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Leading the Way in Cardiovascular Regenerative Medicine

Leading the Way in Cardiovascular Regenerative Medicine. CV disease: US prevalence. Myocardial ischemia 37 million *. Heart failure 5 million. Acute MI 865,000/year. Chest Pain 4.2 million emergency visits/year 6.4 million outpatient visits/year. Peripheral vascular disease 8 million.

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Leading the Way in Cardiovascular Regenerative Medicine

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  1. Leading the Way in Cardiovascular Regenerative Medicine

  2. CV disease: US prevalence Myocardial ischemia 37 million* Heart failure 5 million Acute MI 865,000/year Chest Pain 4.2 million emergency visits/year6.4 million outpatient visits/year Peripheral vascular disease8 million Stroke5.7 million *Symptomatic coronary artery disease (CAD) or angina pectoris. American Heart Association. Heart Disease and Stroke Statistics—2007 Update.

  3. New paradigm for CV disease • Human heart can regenerate • Bone marrow derived stem cells (BMCs) • Circulating progenitor cells (CEPCs) • Circulating hematopoietic stem cells • Resident stem cells • With certain risk conditions (eg, hypertension, diabetes, hypercholesterolemia, aging) and diseases (eg, ischemic heart disease) stem cells are inadequate (number/quality/time) • Can stem cell therapy correct/regenerate blood vessels and/or myocardium?

  4. Cell therapy • Embryonic stem cells • Cord blood stem cells • Adult stem cells • Circulating • Bone marrow (BM) • Hematopoietic • Mesenchymal • Tissue specific • Fat, muscle, etc Gulati R, Simari RD et al. Med Clin N Am. 2007;91:769-85.

  5. CV disease targets for cell therapy clinical trials • CAD • Refractory angina (“no other options”) • Acute myocardial infarction with left ventricular dysfunction (early vs late) • Heart failure (reversible ischemia vs scar) • Peripheral arterial disease • Claudication and critical limb ischemia • Abdominal aortic aneurysm • Ischemic stroke • Nonischemic cardiomyopathy

  6. Some examples of CV disease targets in cell therapy trials in the US • Refractory angina • Baxter: CD 34+ cells post G-CSF: (Phase 1 & 2) • Acute myocardial infarction • Osiris IV mesenchymal cells (Phase 1) • Neuronyx: IM mesenchymal cells • NHLBI-CCTRN: IC BM mononuclear cells (TIME and late TIME) • Heart failure • Bioheart: skeletal myoblasts (MARVEL) • NHLBI-CCTRN: BM mononuclear cells (FOCUS) • Peripheral arterial disease • Baxter: CD34+ cells post G-CSF for claudication and CLI Courtesy of Timothy Henry, MD.

  7. Cell transplantation for cardiac repair and/or inadequate blood supply: Rationale Chronic heart diseases are characterized byirreversible loss of myocytes Although some mitotic activity can be identified, proliferative capacity is inadequate Permanent deficits in number of viable, functioning myocytes promotes development and progression of HF

  8. Damaged myocardium repair: New paradigm Traditional view – no new heart muscle cell formed Usual Outcome: Replacement of heart muscle with SCAR TISSUE New view – replacement of damaged heart cells by new cardiomyocytes Strategy (1): Replicationof endogenous cardiomyocytes Strategy (2): Conversionof stem cells into new cardiomyocytes Grounds MD et al. J Histochem Cytochem. 2002;50:589-610.

  9. Why use adult stem cells? • Readily available • Easy to isolate • Autologous • May be altered to increase gene expression • No ethical concerns

  10. Role of the cell in cardiac regeneration therapy As a cell As a factory As a courier

  11. Cell-mediated CV repair Angiogenesis and re-endothelialization Exercise, VEGF,Estrogen, G-CSFEpo, Statins,SDF-1 Apoptotic bodies, cell-cell contact (?),adhesion (?) SDF-1, VEGF Mobilization Differentiation Homing CV risk factors Re-endothelialization Angiogenesis Werner N, Nickenig G. Arterioscler Thromb Vasc Biol. 2006;26(2):257-66. VEGF = vascular endothelial growth factor.

