Rasmus Sejersten Granulocyte‐Colony Stimulating Factor Ripa Therapy to Induce Neovascularization Granulocyte in Ischemic Heart Disease ‐ Colony Rasmus Sejersten Ripa Stimulating Factor Therapy to Induce Neovascularization in Ischemic Heart Cover: Phoenix depicted in the book Disease Bilderbuch für Kinder by F.J. Bertuch (1747‐1822) ISBN 978‐87‐994776‐0‐9 FACULTY OF HEALTH SCIENCES UNIVERSITY OF COPENHAGEN Granulocyte-Colony Stimulating Factor Therapy to Induce Neovascularization in Ischemic Heart Disease Rasmus Sejersten Ripa Forsvaret finder sted onsdag den 30. november 2011, kl. 14 præcis i Medicinsk Museion, Bredgade 62, København Forsvarsleder: Professor, overlæge, dr.med. Torben V. Schroeder Formand for bedømmelsesudvalget: Professor, overlæge, dr.med. Niels Borregaard Universitetets officielle opponenter: Professor, overlæge, dr.med. Erling Falk og Professor, overlæge, ph.d., dr.med. Hans Erik Bøtker Denne afhandling er i forbindelse med anførte tidligere offentliggjorte artikler af Det Sundhedsvidenskabelige Fakultet ved Københavns Universitet antaget til offentligt at forsvares for den medicinske doktorgrad. København, den 1. august 2011. Ulla Wewer, Dekan This dissertation is based on the following original publications. These publications are referred to by their roman numerals: (I) Ripa RS, Wang Y, Jørgensen E, Johnsen HE, Hesse B, Kastrup J. Intramyocardial Injection of Vascular Endothelial Growth Factor-A165 Plasmid Followed by Granulocyte-Colony Stimulating Factor to Induce Angiogenesis in Patients with Severe Chronic Ischemic Heart Disease. Eur Heart J 2006;27:1785-92. (II) Ripa RS, Wang Y, Goetze JP, Jørgensen E, Johnsen HE, Tägil K, Hesse B, Kastrup J. Circulating Angiogenic Cytokines and Stem Cells in Patients with Severe Chronic Ischemic Heart Disease – Indicators of Myocardial Ischemic Burden? Int J Cardiol 2007;120:181-187. (III) Ripa RS, Jørgensen E, Baldazzi F, Frikke-Schmidt R, Wang Y, Tybjærg-Hansen A, Kastrup J. The Influence of Genotype on Vascular Endothelial Growth Factor and Regulation of Myocardial Collateral Blood Flow in Patients with Acute and Chronic Coronary Heart Disease. Scand J Clin Lab Invest 2009;69:722-728. (IV) Ripa RS, Jørgensen E, Wang Y, Thune JJ, Nilsson JC, Søndergaard L, Johnsen HE, Køber L, Grande P, Kastrup J. Stem Cell Mobilization Induced by Subcutaneous Granulocyte-Colony Stimulating Factor to Improve Cardiac Regeneration After Acute ST- Elevation Myocardial Infarction. Result of the Double-Blind Randomized Placebo Controlled STEMMI Trial. Circulation 2006;113:1983-1992. (V) Ripa RS, Haack-Sørensen M, Wang Y, Jørgensen E, Mortensen S, Bindslev L, Friis T, Kastrup J. Bone Marrow-Derived Mesenchymal Cell Mobilization by Granulocyte-Colony Stimulating Factor after Acute Myocardial Infarction: Results from the Stem Cells in Myocardial Infarction (STEMMI) Trial. Circulation 2007;116(11 Suppl):I24-30. (VI) Ripa RS, Nilsson JC, Wang Y, Søndergaard L, Jørgensen E, Kastrup J. Short- and Long-Term Changes in Myocardial Function, Morphology, Edema and Infarct Mass Following ST-Segment Elevation Myocardial Infarction Evaluated by Serial Magnetic Resonance Imaging. Am Heart J 2007;154:929-36. (VII) Lyngbæk S, Ripa RS, Haack-Sørensen M, Cortsen A, Kragh L, Andersen CB, Jørgensen E, Kjær A, Kastrup J, Hesse B. Serial in vivo Imaging of the Porcine Heart After Percutaneous, Intramyocardially Injected 111Indium-Labeled Human Mesenchymal Stromal Cells. Int J Cardiovasc Imaging 2010; 26:273-84. ISBN 978-87-994776-0-9 - 2 - PREFACE The thesis is based on research performed during my gratitude to Jens Christian Nilsson, MD, PhD and Lars time as research fellow at the Department of Søndergaard, MD, DMSc who taught me cardiovascular Cardiology, Rigshospitalet, from 2003 to 2007. MRI, and to Birger Hesse, MD, DMSc and Andreas Kjær, I am indebted to Jens Kastrup, MD, DMSc who MD, DMSc who introduced me to nuclear medicine. introduced me to cardiac neovascularization therapy. Finally, I want to express my appreciation to all the His continuous interest in my work and never failing research fellows and study nurses at the 14th floor. optimism despite negative results encouraged me to Space was limited, but we have had plenty of coffee, move on. It has always been a pleasure working with fruitful discussions and exiting trips to conferences Jens. Erik Jørgensen, MD has provided brilliant ideas around the world - it was never boring. A special thanks and constructive criticism in all phases of our trials. His to Jens Jakob Thune, MD, PhD who besides conducting clinical approach and broad view of things when I all the STEMMI echoes also was a daily source of tended to be lost into the details has improved the work inspiration, and to Matias Lindholm, MD, PhD who substantially. initiated