constituent of the neural brain cell membrane. It is a modulator for keeping the intracellular environment stabile. We want to investigate whether DHA alone gives neuroprotection through less peroxidation and less apoptosis and whether there is an additional effect to hypothermia treatment.

Experimental set up Newborn piglets (age 12-36h) undergo hypoxia until Base Excess -20m mol/L or mean arterial blood pressure <20mmHg. They are randomly assigned to one out of four groups. A separate sham control group (n=6) goes through the same procedures and observation time (anesthesia, surgery, ventilation and sample collection), but does not undergo hypoxia. Tissues from 5 selected areas of the brain are snap frozen in liquid nitrogen and stored at -80 C until analysis. We also harvest tissues from lung, heart, liver and kidney.

Results The study is ongoing, and is planned to be finalized spring 2014. Post study calculations will include histopathology, magnetic resonance spectroscopy, lipid peroxidation and immunohistochemistry. 70

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Training Courses and Seminars (in Norwegian) 72

Courses

29. - 30.08.2013 Arbeidsmåter Kurset vil ta for seg de vanligst forekommende teknikker og Basalkurs i laparoskopisk kirurgi prosedyrer som brukes i nyfødtmedisinen. For hver teknikk/ prosedyre vil det først bli gitt en kort teoretisk Målgruppe introduksjon. Deretter vil teknikken bli demonstrert av Leger under utdanning i generell kirurgi og gastro- kurslærerne. Demonstrasjonen vil bli fulgt av øvelse på enterologisk kirurgi. modell. For de fleste teknikker vil det bli brukt anesteserte dyr som modeller. Det er lagt opp til at deltakerne skal få flere muligheter til repetisjon av ferdighetene. Kurset vil Læringsmål også omfatte en dag i simuleringslaboratorium for trening i Indikasjoner, operasjonsmetoder, resultater og teamarbeid. kvalitetssikring innen laparoskopisk kirurgi. Kurset vil omhandle teoretisk undervisning og praktiske øvelser på Temaoversikt simulatorer samt D-boks og pop-trainere. Følgende teknikker/prosedyrer vil bli undervist: Temaoversikt • Innleggelse av perifer venenål. Laparoskopets oppbygging og vedlikehold, fysiologiske • Innleggelse av perifer arterienål/-”kran”, inkludert effekter av pneumoperitoneum, anestesi ved laparoskopi, etablering av trykkmåling. laparoskopi ved akutt abdomen og galle. Praktiske øvelser • Kapillær blodprøvetagning. under supervisjon på modeller og simulatorer vil være en • Innleggelse av navlevenekateter. vesentlig del av kurset. • Innleggelse av navlearteriekateter. • Innleggelse av perkutant sentralvenekateter Kurskomite 1.”Peel away” teknikk Trond Buanes og Arne R. Rosseland (kursledere), 2. Seldinger teknikk. Anders Debes, Fredrik Halvorsen, Ronald Mårvik, • Innleggelse av thoraxdren Erik Trondsen, Bjørn Edwin, Ole-Christian Olsen, 1. Vanlig rett dren med mandreng Marianne Berg, Vivi Bull Stubberud. 2. “Pig-tail” dren med Seldingers teknikk 3. Bruk av drenasjekammeret. Kurssted • Spinalpunksjon. Institutt for kirurgisk forskning - Oslo universitetssykehus, • Endotrakeal intubering. Rikshospitalet. • Blærepunksjon (suprapubisk) • Lokalbehandling av ekstravasering. 11. - 15.03.2013 + 21.-24. 10.2013 • Innleggelse av intraossøs nål for infusjon.

Introduksjon til nyfødtmedisinske Kurskomite teknikker og prosedyrer Thor Willy Ruud Hansen og Thomas Rajka (ledere). Komitémedlemmer: Jannicke Andresen, Tor Einar Calisch, Målgruppe Gro Furset Flatekval, Astri Lang, Sverre Medbø, Leger under utdanning i barnesykdommer med særlig Arild Rønnestad, Vigdis Skaug, Inger Elisabeth Silberg, fokus på de helt ferske og uerfarne. Kurset kan også ha Hans Jørgen Stensvold, Vivi Bull Stubberud, interesse for andre kolleger i det barnemedisinske faget Per Arne Tølløfsrud, Bjørn Øglænd. Komitéen utgår fra hhv som arbeider ved avdelinger der visse typer nyfødt- Nyfødtavdelingen, Kvinne- og Barneklinikken; medisinske prosedyrer utføres svært sjelden. Videre vil Utdanningssenteret; Institutt for Kirurgisk Forskning og kurset være relevant for anestesileger som arbeider ved Avdeling for Komparativ Medisin, alle Oslo avdelinger der akuttbehandling av nyfødte ivaretas av Universitetssykehus. anestesiolog. Kurssted Læringsmål Institutt for kirurgisk forskning - Oslo universitetssykehus, Deltakerne skal i løpet av kurset få demonstrert Rikshospitalet. teknikker og selv få utføre praktiske øvelser. Målsettingen er å gi deltakerne grunnleggende kunnskaper og ferdigheter slik at de med større trygghet kan utføre prosedyrer på syke nyfødte. 73

Illustrasjonsfoto, Mikrokirurgikurs

27. - 29.11.2013 Thorako-/laparoskopisk kirurgi

Målgruppe Leger under utdanning i generell kirurgi og gastroenterolo- gisk kirurgi.

Læringsmål Indikasjoner, operasjonsmetoder, resultater og kvalitetssikring innen laparoskopisk kirurgi. Kurset vil om- handle teoretisk undervisning og praktiske øvelser på simu- latorer samt demonstrasjonsoperasjoner.

Temaoversikt Laparoskopets oppbygging og vedlikehold. Fysiologiske effekter av pneumoperitoneum. Anestesi ved laparoskopi. Laparoskopi ved akutt abdomen. Cholecystecomi Antirefluxkirurgi. Laparoskopisk cancerkirurgi. Laparoskopi i urologien. Thoracoskopisk kirurgi. Praktiske øvelser under supervisjon, på modeller og anest- esert gris vil være en vesentlig del av kurset.