  12. Stem-cell homing: Chemoattractive hypothesis Adult stemcells Chemokinereceptors Circulating stem cellsattracted to injury Heart withmyocardialinfarction Area of injurysecretes chemokines Rosenthal N. N Engl J Med. 2003;349:267-74.

  13. Possible routes for cell therapy to the heart RCA CFX Balloon catheter Intracoronary LAD Intravenous Intramyocardial Transendocardial Strauer BE, Kornowski R. Circulation 2003;107:929-34.

  14. Endothelial Progenitor Cells (EPCs)

  15. EPCs in CV diseases CV risk factors Endothelial dysfunction Collaterals Restenosis CV disease EPCs Pathophysiology Therapeutics Atherosclerosis Heart disease Peripheral vascular disease Courtesy of Arshed A. Quyyumi, MD.

  16. Circulating EPCs aid in cardiac repair • CD34+, CD133+, and VEGF2R+ • Circulate in blood stream • Contribute to repair of vascular or myocardial injury and collateral formation Asahara T et al. Science. 1997;275:964-7. Takahashi T et al. Nature Med. 1999;5:434-8.

  17. EPC physiology • Originate in bone marrow • Circulate in blood stream • Number and function (proliferation, migration, homing) modulated by age, CV risk factors, and disease • Release stimulated by organ and vascular injury • Participate in vascular repair (collateralization) and re-endothelialization, partly by paracrine effects • Circulating numbers by exercise and drugs (statins and ACE inhibitors) • Independent predictors of endothelial dysfunction and long-term prognosis in patients with CAD Hill JM et al. N Engl J Med. 2003;348:593-600.

  18. EPC number has prognostic importance N = 519 males with CAD, mean age 66 y 1.00 Group 3 (high EPC level) 0.98 0.96 Group 2 (medium EPC level) Event-free survival 0.94 Group 1 (low EPC level) 0.92 0.90 0 0 100 200 300 365 Days Werner N et al. N Engl J Med. 2005;353:999-1007.

  19. Association between CV risk factors and EPC colony counts N = 45 males without CAD, > 21 years (mean age 50.3) 70 r = –47.0 P = 0.001 60 50 40 EPC colony-forming units 30 20 10 0 -5 0 5 10 15 20 Framingham risk score Hill JM et al. N Engl J Med. 2003;348:593-600.

  20. Mobilization of EPCs after myocardial infarction N = 16 patients with AMI, 8 controls P < 0.001 P < 0.001 P < 0.001 P < 0.05 300 200 MNCCD34+ (/106WBCs) 100 0 Day 1 3 7 14 28 Time after onset Shintani S et al. Circulation. 2001;103:2776-9.

  21. VEGF levels correlate with increase in EPCs 450 r = 0.35 P = 0.01 400 350 300 250 200 MNCCD34+(cells/106 WBCs) 150 100 50 0 0 50 100 150 200 250 300 350 400 450 Plasma VEGF (pg/mL) Shintani S et al. Circulation. 2001;103:2776-9.

  22. EPC activity and coronary collaterals A P = 0.017 0.4 0.3 0.2 CD34/CD133 Dual PositiveCells (% of total lymphocytes) 0.1 0 Colln=13 Colln=10 • 30 patients with isolated left anterior descending disease • Divided into groups with (0.33) and without (0.09) adequate Collateral Flow Index (CFI) B R = 0.75P < 0.0001 0.8 0.6 CD34/CD133 Dual PositiveCells (% of total lymphocytes) 0.4 0.2 0 0 0.1 0.2 0.3 0.4 0.5 CFI Inadequate coronary collateral development associated with ¯ numbers of circulating EPCs and impaired chemotactic and pro-angiogenic activity Lambiase PD et al. Circulation. 2004;109:2986-92.

  23. Decrease in EPCs associated with CV disease Endothelial Progenitor Cells Vasculoprotective agents CV risk factors Atherosclerosis Disease Regression? Disease Progression Improvement of endothelial function Enhanced re-endothelialization Reduced plaque size Improved angiogenesis Myocardial infarction Ischemic stroke Erectile dysfunction Renal insufficiency Peripheral artery disease Werner N, Nickenig G. Arterioscler Thromb Vasc Biol. 2006;26:257-66.

