Leading the Way in Cardiovascular Regenerative Medicine

Transcription

1 Slide 1 Leading the Way in Cardiovascular Regenerative Medicine Leading the Way in Cardiovascular Regenerative Medicine This slide set presents the current work in cell therapy in treating cardiovascular (CV) disease. Types of cells used, how therapy is delivered, results of recent studies in regenerative medicine, and what the future holds for cell therapy research are included. 1

2 Slide 2 2 CV disease: US prevalence Myocardial ischemia 37 million* Heart failure 5 million Chest Pain 4.2 million emergency visits/year 6.4 million outpatient visits/year Acute MI 865,000/year Peripheral vascular disease 8 million Stroke 5.7 million *Symptomatic coronary artery disease (CAD) or angina pectoris. American Heart Association. Heart Disease and Stroke Statistics 2007 Update. CV disease: US prevalence According to the American Heart Association (AHA), approximately 80 million people in the United States have >1 type of cardiovascular (CV) disease. Mortality data from the National Health Center for Statistics (NCHS), 2006 show CV disease as the underlying cause of death in 36.3% of all 2,398,000 deaths in 2004, or one of every 2.8 deaths in the United States. According to the NCHS, if all forms of major CV disease were eliminated, life expectancy would rise by almost 7 years. 2

3 Slide 3 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? New paradigm for CV disease Certain types of stem cells from healthy donors may prove effective in helping the human heart regenerate tissue and muscle. In some settings, however, the quality of stem cells is too low to provide positive results. 3

4 Slide 4 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: Cell therapy While it is not yet clear what the most appropriate cell type for restoration of damaged myocardial tissue will be, cell transplantation has produced some levels of improvement in cardiac function in preclinical models. Cell types employed were embryonic stem cells, skeletal myoblasts, bone marrow cells, and resident cardiac progenitors. Multiple large-scale studies may be the most efficacious method to determine the impact of cell type, dose, and route of delivery on cell engraftment and effect. 4

5 Slide 5 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 CV disease targets for cell therapy clinical trials 37 million people in the US have chronic angina, a disabling manifestation of coronary artery disease (CAD), according to the AHA, and many of those patients continue to have recurrent angina. Many patients continue to endure recurrent angina while on drug therapy and many are not suitable for revascularization procedures. Acute myocardial infarction (MI), heart failure, and peripheral arterial disease also cause quite a lot of suffering and possibly preventable death. 5

6 Slide 6 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. Some examples of CV disease targets in cell therapy trials in the US In patients with refractory ischemia, the goal is to improve coronary blood flow by promoting angiogenesis. The largest experience is with circulating CD34+ cells, and study is ongoing with intramyocardial delivered BM mononuclear cells (BMNCs) into the ischemic zone. In patients with acute MI, trials have utilized intracoronary delivery of BMNCs with considerable variability in the number of cells and the timing of delivery as well as the measurement of the primary endpoint. In patients with coronary heart failure (CHF) following MI, the goal is to promote myogenesis, utilizing skeletal myoblasts delivered into the previously infarcted zone. For peripheral arterial disease, studies are focusing on claudication and critical limb ischemia (CLI). 6

7 Slide 7 7 Cell transplantation for cardiac repair and/or inadequate blood supply: Rationale Chronic heart diseases are characterized by irreversible 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 Cell transplantation for cardiac repair and/or inadequate blood supply: Rationale In the old paradigm, irreversible loss of myocyte number and function and subsequent clinical deterioration were thought to be inevitable. 7

8 Slide 8 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): Replication of endogenous cardiomyocytes Strategy (2): Conversion of stem cells into new cardiomyocytes Grounds MD et al. J Histochem Cytochem. 2002;50: Damaged myocardium repair: New paradigm It has been thought that postnatal cardiac muscle was incapable of tissue repair because, like skeletal myofibers, mature cardiac cells were postmitotic and could not regenerate. Unlike skeletal muscle, there appeared to be no reserve precursor cells, so damaged heart muscle was replaced by scar tissue, which creates a major clinical issue. Replacement of myocytes is the ideal scenario to ameliorate the problem of heart damage after infarction. Cardiomyocyte replacement could occur by stimulating production of endogenous mature cardiomyocytes or stem cells, or by implanting donor cardiomyocytes that may be derived from stem cells. 8

9 Slide 9 9 Why use adult stem cells? Readily available Easy to isolate Autologous May be altered to increase gene expression No ethical concerns Why use adult stem cells? Cell therapy clinical trials are early in development and pose a number of unanswered questions, including the ideal cell, dose, timing and method of delivery, the ideal endpoints, and time points. Adult stem cells as a starting point do at least eliminate ethical concerns. 9