Kurskomite Arne R. Rosseland (kursleder), Trond Buanes, Bjørn Edwin, Ole Christian Olsen, Marianne Berg, Vivi Bull Stubberud, og Stefan Kutzsche

Kurssted Institutt for kirurgisk forskning – Oslo universitetssykehus, Rikshospitalet. 74

Seminars

Date Name Title Department

1/24/2013 Espen Enerly Forebygging av HPV relatert kreft i Norge Kreftregisteret

Neil3 DNA glycosylase improves survival after Avdeling for Mikrobiologi, 3/14/2013 Gunn Hildrestrand induced myocardial infarction in mice Seksjon for forskning, OUH/UiO

HIT it - 4 x 4, 1 x 4 eller 30 sekunder med NTNU, Institutt for sirkulasjon 4/4/2013 Trine Karlsen intervalltrening? og bildediagnostikk

4/18/2013 Hans Nielsen Hauge Hvem vil være våre pasienter i sykehus i fremtiden? Leder eHelse, Helse Sør-Øst

Inflammatory changes in stress and trauma. Anestesiavd., Akershus 4/25/2013 Bård Lundeland Impact on Toll-like receptor 4 signalling Universitetssykehus

9/12/2013 Aphirak Juthajan Fluorescensmikroskopets muligheter Carl Zeiss AS

FSAP; Factor VII activating protease a novel mediator Professor, Inst for medisinske 9/26/2013 Sandip Kanse in fibroproliferative inflammatory diseases” basalfag. Avd. for biokjemi.

A neonatal mouse model for spinal cord injury, Norwegian Center for Stem Cell 10/10/2013 Joel Glover adaptive plasticity, and human stem cell screening Research

Rat Natural Killer Cells, Role in defence against Liste- 10/17/2013 Hamid Shegarfi ISR, RH, OUH ria MONOCYTOGENES.

Spesialist ØNH-sykdommer, 11/28/2013 Karl F. Nordfalk Postoperativ svimmelhet etter cochleaimplantasjon OUH, RH 75

Publications and PhD-Theses 76

Publications 2009 - 2013

2013 9. Gravning J, Smedsrud MK, Omland T, Eek C, Skulstad H, Aaberge L, Bendz B, Kjekshus J, Mørkrid L, Edvardsen T. Sensitive troponin assays and N-terminal 1. Askevold ET, Aukrust P, H Nymo S, Lunde IG, Kaasbøll pro-B-type natriuretic peptide in acute coronary OJ, Aakhus S, Florholmen G, Ohm IK, Strand ME, syndrome: Prediction of significant coronary lesions and Attramadal H, Fiane A, Dahl CP, Finsen AV, Vinge LE, long-term prognosis. Am Heart J. 2013;165(5):716-24 Christensen G, Yndestad A, Gullestad L, Latini R, Masson S, Tavazzi L, The GISSI-HF Investigators, 10. Hasselberg NE, Edvardsen T, Petri H, Berge KE, Leren Ueland T. The cardiokine secreted Frizzled related TP, Bundgaard H, Haugaa KH. Risk prediction of protein 3, a modulator of Wnt-signalling, in clinical and ventricular arrhythmias and myocardial function in Lamin experimental heart failure. J Intern Med (Online A/C mutation positive subjects. Europace December 2013) (Online September 2013) 2. Behnan J, Isakson P, Joel M, Cilio C, Langmoen IA, 11. Haugaa KH, Grenne BL, Eek CH, Ersbøll M, Valeur N, Vik-Mo EO, Badn W. Recruited brain tumor-derived Svendsen JH, Florian A, Sjøli B, Brunvand H, Køber L, mesenchymal stem cells contribute to brain tumor Voigt JU, Desmet W, Smiseth OA, Edvardsen T. Strain progression. Stem Cells (Online December 2013) echocardiography improves risk prediction of ventricular arrhythmias after myocardial infarction. 3. Berg T, Jonsson L. Peripheral neuropathies: JACC Cardiovasc Imaging. 2013;6(8):841-50 corticosteroids and antivirals in Bell palsy. Nat Rev Neurol. 2013;9(3):128-9 12. Joel M, Sandberg CJ, Boulland JL, Vik-Mo EO, Langmoen IA, Glover JC. Inhibition of tumor formation 4. Cornez I, Joel M, Taskén K, Langmoen IA, Glover JC, and redirected differentiation of glioblastoma cells in Berge T. EGF signalling and rapamycin-mediated mTOR a xenotypic embryonic environment. Dev Dyn. inhibition in glioblastoma multiforme evaluated by 2013;242(9):1078-93 phospho-specific flow cytometry. J Neurooncol. 2013;112(1):49-57 13. Kaarbø M, Crane DI, Murrell WG. RhoA Regulation of Cardiomyocyte Differentiation. ScientificWorldJournal. 5. Dannevig I, Solevåg AL, Sonerud T, Saugstad OD, 2013, 491546 Nakstad B. Brain inflammation induced by severe asphyxia in newborn pigs and the impact of alternative 14. Knatten CK, Hviid CH, Pripp AH, Emblem R, Bjørnland resuscitation strategies on the newborn central nervous K. Inflammatory esponser after open and laparoscopic system. Pediatr Res. 2013;73(2):163-70 Nissen fundoplication in children: a randomized study. Pediatr Surg Int (Online November 2013) 6. Estensen ME, Remme EW, Grindheim G, Smiseth OA, Segers P, Henriksen T, Aakhus S. Increased Arterial 15. Lancellotti P, Badano LP, Lang RM, Akhaladze N, Stiffness in Pre-eclamptic Pregnancy at Term and Early Athanassopoulos GD, Barone D, Baroni M, Cardim N, and Late Postpartum: A Combined Echocardiographic Gomez de Diego JJ, Derumeaux G, Dulgheru R, and Tonometric Study. Am J Hypertens. Edvardsen T, Galderisi M, Gonçalves A, Habib G, 2013;26(4):549-56 Hagendorff A, Hristova K, Kou S, Lopez T, Magne J, de la Morena G, Popescu BA, Penicka M, Rasit T, 7. Gravning J, Ahmed MS, Qvigstad E, Krobert KA, Rodrigo Carbonero JD et al. Normal Reference Ranges Edvardsen T, Moe IT, Hagelin EM, Sagave J, Valen G, for Echocardiography: rationale, study design, and Levy FO, Osnes JB, Skomedal T, Attramadal H. methodology (NORRE Study). Eur Heart J Cardiovasc Connective Tissue Growth Factor / Ccn2 Imaging. 2013;14(4):303-8 Attenuates-Adrenergic Receptor Responsiveness and Cardiotoxicity by Induction of G Protein-coupled 16. Lauritzen KH, Morland C, Puchades M, Holm-Hansen Receptor Kinase-5 (Grk5) in Cardiomyocytes. S, Hagelin EM, Lauritzen F, Attramadal H, Mol Pharmacol. 2013;84(3):372-383 Storm-Mathisen J, Gjedde A, Bergersen LH. Lactate Receptor Sites Link Neurotransmission, Neurovascular 8. Gravning J, Ahmed MS, von Lueder TG, Edvardsen T, Coupling, and Brain Energy Metabolism. Cereb Cortex Attramadal H. CCN2/CTGF attenuates myocardial (Online May 2013) hypertrophy and cardiac dysfunction upon chronic pressure-overload. Int J Cardiol. 17. Lekva T, Bollerslev J, Sahraoui A, Scholz H, Bøyum H, 2013;168(3):2049-2056 Evang JA, Godang K, Aukrust P, Ueland T. Thioredoxin 77