  24. Bone Marrow Stem Cells in Cardiac Repair

  25. Stem cells in cardiac repair: Proposed mechanisms of action Cell homing and tissue integration EC differentiation SMC differentiation Cardiac differentiation fusion Paracrine Effects Attraction/ Activation of CSC Angiogenesis Vasculo-genesis Arteriogenesis Cardiomyocyte proliferation Cardio-myogenesis ¯Cardiomyocyte apoptosis Modulation of inflammation Scar remodeling FUNCTIONAL IMPROVEMENT Dimmeler S et al. Arterioscler Thromb Vasc Biol. 2007;Oct. 19 epub.

  26. Bone marrow cells promote myocardial regeneration: Postulated mechanism Infarcted myocardium Transplanted Cells Unknown molecular signal(s) Cell migration to damaged area Proliferation and differentiation Cytoplasmic proteins Nuclear proteins Cardian myosin α-Sarcomeric actin Connexin 43 Csx/Nkx2.5 MEF2 GATA-4 Functional competence Orlic D et al. Nature. 2001;410:701-5.

  27. Cardiac stem cells are derived, in part, from bone marrow Post-mortem analysis of 4 hearts of female recipients of male BM transplants Demonstration of Y-chromosomes in up to 23% of cardiomyocytes Blue, green arrow = Y chromosome–positive true nucleus of BM Red = Derived cardiomyocyte cytoplasm (sarcomeric actin) surrounded by basement membrane laminin (green, arrowhead) Deb A et al. Circulation. 2003;107:1247-9.

  28. Communication between heart and bone marrow signals in repair Heart endosteum Blood vessel endothelium SDF-1 SDF-1 transport CXCR4 Cell Recruitment Stem/progenitor cell Maturing leukocyte Blood vessel endothelium Bone marrow Bone Courtesy of Carl J. Pepine, MD

  29. BMCs regenerate infarcted myocardium in mice Ventricular function LVDP LVEDP 120 100 80 60 40 20 0 40 30 20 10 0 * * † * † mm Hg mm Hg * LV +dP/dt LV –dP/dt 12000 8000 4000 0 12000 8000 4000 0 * † * † mm Hg s-1 mm Hg s-1 * * SO MI MI + BM SO MI MI + BM Orlic D et al. Nature. 2001;410:701-5.

  30. BMCs reduce perfusion defect in ischemic pig hearts Kamihata H et al. Circulation. 2001;104:1046-52.

  31. BMCs enhance collaterals to infarct region LAD Ligation BM-MNC after 3 weeks Kamihata H et al. Circulation. 2001;104:1046-52.

  32. BMC therapy increases angiogenesis in ischemic pig hearts BM-MNC (Factor-VII) Control Medium (Factor-VIII) In part via enhanced synthesis of angiogenic factors in the infarcted region (ie, VEGF) Kamihata H et al. Circulation. 2001;104:1046-52.

  33. Infarcted myocardium repair via autologous intracoronary mononuclear BMC transplantation Human model Strauer BE, et al. Circulation. 2002;106:1913-8.

  34. BMCs minimize infarcted myocardium region 25 20 15 10 5 0 * P = 0.04 At 3 months: • SV index 49  56, P = 0.01 • Left ventricular end-systolic volume 8267, P = 0.01 • Thallium scintigraphy, cm2 174128 Infarct region (%) Cell therapy Standard therapy Strauer BE et al. Circulation. 2002;106:1913-8.

  35. Assessment of intracoronary cell therapy in AMI PMC = peripheral mononuclear cells; RCT= randomized controlled trial; WMSI = wall motion score index. Lipinsky MJ et al. J Am Coll Cardiol.2007;50:1761-7.

  36. Effects of intracoronary cell therapy on LVEF Study EF change % (random) EF change %or sub-category 95% CI 95% CI ASTAMI, 2005 -1.49 (-2.81, 0.01) Bartunek, 2005 -1.10 (-9.14, 2.94) BOOST, 2004 -2.83 (-3.00, -0.60) Jannsens, 2004 -1.10 (-2.68, 0.68) MAGIC-3, 2006 -2.20 (-7.18, 1.23) Meluzin, 2006 -2.03 (-2.94, -1.04) REPAIR-AM, 2006 -2.59 (-1.54, -1.44) Strauer, 2002 -1.03 (-4.06, 2.04) TCT-STAMI, 2006 -6.70 (-1.89, -3.51) Zhan-Quan, 2006 -5.50 (-3.19, -2.83) Total-2.97 (-1.04, -1.22) -10 -5 0 5 10 Favors cell therapy Favors control Test for heterogenicity, Chi2 = 33.62, af = 9 (P = 0.0001), P = 73.2%Test for overall effect: Z = 5.35 (P = 0.00001) Lipinsky MJ et al. J Am Coll Cardiol. 2007;50:1761-7.