10 Slide Role of the cell in cardiac regeneration therapy As a cell As a factory As a courier Roll of the cell in cardiac regeneration therapy Cell-based therapy may be approached from 3 different perspectives, as summarized here. 10

11 Slide Cell-mediated CV repair Angiogenesis and re-endothelialization Exercise, VEGF, Estrogen, G-CSF Epo, Statins, SDF-1 Apoptotic bodies, cellcell contact (?), adhesion (?) SDF-1, VEGF Mobilization Differentiation Homing CV risk factors Angiogenesis Re-endothelialization VEGF = vascular endothelial growth factor. Werner N, Nickenig G. Arterioscler Thromb Vasc Biol. 2006;26(2): Cell-mediated CV repair Cell-mediated angiogenesis and re-endothelialization need efficient mobilization, differentiation, and homing of cells. Various agents, as seen at the top of the graphic, have been shown to positively influence these steps. CV risk factors, however, impose a significant negative impact on the mobilization, differentiation, and homing processes. 11

12 Slide Stem-cell homing: Chemoattractive hypothesis Adult stem cells Chemokine receptors Circulating stem cells attracted to injury Heart with myocardial infarction Area of injury secretes chemokines Rosenthal N. N Engl J Med. 2003;349: Stem-cell homing: Chemoattractive hypothesis Cells with chemokine receptors circulate to various tissues that secrete chemoattractive molecules. The injured area secretes large amounts of chemokines, attracting circulating stem cells. Using this theory, specific chemoattractants might be identified and protocols developed to enhance the passage of stem cells into lesions. 12

13 Slide Possible routes for cell therapy to the heart RCA CFX Balloon catheter LAD Intravenous Intracoronary Intramyocardial Transendocardial Strauer BE, Kornowski R. Circulation 2003;107: Possible routes for cell therapy to the heart In red at the bottom of the heart on the image is a representative apical lesion of the left ventricle by MI. The balloon catheter is localized in the infarct-related artery and is situated above the border zone of the infarction. The possible route of cell infusion and migration into the infarct are represented by the blue and green arrows. The 2 small figures at bottom right and left depict the transendocardial and intramyocardial route of cell therapy. 13

14 Slide 14 Endothelial Progenitor Cells (EPCs) Endothelial Progenitor Cells (EPCs) The following section examines endothelial progenitor cells and their role in cardiovascular regenerative medicine. 14

15 Slide EPCs in CV diseases EPCs Pathophysiology Therapeutics CV risk factors Endothelial dysfunction Collaterals Restenosis CV disease Atherosclerosis Heart disease Peripheral vascular disease Courtesy of Arshed A. Quyyumi, MD. EPCs in CV diseases CV risk factors contribute to atherogenesis by inducing endothelial cell injury and dysfunction. Many studies have been investigating EPCs for their potential role in angiogenesis, and due to their relatively high frequency in the circulation and the limited degree of neovascularization that occurs in healthy adults, EPCs may well have an important role beyond angiogenesis. 15

16 Slide 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: Takahashi T et al. Nature Med. 1999;5: Circulating EPCs aid in cardiac repair Neovascularization is considered to result from the proliferation, migration, and remodeling of fully differentiated EPCs derived from pre-existing blood vessels. Circulating EPCs are mobilized endogenously in response to tissue ischemia or exogenously by cytokine therapy and thereby contribute to neovascularization of ischemic tissues. As seen in the photo, EPC colonies are characterized by a central cluster of rounded cells surrounded by radiating thin, flat cells. 16

17 Slide 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: EPC physiology In one study, the levels of circulating EPCs were found to be a better predictor of vascular reactivity than the presence or absence of conventional risk factors. Also, EPCs from subjects with a high CV disease risk had higher rates of in vitro senescence than cells from subjects at low risk. Levels of EPCs may be a surrogate biologic marker for vascular function and cumulative cardiovascular risk. These findings suggest that endothelial injury in the absence of sufficient circulating progenitor cells may increase the risk of CV disease. 17

18 Slide EPC number has prognostic importance N = 519 males with CAD, mean age 66 y 1.00 Group 3 (high EPC level) 0.98 Event-free survival Group 2 (medium EPC level) Group 1 (low EPC level) Days Werner N et al. N Engl J Med. 2005;353: EPC number has prognostic importance In a recent analysis of death from CV causes, higher levels of CD34+KDR+ EPCs were associated with a decreased risk of death from CV causes. Cumulative event-free survival increased dramatically with increasing levels of baseline EPCs. The risk of death from CV causes was increased more than 3 times among males with low EPCs levels, as compared with males having high levels. 18