Interacting Protein Is a Potential Regulator of Glucose Huitfeldt HS, Zhang SJ, Line PD. RAF-targeted therapy and Energy Homeostasis in Endogenous Cushing’s for hepatocellular carcinoma in the regenerating liver. Syndrome. PLoS One. 2013;8(5):e64247 J Surg Oncol. 2013;107(4):393-401

18. Moe IT, Pham TA, Hagelin EM, Ahmed MS, Attramadal 27. Shi JH, Scholz H, Huitfeldt HS, Line PD. The effect H. CCN2 exerts direct cytoprotective actions in adult of hepatic progenitor cells on experimental hepatocellu cardiac myocytes by activation of the PI3-kinase/ lar carcinoma in the regenerating liver. Akt/GSK-3 signaling pathway. J Cell Commun Signal. Scand J Gastroenterol (Online November 2013) 2013;7(1):31-47 28. Solberg R, Escobar J, Arduini A, Torres-Cuevas I, Lahoz 19. Murrell W, Palmero E, Bianco J, Stangeland B, Joel M, A, Sastre J, Saugstad OD, Vento M, Kuligowski J, Paulson L, Thiede B, Grieg Z, Ramsnes I, Skjellegrind Quintás G. Metabolomic Analysis of the Effect of HK, Nygård S, Brandal P, Sandberg C, Vik- Postnatal Hypoxia on the Retina in a Newly Born Piglet Mo E, Palmero S, Langmoen IA. Expansion of Model. PLoS One. 2013;8(6):e66540 multipotent stem cells from the adult human brain. PLoS One. 2013;8(8):e71334 29. Vik-Mo EO, Nyakas M, Mikkelsen BV, Moe MC, Due-Tønnesen P, Suso EM, Sæbøe-Larssen S, 20. Roy S. Focal hydrothermal ablation: preliminary Sandberg C, Brinchmann JE, Helseth E, investigation of a new concept. Cardiovasc Intervent Rasmussen AM, Lote K, Aamdal S, Gaudernack G, Radiol. 2013;36(4):1112-9 Kvalheim G, Langmoen IA. Therapeutic vaccination against autologous cancer stem cells with mRNA- 21. Russell K, Eriksen M, Aaberge L, Wilhelmsen N, transfected dendritic cells in patients with glioblastoma. Skulstad H, Gjesdal O, Edvardsen T, Smiseth OA. Cancer Immunol Immunother. 2013;62(9):1499-509 Assessment of wasted myocardial work - A novel method to quantify energy loss due to un-coordinated left ventricular contractions. Am J Physiol Heart Circ 2012 Physiol. 2013;305(7):H996-H1003

22. Ruud TE, Gundersen Y, Krohn CD, Sveen O, Aasen AO. 1. Axelsson S, Berg T, Jonsson L, Engström M, Kanerva Effects of Infliximab and Hydrocortisone on In Vitro M, Stjernquist-Desatnik A. Bell’s palsy - the effect of Cytokine Responses after Stimulation with Lipopo- prednisolone and/or valaciclovir versus placebo in lysaccharide. Surg Infect (Larchmt). 2013;14(1):30-4 relation to baseline severity in a randomised controlled trial. Clin Otolaryngol 2012;37(4):283-90 23. Sahraoui A, Jensen KK, Ueland T, Korsgren O, Foss A, Scholz H. Anakinra and tocilizumab enhance survival 2. Berg T, Bylund N, Marsk E, Jonsson L, Kanerva M, and function of human islets during culture: implications Hultcrantz M, Engstrom M. The Effect of Prednisolone for clinical islet transplantation. Cell Transplant (Online on Sequelae in Bell’s Palsy. Arch. Otolaryngol. April 2013) Head Neck Surg. 2012;138(5):443-447

24. Sandberg CJ, Altschuler G, Jeong J, Strømme KK, 3. Bilbija D, Gravning JA, Haugen F, Attramadal H, Valen Stangeland B, Murrell W, Grasmo-Wendler UH, G. Protecting the heart through delivering DNA Myklebost O, Helseth E, Vik-Mo EO, Hide W, Langmoen encoding for heme oxygenase-1 into skeletal muscle. IA. Comparison of glioma stem cells to neural stem cells Life Sci 2012;91(17-18):828-36 from the adult human brain identifies dysregulated Wnt- signaling and a fingerprint associated with clinical 4. Boulland JL, Leung DS, Thuen M, Vik-Mo E, Joel M, outcome. Exp Cell Res. 2013;319(14):2230-43 Perreault MC, Langmoen IA, Haraldseth O, Glover JC. Evaluation of intracellular labeling with micron-sized 25. Sarvari SI, Haugaa KH, Zahid W, Bendz B, Aakhus S, particles of iron oxide (MPIOs) as a general tool for in Aaberge L, Edvardsen T. Layer-Specific Quantification vitro and in vivo tracking of human stem and progenitor of Myocardial Deformation by Strain Echocardiography cells. Cell Transplant 2012;21(8):1743-59 May Reveal Significant CAD in Patients With Non-ST Elevation Acute Coronary Syndrome. JACC Cardiovasc 5. de Vecchi A, Nordsletten DA, Remme EW, Imaging. 2013;6(5):535-44 Bellsham-Revell H, Greil G, Simpson JM, Razavi R, Smith NP. Inflow typology and ventricular geometry 26. Shi JH, Liu SZ, Wierød L, Scholz H, Anmarkrud JA, determine efficiency of filling in the hypoplastic left heart. Ann Thorac Surg 2012;94(5):1562-9 78

