Mechanisms of chronic allograft injury in a heart transplant model

1. Mechanisms of chronic allograft injury in a heart transplant model Karl Lemström*, Antti Nykänen*, Jussi Tikkanen, Rainer Krebs, Mikko Keränen, Raimo Tuuminen, Alireza Raissadati, Simo Syrjälä, Janne Jokinen*, Jussi Ropponen*, Petri Koskinen Transplantation Laboratory, Haartman Institute, University of Helsinki & HUSLAB, Helsinki University Central Hospital. *Department of Cardiothoracic Surgery, Helsinki University Central Hospital.

2. Leading causes of death for adult heart transplants performed between 1992-2008 Stehlik et al. J Heart Lung Transplant 2010;29:1089

3. Graft failure - loss of functional microvasculature • Primary graft failure • immediate loss of functional microvasculature • brain-death • hypothermic preservation • warm ischemia • “marginal donors” • reperfusion injury • Late graft failure • gradual loss of functional microvasculature • acute rejection, cellular and antibody-mediated • CMV infection

4. Loss of microvascular circulation may be fundamental cause of chronic rejection

5. Pre-operative ischemia-reperfusion injury enhances acute and chronic rejection and impairs allograft survival new immunosuppressive drugs effectively prevent acute rejection however, they do not prevent microvascular dysfunction and signals that mediate IRI and that bridge innate and adaptive immune responses therefore, new strategies that target at other pathways than T cell proliferation are required to prevent allograft failure

6. Normal microvasculature maintains tissue homeostasis • Quiescent endothelial cells (EC) are tightly connected to neighbouring EC by junction proteins • This prevents leakage of plasma proteins and fluids and extravasation of leukocytes

7. Microvascular dysfunction - role of endothelial cells loss of EC integrity dissociation of junctional complex protein like VE-cadherin from interendothelial junctions relocation of actin to form cellular stress fibers, and EC contraction Players: hypoxia, IRI, VEGF, thrombin, activation of RhoA GTPase, Angiopoietin-2, LPS Antagonists: Angiopoietin-1

8. Microvascular dysfunction - role of pericytes Pericytes Supporting cells for ECs Regulation of vascular tone Pericyte – EC interactions Pericyte contraction impairs capillary reflow after cerebral ischemia-reperfusion injury Yemisci et al. Nature Medicine 2009 Pericyte Requirement for Anti-Leak Action of Angiopoietin-1 and Vascular Remodeling in Sustained Inflammation Fuxe et al. Am J Pathol 2011;178:2897

9. Angiogenic and Lymphangiogenic Growth Factors • have important roles in tissue hypoxia and injury and acute and chronic inflammation • strictly regulated in normal vasculogenesis • dysregulated in • vascular leakage • angiogenesis • diabetic retinopathy • tumour growth • arteriosclerosis

10. Ang1 VEGF PDGFR-ß TGF-ß Tie2 PDGF-B VEGFR PDGF-B Ang1 Recruitment of pericytes/SMC Endothelial cells form patent tubes Blood vessel maturation Angioblast Angiogenic growth factors regulate endothelial cell and pericyte assembling in vasculogenesis Modified from Cleaver&Melton Nature Medicine 2003;9:661

11. HIF and angiogenic and lymphangiogenic growth factors have an important role in chronic rejection in cardiac allografts without ischemia Adenovirus  Ang1  VEGF,  VEGFR3-Ig EC Injury and Inflammation AAV TxCAD Early response  Ang1/2  PDGF-A/C/  HIF SMC proliferation Abs, RTKIs •  VEGFR-1/-2 •  PTK797 •  imatinib Late response Circulation 2002;105:2524; Circulation 2003;107:1308; Circ Res 2006;98:1373; Arterioscler Thromb Vasc Biol, 2007;27:819; J Am Coll Cardiol 2002;39:710; Transplantation 2003;75:334; Transplantation 2005;79:182; Arterioscler Thromb Vasc Biol, 2009;29:691

12. Effect of imatinib on allograft arteriosclerosis in cardiac allografts without any ischemia and with 4-hour cold ischemia No ischemia 4-h ischemia Billingham score 0-4 Billingham score 0-4 Ctrl Imatinib Ctrl Imatinib Sihvola et al. Circulation 1999;99:2895 Tuuminen et al. Unpublished

13. Modification of cardiac allograft model

14. Hypothermic preservation increases innate immune ligands and receptors during IRI Syrjala et al. J Heart Lung Transplant 2010;29;1047

15. Hypothermic preservation increases DC activation and proinflammatory cytokine production during IRI Syrjala et al. J Heart Lung Transplant 2010;29;1047

16. Preoperative ischemia enhances cardiac fibrosis and arteriosclerosis 10 days after Tx 2 months after Tx Syrjala et al. J Heart Lung Transplant 2010;29;1047