19 Slide Association between CV risk factors and EPC colony counts N = 45 males without CAD, > 21 years (mean age 50.3) r = 47.0 P = EPC colonyforming units Framingham risk score Hill JM et al. N Engl J Med. 2003;348: Association between CV risk factors and EPC colony counts A strong association was found between CV risk factors and EPC colony counts. The number of colony-forming units was strongly correlated with the subjects Framingham risk score, with a significant inverse correlation between the score and EPC counts. 19

20 Slide Mobilization of EPCs after myocardial infarction N = 16 patients with AMI, 8 controls P < P < P < P < 0.05 MNC CD34+ (/10 6 WBCs) Day Time after onset Shintani S et al. Circulation. 2001;103: Mobilization of EPCs after myocardial infarction EPCs circulate in adult peripheral blood (PB) and contribute to neovascularization. However, little is known whether EPCs and their putative precursor, CD34+ mononuclear cells (MNC CD34+ ), are mobilized into PB in acute ischemic events. Flow cytometry revealed that circulating MNC CD34+ counts significantly increased in patients with acute MI, peaking on day 7 after onset. During culture, PB-MNCs formed multiple cell clusters, and EPC-like attaching cells with endothelial cell lineage markers sprouted from the clusters. In patients with acute MI, more cell clusters and EPCs developed from cultured PB-MNCs obtained on day 7 than those on day 1. Plasma levels of vascular endothelial growth factor (VEGF) significantly increased, peaking on day 7, positively correlating with circulating MNC CD34+ counts, the first clinical demonstration showing that EPCs and MNC CD34+ are indeed mobilized during an acute ischemic event in humans. 20

21 Slide VEGF levels correlate with increase in EPCs r = 0.35 P = MNC CD34+ (cells/10 6 WBCs) Plasma VEGF (pg/ml) Shintani S et al. Circulation. 2001;103: VEGF levels correlate with increase in EPCs The potential relationship between MNC CD34+ counts and plasma cytokine levels in acute MI were examined. A simple regression analysis revealed that the number of circulating MNC CD34+ positively correlated with the plasma levels of VEGF. In addition, a recent report has shown that VEGF functions as a mobilizer for EPCs in patients with CAD receiving therapeutic VEGF gene transfer. 21

22 Slide EPC activity and coronary collaterals 30 patients with isolated left anterior descending disease Divided into groups with (0.33) and without (0.09) adequate Collateral Flow Index (CFI) A CD34/CD133 Dual Positive Cells (% of total lymphocytes) P = Coll n=13 Coll n=10 B CD34/CD133 Dual Positive Cells (% of total lymphocytes) R = 0.75 P < 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: EPC activity and coronary collaterals The mechanisms underlying the variation in collateral formation between patients, even with similar patterns of CAD, remain unclear. This study sought to determine whether circulating humoral or cellular factors could provide additional information. There was an inverse correlation between serum mitogenicity and CFI (r = 0.61, P < 0.01). No significant differences were detected between the 2 groups in plasma levels of total VEGF, VEGF165, or placental growth factor. There was a strong positive correlation between numbers of CD34/CD133+ circulating hemopoietic precursor cells and CFI (r = 0.75, P < 0.001). In patients with inadequate CFI, the numbers of differentiated EPCs appearing in the circulation and in culture were significantly reduced by 75% (P < 0.05) and 70% (P < 0.05), respectively. Angiogram provided by Carl J. Pepine, MD. 22

23 Slide Decrease in EPCs associated with CV disease Endothelial Progenitor Cells Vasculoprotective agents CV risk factors Disease Regression? Atherosclerosis 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: Decrease in EPCs associated with CV disease Vasculoprotective agents increase the number and function of EPCs, improving endothelial function and preventing the progression of atherosclerosis. CV risk factors and several CV diseases have been associated with impaired number and function of circulating EPCs. For example, all conditions of discernable atherosclerotic disease are accompanied by reduced EPC counts and migratory capacity. 23

24 Slide 24 Bone Marrow Stem Cells in Cardiac Repair Bone Marrow Stem Cells in Cardiac Repair This section illustrates the mechanism of action for cardiac repair using bone marrow stem cells and shows some actual results of cell therapy. 24