6. Edvardsen T. The continued discovery of left atrial increases their ability to induce classical monocyte function. Eur Heart J Cardiovasc Imaging migration. J Innate Immun 2012;4(2):176-86 2012;13(3):203-4 16. Kolseth IB, Førland DT, Risøe PK, Flood-Kjeldsen S, 7. Edvardsen T, Haugaa KH. Republished article: Imaging Ågren J, Reseland JE, Lyngstadaas SP, Johnson E, assessment of ventricular mechanics. Dahle MK. Human monocyte responses to Postgrad Med J 2012;88(1036):105-12 lipopolysaccharide and 9-cis retinoic acid after laparoscopic surgery for colon cancer. Scand J Clin 8. Estensen ME, Grindheim G, Remme EW, Swillens A, Lab Invest 2012;72(8):593-601 Smiseth OA, Segers P, Henriksen T, Aakhus S. Systemic arterial response and ventriculo-arterial 17. Marsk E, Bylund N, Jonsson L, Hammarstedt L, interaction during normal pregnancy. Engström M, Hadziosmanovic N, Berg T, Hultcrantz M. Am J Hypertens 2012;25(6):672-7 Prediction of nonrecovery in Bell’s palsy using Sunnybrook grading. Laryngoscope 2012;122(4):901-6 9. Fossdal G, Vik-Mo EO, Sandberg C, Varghese M, Kaarbø M, Telmo E, Langmoen IA, Murrell W. Aqp 9 18. Nguyen TT, Espinoza AW, Hyler S, Remme EW, and brain tumour stem cells. ScientificWorldJournal D’hooge J, Hoff L. Transmural strain distribution across 2012; 15176 the cardiac wall and its dependency on measurement site. IEEE INTERNATIONAL ULTRASONICS 10. Gravning J, Orn S, Kaasbøll OJ, Martinov VN, SYMPOSIUM (IUS) 2012 Manhenke C, Dickstein K, Edvardsen T, Attramadal H, Ahmed MS. Myocardial Connective Tissue Growth 19. Nguyen TT, Espinoza AW, Hyler S, Remme EW, Factor (CCN2/CTGF) Attenuates Left Ventricular D’hooge J, Hoff L. Effect of myocardial perfusion on Remodeling after Myocardial Infarction. end-systolic radial strain at the apex. IEEE PLoS One 2012;7(12): e52120 INTERNATIONAL ULTRASONICS SYMPOSIUM (IUS) 2012 11. Harvey E, Audigé L, Herscovici D, Agel J, Madsen JE, Babst R, Nork S, Kellam J. Development and validation 20. Nguyen TT, Espinoza AW, Remme EW, D’hooge J, Hoff of the new international classification for scapula L. Transmural Myocardial Strain Distribution Measured fractures. J Orthop Trauma2012;26(6):364-9 at High Spatial and Temporal Resolution. 2011 IEEE INTERNATIONAL ULTRASONICS SYMPOSIUM (IUS) 12. Haugaa KH, Goebel B, Dahlslett T, Meyer K, Jung C, 2012;696-699 Lauten A, Figulla HR, Poerner TC, Edvardsen T. Risk assessment of ventricular arrhythmias in patients with 21. Odland HH, Brun H, Sejersted Y, Dalen M, nonischemic dilated cardiomyopathy by strain Edvardsen T, Saugstad OD, Thaulow E. Myocardial echocardiography. J Am Soc Echocardiogr longitudinal peak systolic acceleration (pSac): 2012;25(6):667-73 relationship to ejection phase, pressure, and contractility. Echocardiography 2012;29(5):541-53 13. Heir S, Årøen A, Løken S, Holme I, Engebretsen L, Reinholt FP. Cartilage repair in the rabbit knee: 22. Odland HH, Kro GA, Edvardsen T, Thaulow E, mosaic plasty resulted in higher degree of tissue filling Saugstad OD. Impaired diastolic function and but affected subchondral bone more than disruption of the force-frequency relationship in the microfracture technique: a blinded, randomized, right ventricle of newborn pigs resuscitated with 100% controlled, long-term follow-up trial in 88 knees. Knee oxygen. Neonatology 2012;101(2):147-53 Surg Sports Traumatol Arthrosc 2012;20(2):197-209 23. Opdahl A, Remme EW, Helle-Valle T, Edvardsen T, 14. Hviid CV, Erdem JS, Kunke D, Ahmed SM, Kjeldsen SF, Smiseth OA. Myocardial relaxation, restoring Wang YY, Attramadal H, Aasen AO. The matri-cellular forces, and early-diastolic load are independent proteins ‘cysteine-rich, angiogenic-inducer, 61’ and determinants of left ventricular untwisting rate. ‘connective tissue growth factor’ are regulated in Circulation 2012;126(12):1441-51 experimentally-induced sepsis with multiple organ dysfunction. Innate Immun 2012;18(5):717-26 24. Remme EW, Hoff L, Halvorsen PS, Opdahl A, Fosse E, Elle OJ. Simulation model of cardiac three dimensional 15. Kolseth IB, Agren J, Sundvold-Gjerstad V, Lyngstadaas accelerometer measurements. SP, Wang JE, Dahle MK. 9-cis retinoic acid inhibits Med Eng Phys 2012;34(7):990-8 inflammatory responses of adherent monocytes and 79