the “kageklub” and explored Boston shopping The remaining members of the “Cardiac Stem Cell with me. Laboratory”, Wang Yongzhong, MD, PhD; Mandana This work would not have been possible without the Haack-Sørensen, MSc; Lene Bindslev, MSc, PhD; Tina generous donations by: The Danish Heart Foundation; Friis, MSc, PhD; Stig Lyngbæk, MD; Steen Mortensen, Danish Stem Cell Research Doctoral School; Faculty of Tech; and Sandra Miran, RN have provided an Health Sciences, Copenhagen University; Hjertecentrets innovative and enthusiastic atmosphere. Forskningsfond, Rigshospitalet; The Danish Medical I was originally introduced to research during medical Research Council; Lundbeck Fonden; Raimond og school by Marie Luise Bisgaard, MD. The years of basic Dagmar Ringgård-Bohn’s Fond; Aase og Ejnar laboratory work has been a steady foundation for my Danielsens Fond; Erik og Martha Scheibels Legat; Arvid continued interest in research. Later, I became involved Nilssons Fond; Fonden af 17.12.1981; and Georg in clinical research during my stay as research fellow at Bestles Fond. Duke University Medical Center, North Carolina, USA. Galen S. Wagner, MD; Peter Clemmensen, MD, DMSc; Rasmus Sejersten Ripa and Peer Grande, MD, DMSc were devoted mentors February 2011 (and pleasant travel companions) and have since been following my work with great interest and inspiring comments. Til Maria, I wish to thank all my co-authors who contributed with Alvin og Josefine their expertise to complete the studies. A special - 3 - CONTENTS BACKGROUND AND AIM G-CSF FOR STEMI Ischemic heart disease 7 Clinical trials 25 Biological intervention in myocardial Safety of treatment with G-CSF regenerative medicine 7 after STEMI 26 Protein therapy 7 Why does G-CSF not improve Gene therapy 8 myocardial function following STEMI? 27 Stem cell therapy 8 Time to G-CSF 27 Granulocyte-Colony Stimulating Factor 9 The G-CSF dose 28 Pathogenesis of myocardial regeneration 11 Differential mobilization of cells types 28 Bone marrow-derived stem- and Homing of mobilized cells progenitor cells 13 to the myocardium 29 Potential mechanisms of Conclusion 30 cell-based therapy 15 Aim and hypothesis 16 MYOCARDIAL RECOVERY AFTER STEMI 31 TRIAL DESIGN FROM BED TO BENCH 32 Measures of efficacy 16 GENERAL CONCLUSIONS AND FUTURE Myocardial volumes and function 16 DIRECTIONS 34 Myocardial perfusion 17 Conclusion 18 SUMMARY IN DANISH – DANSK RESUMÉ 36 Ethical considerations in trial design 18 REFERENCES 37 G-CSF FOR CHRONIC ISCHEMIA G-CSF and VEGF gene therapy 19 Safety of G-CSF to patients with chronic ischemia 21 Does G-CSF reduce myocardial ischemia? 21 Angiogenesis or arteriogenesis 21 Patient population 22 Homing of bone marrow-derived cells into ischemic myocardium 23 - 4 - Granulocyte-Colony Stimulating Factor Therapy to Induce Neovascularization in Ischemic Heart Disease Rasmus Sejersten Ripa ABSTRACT Cell based therapy for ischemic heart disease has the compared to placebo treatment. In subsequent potential to reduce post infarct heart failure and chronic analyses, we found significant differences in the types ischemia. of cells mobilized from the bone marrow by G-CSF. This Treatment with granulocyte-colony stimulating factor could explain why intracoronary injections of (G-CSF) mobilizes cells from the bone marrow to the unfractionated bone marrow-derived cells have more peripheral blood. Some of these cells are putative stem effect that mobilization with G-CSF. or progenitor cells. G-CSF is injected subcutaneously. A number of other factors could explain the neutral This therapy is intuitively attractive compared to other effect of G-CSF in our trial compared to previous cell based techniques since repeated catheterizations studies. These factors include timing of the treatment, and ex vivo cell purification and expansion are avoided. G-CSF dose, and study population. It is however, Previous preclinical and early clinical trials have remarkable that the changes in our G-CSF group are indicated that treatment with G-CSF leads to improved comparable to the results of previous non-blinded myocardial perfusion and function in acute or chronic studies, whereas the major differences are in the ischemic heart disease. control/placebo groups. We found that ejection fraction, The hypothesis of this thesis is that patient with wall motion, edema, perfusion, and infarct size all ischemic heart disease will benefit from G-CSF therapy. improve significantly in the first month following ST- We examined this hypothesis in two clinical trials with segment myocardial infarction with standard guideline G-CSF treatment to patients with either acute treatment (including acute mechanical re- myocardial infarction or severe chronic ischemic heart vascularization), but without cell therapy. This