25 Slide Stem cells in cardiac repair: Proposed mechanisms of action Cell homing and tissue integration EC differentiation SMC differentiation Paracrine Effects Cardiac differentiation fusion Angiogenesis Attraction/ Activation of CSC Arteriogenesis Cardiomyocyte proliferation Vasculogenesis Cardiomyogenesis Cardiomyocyte apoptosis Modulation of inflammation Scar remodeling FUNCTIONAL IMPROVEMENT Dimmeler S et al. Arterioscler Thromb Vasc Biol. 2007;Oct. 19 epub. Stem cells in cardiac repair: Proposed mechanisms of action It is vital to distinguish between the target goals, eg, acute vs chronic ischemia, when considering therapies to improve functional recovery, because fundamentally different pathophysiological processes are targeted. For example, in patients with acute MI, the goal of progenitor cell transplantation is to prevent or ameliorate postinfarction left ventricular remodeling in an effort to reduce postinfarction heart failure. This could even be achieved by enhanced neovascularization and reduced cardiomyocyte apoptosis, regardless of long-term engraftment and transdifferentiation. Thus, the assumed mechanisms underlying cell therapy mediated functional recovery that are illustrated may differ with respect to the clinical relevance in different types of cardiac failure. 25

26 Slide 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 Cardian myosin α-sarcomeric actin Connexin 43 Nuclear proteins Csx/Nkx2.5 MEF2 GATA-4 Functional competence Orlic D et al. Nature. 2001;410: Bone marrow cells promote myocardial regeneration: Postulated mechanism Locally transplanted Lin-c-kit POS bone marrow cells have shown a high capacity for cardiac tissue differentiation. Here, they led to the partial repair of the infarcted heart in the mice used, implying that the transplanted cells responded to signals from the injured myocardium that promoted migration, proliferation, and differentiation within the necrotic area. These differentiating myocytes expressed nuclear and cytoplasmic proteins that are typical of cardiac tissue, and the presence of connexin 43 indicates cellular coupling and functional competence of the restored myocardium. 26

27 Slide 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: Cardiac stem cells are derived, in part, from bone marrow Hearts of 4 female subjects who had undergone sex-mismatched bone marrow transplantation were analyzed for the presence of Y chromosome positive cardiomyocytes, with 4 female gender-matched transplant subjects as controls. The mean percentage of Y chromosome positive cardiomyocytes in patients with sex-mismatched BMT was 0.23±0.06%. No single Y chromosome positive cardiomyocyte was identified in any of the control patients. These data establish that human bone marrow is a source of extracardiac progenitor cells capable of de novo cardiomyocyte formation. 27

28 Slide Communication between heart and bone marrow signals in repair endosteum Blood vessel endothelium Heart SDF-1 SDF-1 transport CXCR4 Cell Recruitment Stem/progenitor cell Maturing leukocyte Blood vessel endothelium Bone Bone marrow Courtesy of Carl J. Pepine, MD Communication between heart and bone marrow signals in repair Immature and maturing cell recruitment mediated by SDF-1 transport can be seen as a model for communication between the injured organ and bone marrow. Organ injury induces increased local production of SDF-1. Endothelial cells of the blood vessels translocate SDF-1 from the damaged tissue via the circulation into the bone marrow in a CXCR4-dependent manner. Presentation of the translocated SDF-1 by bone marrow endothelial and other stromal cells recruits CXCR4-expressing immature progenitors and stem cells as well as maturing leukocytes to the injured organ as part of host defense and organ repair. 28

29 Slide BMCs regenerate infarcted myocardium in mice Ventricular function 40 LVEDP 120 LVDP mm Hg * * mm Hg * * LV +dp/dt LV dp/dt mm Hg s * * mm Hg s * * 0 SO 0 MI MI + BM SO MI MI + BM Orlic D et al. Nature. 2001;410: BMCs regenerate infarcted myocardium in mice Infarcted myocardium injected with Lin-c-kit POS cells from bone marrow indicates regenerating myocardium at the upper left. At bottom left, b: Same MI at higher magnification; c and d: Low and high magnifications of MI injected with Lin-c-kit POS cells; e: MI injected with Lin-c-kit NEG cells; only healing is apparent. Asterisk indicates necrotic myocytes. To determine if developing myocytes derived from the Lin-c-kit POS cells had an impact on function, hemodynamic parameters were obtained before death, and results from infarcted mice noninjected or injected with Lin-c-kit NEG cells were combined. In comparison with sham-operated mice, the infarcted groups revealed indices of cardiac failure. In mice treated with Lin-c-kit POS cells, LV end-diastolic pressure (LVEDP) was 36% lower, and developed pressure (LVDP), LV + dp/dt and LV - dp/dt were 32%, 40%, and 41% higher, respectively. 29