25. Russell K, Eriksen M, Aaberge L, Wilhelmsen N, 2011 Skulstad H, Remme EW, Haugaa KH, Opdahl A, Fjeld JG, Gjesdal O, Edvardsen T, Smiseth OA. A novel 1. Ahmed MS, Gravning J, Martinov VN, von Lueder TG, clinical method for quantification of regional left Edvardsen T, Czibik G, Moe IT, Vinge LE, Øie E, Valen ventricular pressure-strain loop area: a non-invasive G, Attramadal H. Mechanisms of novel cardioprotective index of myocardial work. functions of CCN2/CTGF in myocardial Eur Heart J 2012;33(6):724-33 ischemia-reperfusion injury. Am J Physiol Heart Circ Physiol 2011;300(4):H1291-302 26. Sarvari SI, Gjesdal O, Gude E, Arora S, Andreassen AK, Gullestad L, Geiran O, Edvardsen T. Early postoperative 2. Andersen GØ, Ueland T, Knudsen EC, Scholz H, left ventricular function by echocardiographic strain is a Yndestad A, Sahraoui A, Smith C, Lekva T, Otterdal K, predictor of 1-year mortality in heart transplant Halvorsen B, Seljeflot I, Aukrust P. Activin A levels are recipients. J Am Soc Echocardiogr 2012;25(9):1007-14 associated with abnormal glucose regulation in patients with myocardial infarction: potential counteracting 27. Smedsrud MK, Sarvari S, Haugaa KH, Gjesdal O, effects of activin A on inflammation. Ørn S, Aaberge L, Smiseth OA, Edvardsen T. Duration Diabetes 2011;60(5):1544-51 of myocardial early systolic lengthening predicts the presence of significant coronary artery disease. 3. Bjørnstad JL, Sjaastad I, Nygård S, Hasic A, Ahmed J Am Coll Cardiol 2012;60(12):1086-93 MS, Attramadal H, Finsen AV, Christensen G, Tønnessen T. Collagen isoform shift during the early 28. Smiseth OA, Russell K, Skulstad H. The role of phase of reverse left ventricular remodelling after relief echocardiography in quantification of left ventricular of pressure overload. Eur Heart J 2011;32(2):236-45 dyssynchrony: state of the art and future directions. Eur Heart J Cardiovasc Imaging 2012;13(1):61-8 4. Czibik G, Gravning J, Martinov V, Ishaq B, Knudsen E, Attramadal H, Valen G. Gene therapy with hypoxia- 29. Stanisic M, Lyngstadaas SP, Pripp AH, Aasen AO, inducible factor 1 alpha in skeletal muscle is Lindegaard KF, Ivanovic J, Ilstad E, Konglund A, cardioprotective in vivo. Life Sci 2011;88(11-12):543-50 Sandell T, Ellingsen O, Saehle T. Chemokines as markers of local inflammation and angiogenesis in 5. Dannevig I, Solevåg AL, Wyckoff M, Saugstad OD, patients with chronic subdural hematoma: Nakstad B. Delayed onset of cardiac compressions in a prospective study. Acta Neurochir (Wien) cardiopulmonary resuscitation of newborn pigs with 2012;154(1):113-20; discussion 120 asphyctic cardiac arrest. Neonatology 2011;99(2):153-62 30. Stanisic M, Aasen AO, Pripp AH, Lindegaard KF, Ramm-Pettersen J, Lyngstadaas SP, Ivanovic J, 6. Edvardsen, T. Can modern echocardiographic Konglund A, Ilstad E, Sandell T, Ellingsen O, Sæhle T. techniques predict drug-induced cardiotoxicity?. Local and systemic pro-inflammatory and J Am Coll Cardiol 2011;57(22):2271-2 anti-inflammatory cytokine patterns in patients with chronic subdural hematoma: a prospective study. 7. Edvardsen T, Haugaa KH. Imaging assessment of Inflamm Res 2012;61(8):845-52 ventricular mechanics. Heart 2011;97:1349-1356

31. von Lueder TG, Gravning J, How OJ, Vinge LE, Ahmed 8. Gjesdal O, Remme EW, Opdahl A, Skulstad H, Russell MS, Krobert KA, Levy FO, Larsen TS, Smiseth OA, K, Kongsgaard E, Edvardsen T, Smiseth OA. Aasum E, Attramadal H. Cardiomyocyte-restricted Mechanisms of abnormal systolic motion of the inhibition of G protein-coupled receptor kinase-3 interventricular septum during left attenuates cardiac dysfunction after chronic bundle-branch block. Circ Cardiovasc Imaging pressure overload. Am J Physiol Heart Circ Physiol 2011;4(3):264-73 2012;303(1):H66-74 9. Haugaa KH, Bergestuen DS, Sahakyan LG, 32. Østerholt HC, Dannevig I, Wyckoff MH, Liao J, Skulstad H, Aakhus S, Thiis-Evensen E, Edvardsen T. Akgul Y, Ramgopal M, Mija DS, Cheong N, Longoria C, Evaluation of right ventricular dysfunction by myocardial Mahendroo M, Nakstad B, Saugstad OD, Savani RC. strain echocardiography in patients with intestinal Antioxidant protects against increases in low molecular carcinoid disease. J Am Soc weight hyaluronan and inflammation in asphyxiated Echocardiogr. 2011;24(6):644-50 newborn pigs resuscitated with 100% oxygen. PLoS One 2012;7(6):e38839 80

10. Haugaa KH, Edvardsen T, Amlie JP. Prediction of timing of conversion from external fixation to Life-Threatening Arrhythmias – Still an Unresolved secondary intramedullary nailing in experimental tibial Problem. Cardiology 2011;118:129–137 fractures. J Orthop Res. 2011;29(1):126-30

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PhD-Theses 2013

Petter K. Risøe Petter K. Anders Opdahl Marit Kristine Smedsrud Jørgen Gravning Petter K. Risøe Anders Opdahl Marit Kristine Smedsrud Jørgen Gravning January 31, 2013 March 8, 2013 April 10, 2013 May 23, 2013 Inflammatory Modulation in Mechanisms of left Assessment of incipient Cardioprotective Mechanisms in Sepsis – Role of Adenylyl ventricular early-diastolic global myocardial Ischemia-Reperfusion Injury and Cyclases. untwisting and lengthening. dysfunction by speckle Heart Failure - Experimental and tracking echocardiography. Clinical Studies Dissecting the Functional Effects of Connective Tissue Growth Factor (CTGF/CCN2) in the Heart.

Mrinal Joel Thomas Helle-Valle Sebastian Imre Sarvari Thomas Helle-Valle Sebastian Imre Sarvari Mrinal Joel May 24, 2013 September 18, 2013 December 10, 2013 New Non-invasive Methods Detection of subtle Characterization of stem for Quantification of Regional myocardial alterations by cell-enriched glioblastoma and Global Myocardial Func- echocardiographic cells in vitro and in xeno- tion in the Normal and Isch- techniques for improved typic tissue environments. emic Left Ventricle. prognostic information in patients with heart disease.

2012 ÅGREN Joanna Sara Maria VARTDAL Trond Modulation of Monocyte and macrophage endotoxin Early Assessment of Infarct Size and Left Ventricular responses; Focus on nuclear receptor LXR. Remodeling by Strain Echocardiography.