  37. Autologous CD34+ cells for intractable angina • N = 24 patients with CCS class 3/4 angina • G-CSF 5 μg/kg/day x 5 days • Leukapheresis performed on Day 5 • CD34+ cell selection • NOGA-guided transplantation to zones of myocardial ischemia • Phase I/IIa double-blind, 3:1 randomization, with crossover of placebo patients using frozen cells Losordo DW et al. Circulation.2007;115:3165-72.

  38. Decrease in angina frequency with CD34+ cell therapy 3 6 Months Losordo DW et al. Circulation.2007;115:3165-72.

  39. Skeletal Myoblast Cells

  40. Skeletal myoblasts • Derived from satellite cells in skeletal muscle • With appropriate stimulus, satellite cells differentiate into muscle fibers • Highly resistant to ischemia • Do not contract spontaneously • Do not differentiate into cardiomyocytes

  41. Skeletal myoblasts 2-3 cm biopsy sample of thigh vastus lateralis (12-18 g) explanted under local anesthesia Human skeletal myoblasts after 3-wk in vitro culture period (magnification ×40) Courtesy of Arshed A. Quyyumi, MD.

  42. Skeletal myoblast transplantation in post-MI HF patients Before surgery After surgery Menasché P et al. Lancet. 2001;357:279-80.

  43. Autologous skeletal myoblast injection for ischemic cardiomyopathy trial (MAGIC) • Patients: • Moderate to severe LVSD referred for CABG • Cells: • Muscle Bx from thigh • Skeletal muscle myoblasts cultured • Delivery: • Direct injection into scar at surgery • Results: • Stopped early by DSMB due to low enrollment rate • Adverse event rate similar (25% cells vs 20% controls) • ICD Therapy in 15% • No improvement in LVEF by TTE (primary outcome)* • LVEF improved by SPECT • Highly significant dose-dependent LV size decrease Cleland JGF et al. Eur J Heart Failure 2007;9:92-97.

  44. Cell Therapy in CV Disease:Newest Evidence

  45. Induction of pluripotent stem cells from human fibroblasts Transcription factors introduced via retroviral transduction Takahashi K et al. Cell. 2007.Yu J et al. Science. 2007. iPS = induced pluripotent stem cells

  46. Evidence for successful reprogramming of human fibroblasts* • Morphologic similarities with human embryonic stem (ES) cells • Expression of surface antigens found in ES • Telomerase activity • Ability to sustain continuous culture • Expression of pluripotency-associated genes • Pluripotency demonstrated in vivo via teratoma formation in mice Takahashi K et al. Cell. 2007.Yu J et al. Science. 2007. *Demonstrated in both studies.

  47. Effects of composite transcription factor modulation of human fibroblasts: Conclusions • Induced pluripotent stem cells can be generated from human fibroblasts by retroviral transduction of transcription factors • Use of vectors that may integrate into genome, introducing mutations, precludes any clinical application at present Takahashi K et al. Cell. 2007.Yu J et al. Science. 2007.

  48. Cell Therapy in CV Disease:Future Directions

  49. Cardiac renewal: Is the goal in sight? “Remaining young at heart is a desirable but elusive goal. Myocyte regeneration may accomplish just that. Continuous cell renewal in adult myocardium was thought to be impossible; multipotent stem cells may be able to renew myocardium and, under certain circumstances, can be coaxed to repair the broken heart after infarction.” Anversa P, Nadal-Ginard B et al. Nature. 2002;415:240-3.

  50. Stem cell therapy: More questions than answers? Timing of delivery Acute vs chronic Routes of delivery Surgical vs percutaneous ? Cell type and marker Myoblast vs BM CD 34/ AC133/ SP Target patient population Best criteria Cell origin Embryonic vs adult BM vs peripheral Culture expanded vs fresh Dose Dose response Courtesy of Timothy Henry, MD.

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