17. Hypothesis:HIF, angiogenic, and lymphangiogenic growth factors may have a critical role microvascular dysfunction

18. Statins and microvascular dysfunction

19. Pleiotropic effects of statins through RhoA inhibition - cytoskeleton and intracellular signalling

20. Microvascular endothelial cells and pericytes express HMG-CoA reductase in normal hearts Cardiomyocyte EC Pericyte SMC Tuuminen et al Circulation 2011;124

21. Effect of donor simvastatin treatment on preservation injury of cardiac allografts Simvastatin (5mg/kg p.o.) to donor 2 hours before graft removal DA rat 4-h cold and 1-h warm ischemia WF rat 0 30 min 6 h 10 d 2 months VascuIar integrity Graft mRNA IRI Chronic rejection Permeability Perfusion Alloimmunity Fibrotic cascades Tuuminen et al Circulation 2011;124

22. During preservation donor simvastatin treatment prevents phosphorylation of adducin - downstream of RhoA/ROCK activation Tuuminen et al Circulation 2011;124

23. Donor simvastatin treatment prevents EC-EC gap formation during preservation Transmission electron microscopy EC-EC gaps Intracoronary lectin perfusion: EC + exposed BM

24. Donor simvastatin decreases HIF-1, iNOS and ET-1 and increases HO-1 mRNA during preservation

25. Donor simvastatin prevents vascular leakage and maintains capillary perfusion 30 min after re-perfusion Extravasation of Evans blue dye L. Esculentum lectin Tuuminen et al Circulation 2011;124

26. Donor simvastatin treatment restores immediate microvascular circulation and prevents cardiac edema after reperfusion laser doppler MRI Tuuminen et al Circulation 2011;124

27. Effect of donor simvastatin treatment on ischemia-reperfusion injury Simvastatin to donor, recipient, or both DA rat 4-h cold and 1-h warm ischemia WF rat 0 30 min 6 h 10 d 2 months VascuIar integrity Graft mRNA IRI Chronic rejection Permeability Perfusion Alloimmunity Fibrotic cascades Tuuminen et al Circulation 2011;124

28. Donor but not recipient simvastatin treatment prevents ischemia-reperfusion injury Macrophages Neutrophils Troponin-t D – Donor simvastatin treatment R – Recipient simvastatin treatment D/R – Donor and Recipient simvastatin treatment Tuuminen et al Circulation 2011;124

29. Donor simvastatin treatment increases Ang-1 and Hsp27 and decreases HIF-1, bFGF and TGF-ß during IRI Tuuminen et al Circulation 2011;124

30. Beneficial effects of donor simvastatin treatment on microvascular stability and perfusion are mediated through NO and RhoA Vascular leakage Perfused vessels Troponin T Macrophages Neutrophils CD4+ T cells CD8+ T cells OX62+ DC L-NAME - NOS inhibition ZnPP - HO-1 inhibition GGPP - RhoA pathway supplementation Tuuminen et al Circulation 2011;124

31. Donor simvastatin treatment has sustained anti-inflammatory effects 10 days after TX VCAM-1 Macrophages Neutrophils CD4+ T cells CD8+ T cells OX62+ DC Tuuminen et al Circulation 2011;124

32. Donor simvastatin treatment decreases fibrosis and TGF-b signaling 10 days after TX Fibroblasts TGF-ß activation Tuuminen et al Circulation 2011;124

33. Simvastatin inhibits TGF-b-induced endothelial-mesenchymal transition of human cardiac microvascular endothelial cells Tuuminen et al Circulation 2011;124

34. Simvastatin treatment of both donor and recipient maintains microvascular density and reduces fibrosis and arteriosclerosis in cardiac allografts Capillary density Arterial occlusion Fibrosis

35. Transplantation Laboratory Antti Nykänen, MD Jussi Tikkanen, MD, PhD Petri Koskinen, MD, PhD Rainer Krebs, MSc Mikko Keränen, MD Raimo Tuuminen, MD Simo Syrjälä, MB Alireza Raissadati, MB Janne Jokinen, MD, PhD Jussi Ropponen, MD Eeva Rouvinen, RN Eriika Wasenius, RN Risto Renkonen, MD Heikki Helin, MD Molecular Cancer Biology Laboratory, Biomedicum Helsinki, Helsinki, Finland Kari Alitalo, MD, PhD A.I. Virtanen Institute for Molecular Sciences, University of Kuopio, Kuopio, Finland Seppo Ylä-Herttuala, MD, PhD Imclone, New York Jan Wy, PhD Novartis Pharma, Basle, Switzerland Elisabeth Buchdunger, PhD Jeanette Wood, PhD Acknowledgments

36. Acknowledgments - Sources of funding • Academy of Finland • Sigrid Juselius Foundation • Helsinki University Central Hospital Research Funds • Finnish Foundation for Cardiovascular Research • Päivikki and Sakari Sohlberg Foundation • University of Helsinki

37. Thank you!