RUSSELL Kristoffer Engh 2011 Novel methods for assessing left ventricular dyssynchrony SIGURDSEN, Ulf Eirik Wangsvik: Tibial bone healing: and myocardial function. Experiments with external fixation and intramedullary nailing. 86

SOLBERG, Rønnaug: Resuscitation of the newborn. 2007 An experimental study of toxic effects of supplementary ØVERLAND, Gunhild: Kupffer cell activation in systemic oxygen in newborn piglets. inflammation.

HEIR, Stig: Focal Cartilage Defects in the Knee. MYHRE, Anders E: Gram-positive endotoxemia and modulation of the innate immune response. VIK-MO, Einar Osland: Propagation and characterization of stem like cells from brain tumors - establishment of a clinical protocol for immunotherapeutic targeting of tumor 2006 stem cells in glioblastomas. BØRKE, Wenche Bakken: Myocardial injury and performance in hypoxaemic neonates: Effects of oxygen DALEN, Marit Lunde: Hypothermia and room air resus- and carbon dioxide during reoxygenation. An experimental citation in NT2-N neurons, immature rats and newborn pigs. study in newborn pigs.

LARSEN, Geir Arne: Early mechanisms of brain ischemia. 2010 An experimental study in isolated rat neurons. DIMMEN, Sigbjørn: Effects of cox inhibitors on bone and tendon healing. LYSEGGEN, Erik: Assessment of myocardial viability and reperfusion by tissue Doppler echocardiography. HAUGAA, Kristina Hermann: Prediction of cardiac ventri- cular arrhythmias by echocardiography in patients at risk. LINDENSKOV, Paal HH: Inflammation in experimental meconium aspiration syndrome.

2009 VINGE, Leif Erik: Myocardial G protein-coupled receptor VON LUEDER, Thomas G: Molecular Mechanisms of kinases – Distinct isoform specificities at receptors as basis Myocardial Hypertrophy and Heart Failure. for diverse roles in regulation of cardiac function. Experimental Studies on Cardiac G Protein-Coupled Receptor Signaling with Emphasis on Endothelin-1. PAASCHE, Joachim Dericq: Molecular Mechanisms of Regulation and Intracellular Trafficking of the Endothelin LUND, Tormod: Strategies to counteract-cell loss in Receptors. pancreatic islet transplantation. AHMED, Muhammad Shakil: Autocrine/Paracrine BAINS, Ravi: Isoflurane and sevoflurane: Effects on Regulators of Myocardial and Pulmonary Remodeling in mitochondrial function in the rat and human brain. Heart Failure.

ØLSTØRN, Håvard: Neural stem, cells from the adult SKULSTAD, Helge: New insights into the function of normal human central nervous system and brain tumors. and ischaemic myocardium. Properties in vivo following xenotransplantation. LYNG, Kristin: Experimental induction of fetal brain damage in pigs as a model of infection-induced brain damage 2008 in humans. VARGHESE, Mercy: Isolation and Characterization of Stem Cells from the Adult Human Central Nervous System and Brain Tumors. 2005 CASTELLHEIM, Albert: Meconium aspiration syndrome, ANDRESEN, Jannicke: Neuroprotective effects of systemic inflammatory response, and the complement intravenous nicotine administration. An experimental study system. in newborn piglets. MUNKEBY, Berit Holthe: Resuscitation of the newborn with WANG, Yun Young: Novel targets in modulation of inflam- 100% O2 - detrimental effects on the brain, lungs and matory responses in experimental endotoxemia and sepsis: heart. focus on the liver X receptor. 87

SOLÅS, Anne-Beate: Resusciation of the newborn – with MEDBØ, Sverre: Pulmonary hemodynamics during or without supplemental oxygen? neonatal hypoxemia and reoxygenation. Role of oxygen, endothelin-1 and nitric oxide in piglets. URHEIM, Stig: Quantification of myocardial function by Doppler echocardiography. HYSTAD, Marit: Origins of increased plasma concentrations of Atrial –and Brain natriuretic peptid in heart failure. ÅRØEN, Asbjørn: Cartilage Injuries and The Repair Process. 2000 ROY, Sumit: Interventional radiologic management of deep 2004 vein thrombosis. TØLLØFSRUD, Per Arne: Meconium Aspiration Syndrome – An experimental study in newborn piglets. ØIE, Erik: Autocrine/Paracrine Regulatory mechanisms and myocardial remodeling during ischemic heart failure. MOE, Morten Carstens: Isoflurane and Sevoflurane An experimental study in rats with special emphasis on – Mechanisms of action in the presynaptic terminal the role of myocardial endothelin and adrenomedulin. – An experimental study in the rat and human brain. KJEKSHUS, Harald: Vasoactive mediators and vascular function. 2002 EDVARDSEN, Thor: Assessment of regional myocardial KLINGE, Randi: Cardiac natriuretic peptides as endocrine dysfunction by tissue Doppler imaging and strain Doppler markers of cardiac function. A study of changes in plasma echocardiography. concentrations during development and treatment of heart failure and angina pectoris KUTZSCHE, Stefan P: Effects of hypoxemia and reoxygenation on cerebral microcirculation, nitric oxide, hydrogen peroxide and excitatory amino acid 1999 concen-trations. An experimental study in newborn pigs. BJØRNLAND, Kristin: Importance of proteolytic enzymes in tumor cell invasion. SCHOLZ, Tim: Studies on activation of the humoral defence system and inflammatory cells in liver MADSEN, Jan Erik: Neural regulation of bone repair. transplantation. Experimental studies of fracture healing, posttraumatic osteopenia and bone graft incorporation in the rat. JØRGENSEN, Pål Foyn: Immunomodulation and innate immune responses in experimental sepsis. A study with GUNDERSEN, Yngvar: Modulation of nitric oxide values emphasis on the monocytemacrophage system. in endotoxaemia. An experimental study with special emphasis on hepatic circulation and function. QI, Wei: A-and B-type cardiac natriuretic peptides-markers of atrial pressure and left ventricular hypertrophy. HOSAKA, Junro: Preclinical evaluation of the temporary FRØEN, Fredrik: Effects on inflammation and nicotine on venous “spring filter” for prevention of pulmonary embolism. apnea, respiratory response and hypoxic-ischemic brain damage in the newborn piglet. ODDEN, Jan Petter: Cerebral and Ocular Blood Flow responses in the posthypoxemic newborn piglet.

2001 FIANE, Arnt: Pig-to-human xenotransplantation. WANG, Jacob: Cellular responses involved in sepsis Inhibition of hyperacute rejection. caused by gram-positive bacteria and fungal pathogens. LEHNE, Gustav : P-glycoprotein- a target for circumvention BORTHNE, Kjell: The role of 1- og -adenoceptors in of multidrug resistance in cancer. sympathetic inotropic myocardial control. HVAAL, Kjetil: Attenuation of ischaemic skeletal muscle necrosis. 88

KAASTAD, Trine Sand: Osteoporotic fractures relative to TRONDSEN, Erik: Treatment of gallstone disease. Aspects strength of bone and muscle. Experimental rat models for of Surgical Treatment in the early laparoscopic era. studying means of prevention. LARSEN, Morten: Isoflurane: Effects on synaptic release ÅSBERG, Anders: Effects of cyclosporine A on micro- and uptake of glutamate and GABA. *vascular function and endothelin-1 in renal transplant recipients. 1996 YU, Xiang-Qing: Hemodynamic and metabolic effects of BENTDAL, Øystein H.: Immune responses in protein- nitric oxide. An experimental study in newborn piglets with energy-malnutrition. A study in patients with anorexia acute lung injury. nervosa, carcinoma of cardia or oesophagus.

SATAS, Saulias: Pharmacokinetics, posthypoxic STORM, Hanne: Beta-endorphin in victims of sudden infant hypothermia and cerebral microdialysis in the newborn pig. death syndrome.

KROHN, Claus D: Pathophysiological aspects on KONGSGÅRD, Erik: Charateristics of radiofrequency postoperatively, drained, untreated blood for autologous current catheter ablation. An experimental study in vitro transfusion. and in vivo.

STEINE, Kjetil: Mechanisms of early diastolic left ventricular BUØ, Laila: Proteolytic mechanisms in tumor spread. intracavitary flow: a study of flow patterns by colour Studies on the role of serine proteinases in human M-mode doppler echocardiography and measurements gastrointestinal cancer. of intraventricular driving pressures. HAUGSTAD, Tor: Mechanisms of amino acid release during ANFINSEN, Ole-Gunnar: Radiofrequency catheter ablation: cerebral ischemia. a study concerning electrode configuration, lesion size and potential complications. AUNE, Arne Kristian: The muscle stabilized knee. Significance for anterior cruciate ligament injury and HAALAND, Kirsti: Hypothermia as treatment of asphyxia in reconstruction. a newborn piglet model. LINE, Pål-Dag: Laser Doppler assessment of limb RISE, Ingunn: Cerebral blood flow during high intracranial perfusion. Physiological and methodological aspects. pressure and blood loss.

1995 1998 STUGAARD, Marie: Intraventricular filling pattern and FEET, Bjørn: Resucitation of hypoxic newborn piglets with diastolic function. Clinical and experimental studies by color different oxygen concentration M-mode doppler echocardiography.

STEEN, Torkel: Noninvasive evaluation of left ventricular MOEN, Atle: Circulatory effects of surfactant replacement. diastolic function. A critical investigation of two proposed An experiental study in depleted newborn piglets. methods.

GRØNDAHL, Tor Ø.: Ion fluxes during cerebral ischemia SANDERUD, Jon: Oxygen radical induced pulmonary with special emphasis on measurements of intracellular hypertension. calcium. An experimental study in vitro. ROOTWELT, Terje: Resuscitation with 21% or 100% oxygen.

1997 HEGSTAD, Elisabeth: Amino acids in the human brain. KARLSRUD, Tove Sigstad: Human kininogens as A study of possible roles in synaptic transmission and multifunctional proteins. Studies focusing on their function ischemia with special reference to glutamate and GABA. as cysteine protease inhibitors. 89

1994 BØRSUM, Tone: Investigations on cultured human en- VEDDENG, Odd J.: Cardiac mechanical effects of selective dothelial cells. Cytotoxic effects of low density lipoprotein. positive end-expiratory pressure ventilation. Protein composition and surface structure.

SKJELDAL, Sigmund: Acute Skeletal Muscle Ischemia. KARLSEN, Steinar Johan: Acute effects of extracorporeal Microcirculation and histological changes in rat hindlimbs. shock waves on kidney function and morphology.

NORDSLETTEN, Lars: Muscle protection against fracture. A biomechanical study in the rat tibia. 1990 EMBLEM, Ragnhild: Straight ileonal anastomosis in SAMDAL, Frode: Suction-assisted lipectomy. A clinical and patients with familial poliposis and ulcerative colitis. experimental study. MYRENG, Yngvar: Relaxation and filling of the left RISØE, Cecilie: Changes in splanchnic vascular ventricle assessed by doppler echocardiography. capacitance during experimental and clinical heart failure. A clinical and experimental investigation.

STOLTENBERG, Lauritz: Biochemical, immunological and experimental studies of sudden infant death syndrome 1989 (SIDS). HALL, Christian: Experimentally induced acute ischemic heart failure. A study in dogs of some endocrine and tissue blood flow responses, with special reference to effects of 1993 angiotensin converting enzyme inhibition. SOLHEIM, Erik: Effects of Bioerodible Polyorthoester on Heterotopic and Orthopic Bone Induction in rats. STORDAHL, Arvid: Water-soluble contrast media in obstructed and in ischemic small intestine. A clinical and KIRKEBY, Ole Jørgen: Incorporation of Bone Grafts. experimental study. An experimental study in rats.

POULSEN, Jan Petter: Extracelluar oxypurines during 1988 hypoxia and reoxygenation. ROSSELAND, Arne R: Endoscopic papillotomy. A clinical and experimental study. KROHG-SØRENSEN, Kirsten: Laser Doppler Flowmentry in Evaluation of Colonic Perfusion. 1987 QIAO, Zhi-Gui: Measurement of electroderman and BRØNDBO, Kjell: Reinnervation of laryngeal muscles. micro-circulatory activities and responses in the human palm. An experimental study in dogs.

1992 SOLHEIM, Ludvig Fjeld: Influence of nonsteroidal anti-in- KONGSGAARD, Ulf E: Changes in the plasma protease flammatory drugs on bone. A mechanical and biochemical systems during open heart surgery. study with acetylsalicylic acid and naproxen in rats. PINHOLT, Else Marie: Experimental alveolar ridge augmen- tation studies. MATHISEN, Svein R: Healing of synthetic and biological protheses in the cardiovascular system. An experimental STIRIS, Tom Arne: Ocular blood flow in the newborn. study. Effects of lights and blood gas variations on retinal and choroidal blood flow in the new born piglet. 1986 RØNNINGEN, Helge: Bone ingrowth in porous fiber 1991 titanium Experimental investigation of ingrowth for the RIDDERVOLD, Fridtjov: Atrial mechanical and endocrine purpose of fixation of weight-bearing skeletal implants. function during changes in heart rate and loading. Clinical and experimental studies. GLENNÅS, Anne: Experimental studies on resistance to certain cytotoxic effects of three gold-compounds, with special reference to metallothionein. 90

BARTH, Elin: Bioactive, non-porous and bioinert, porous HJORT, Erling F: An experimental study on hematogenous implant materials: comparison of two principles for pyelonephritis in the rat and its possible bearing on human cementless prosthesis fixation. pathology.

1985 1979 ENDRESEN, Liv: Metallothionein. Experimental studies on HENRIKSEN, Tore: Interactions of serum lipoproteins with its protective function against certain anticancer agents. human endothelial cells in culture.

ENDRESEN, Gerhard KM: Immunological studies on HOLEN, Jarle: The quantification of flow obstructions in human platelet proteins. Findings in relation to the mitral flow channel with doppler echo-cardiography. auto-immune diseases. ENGESÆTER, Lars B: Biomechanical and biochemical SMITH-ERICHSEN, Nils: Septicemia evaluated by means effects of antibiotics on bone and skin. of chromogenic peptide substrate assays. A retrospective study in man. LILLEAASEN, Per: Hemodilution in open-heart surgery. WILLE, Sven Øivind: Numerical models of arterial blood SCHRADER, Harald: Dynamics of intracranial expanding flow. masses, An experimental study with particular reference to the Cushing response. 1978 AASEN, Ansgar O: Activities and inhibition of proteases 1983 found in plasma during endotoxin shock. An experimental LÆRUM, Frode: In vivo and in vitro studies of contrast study in dogs. media effects in lower limb phlebography. BERG, Knut Joachim: Effects of acetylsalicylic acid on renal BAKKA, Arne: Studies on possible functions of metallothionein. function.

SLAATTELID, Olav: Studies in hypothermic kidney 1982 perfusion. BREKKE, Inge B: Transplantation of the duct-occluded rat pancreas. Long-term endocrine function and metabolic effects. 1977 SAUGSTAD, Ola Didrik: Hypoxanthine as an indicator of tissue hypoxia. A study of plasma, cerebro-spinal fluid and 1981 brain tissue concentrations. AMLIE, Jan P: Mode of actions of digitoxin and digoxin on cardiac electrophysiology and inotropy. KVEIM, Morten: The acetate ion as a source of base. Experimental and clinical studies. GULDVOG, Ivar: Gastric acid and pepsin secretion in dogs. The role of vagal innervation. ENGE, Ivar: Angiography in regeneration of the liver. An experimental and clinical study. FRENG, Atle: Transversal growth of the maxillary base fol- lowing resections of the mid-palatial suture - a biometrical and morphological study in the man and the cat. 1976 BERGAN, Anstein: Aspects of bilirubin metabolism in EKELAND, Arne: Influence of calcitonin on healing and normal and cholestatic dogs. intact bone and skin. A biomechanical and biochemical study in rats. 1975 BLIX, Arnoldus Schytte: The elicitation and regulation of 1980 the cardiovascular responses to diving. WIE, Henrik: Effects of cyclophosphamide on connective tissues. An experimental study in rats. 91

SUDMANN, Einar: Vital microscopy of bone remodelling in 1969 rabbit ear chambers. SANDER, Sten: The uptake of oestradiol in normal breast tissue and in induced breast cancer of the rat. HØIE, Johan: Hemodynamic changes following acute thrombininduced intravascular coagulation. An experimental study in dogs.

LANGELAND, Norvald: Effects of oestradiol on bone collagen metabolism. An experimental study in female rats.

1974 NUSTAD, Kjell: The relationship between urinary and kidney kallikrein in the rat.

BENUM, Pål: Autogenous transplantation of apophyseal cartilage to osteochondral defects of joints. An experimental study in dogs.

1973 JAKOBSEN, Arnt: Rabbit anti-rat lymphocyte serum. A study on the influence of the antigen dose and immunization schedule on some in vitro characteristics and in vivo immunosuppressive potency.

NESBAKKEN, Ragnar: Aspects of free fatty acid metabolism during induced hypothermia.

1972 SEMB, Bjarne KH: Cardiac transplantation. An experimental study in dogs.

1970 OFSTAD, Egil: Formation and destruction of plasma kinins during experimental acute hemorrhagic pancreatitis in dogs.

TVETER, Kjell: Studies on selective uptake and metabolism of testerone-3H in the prostate and the seminal vesicles of the rat.

TRIPPESTAD, Arne: Aspects of host defence reactions during experimental intestinal strangulation obstruction in rats.

UNHJEM, Olav: Studies on the interaction between androgen and macromolecular components of the rat ventral prostate. 92

Egil Amundsen Lecture

Professor Markus W. Büchler has been awarded the Amundsen medal and diploma by professor Ansgar O. Aasen

22.09.06 | Professor Iver A. Langmoen 11.04.11 | Professor Wolfgang G. Junger Department of Neurosurgery, Ullevål Hospital, Beth Israel Deaconess Medical Center, Harvard Medical University of Oslo, Norway School, Boston, USA “Purinergic control of immune cell ”Stem cells in the Adult Human Brain” function”

23.04.12 | Professor Paolo Macchiarini 27.04.09 | Professor Olle Korsgren Advanced Center For Translational Regenerative Medicine, Department of Immunology, Genetics and Pathology, Karolinska Institutet, Karolinska University Hospital, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden ”Clinical Islet Transplantation: an emerging therapy for Stockholm, Sweden patients with type I diabetes” “Stem cells based organ transplantation”

18.10.10 | Professor Henrik Kehlet 13.05.13 | Professor Dr. Dr. h.c. Markus W. Büchler Section of Surgical Pathophysiology, Rigshospitalet, Department of Surgery, Heidelberg University, Germany Copenhagen University. ”Fast-track surgery – what is in it “Clinical trials in surgery; towards evidence” and why should we do it?”