30 Slide BMCs reduce perfusion defect in ischemic pig hearts Kamihata H et al. Circulation. 2001;104: BMCs reduce perfusion defect in ischemic pig hearts To see the effects of BMCs on regional myocardial blood flow, BM-MNCs (n = 58), CMECs (n = 55), or medium alone (n = 58) was injected into the left anterior descending (LAD) risk area, and myocardial contrast echocardiography was performed ~60 minutes (baseline) and 3 weeks after LAD ligation. The perfusion defect did not vary from the baseline value 3 weeks after injection of CMEC or medium, but in the BM-MNC group, the perfusion defect was notably reduced by up to 83% compared with baseline values. 30

31 Slide BMCs enhance collaterals to infarct region LAD Ligation BM-MNC after 3 weeks Kamihata H et al. Circulation. 2001;104: BMCs enhance collaterals to infarct region In this study, angiographically visible branches extend from LCx and patent LAD, and the distal portion of the LAD was visible in all animals treated by BM-MNC, CMEC, or medium injection 3 weeks after ligation. This suggests that collateral capillaries which supply blood flow to the LAD were formed even in the control group. However, the number of visible collateral vessels (> ~100 µm in diameter) branching from the distal portion of the LCx in the direction of the infarct were markedly increased in BM-MNC treated animals compared with CMEC controls. 31

32 Slide 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: BMC therapy increases angiogenesis in ischemic pig hearts Tissue sections were stained for antifactor VIII antibody to detect endothelial cells. The numbers of capillaries were 2.1-fold greater in the BM-MNC group than in CMEC control. Total numbers of vessels in 25 U areas were evaluated in each animal. 32

33 Slide Infarcted myocardium repair via autologous intracoronary mononuclear BMC transplantation Human model Strauer BE, et al. Circulation. 2002;106: Infarcted myocardium repair via autologous intracoronary mononuclear BMC transplantation In this procedure of cell transplantation into infarcted myocardium in humans, the balloon catheter enters the infarct-related artery and is placed above the border zone of the infarction. It is then inflated and the cell suspension infused at high pressure under stopflow conditions, transplanting cells into the infarcted zone via the infarct-related vasculature. Cells infiltrate the infarcted zone, where a supply of blood flow exists and the cells are therefore able to reach both the border and the infarcted zone. 33

34 Slide BMCs minimize infarcted myocardium region 25 * P = Infarct region (%) Cell therapy Standard therapy At 3 months: SV index 49 56, P = 0.01 Left ventricular end-systolic volume 82 67, P = 0.01 Thallium scintigraphy, cm Strauer BE et al. Circulation. 2002;106: BMCs minimize infarcted myocardium region Comparison of cell therapy and standard therapy groups after 3 months of therapy showed several significant differences in LV dynamics, according to the global and regional analysis of left ventriculogram. The infarct region of the left ventricle decreased significantly in the cell therapy group, significantly smaller compared with the standard therapy group after 3 months. Within the standard therapy group, only a statistically nonsignificant decrease could be seen. 34

35 Slide Assessment of intracoronary cell therapy in AMI Study Design N enrolled (follow-up) Cell type Follow-up (months) Primary endpoint Strauer, 2002 Non-RCT 20(20) BMC 3 LVEF Bartunek, 2005 Non-RCT 35 (35) BMC 4 Safety, LVEF Jannsens, 2006 RCT 67 (66) BMC 4 LVEF BOOST, 2006 RCT 60 (60) BMC 18 LVEF, safety Zhan-Quang, 2006 Non-RCT 70 (58) PMC 6 LVEF, LV vol, WMSI MAGIC CELL-3- DES, 2006 RCT 56 (50) PMC 6 LVEF TCT-STAMI, 2006 RCT 20 (20) BMC 6 LVEF ASTAMI, 2006 RCT 100 (97) BMC 6 LVEF, EDV, infarct size REPAIR-AMI, 2006 RCT 204 (197) BMC 12 LVEF Meluzin, 2006 RCT 66 (66) BMC 3 Infarct zone systolic function PMC = peripheral mononuclear cells; RCT= randomized controlled trial; WMSI = wall motion score index. Lipinsky MJ et al. J Am Coll Cardiol. 2007;50: Assessment of intracoronary cell therapy in AMI Ten studies (698 patients, median follow-up 6 months) have assessed the effects of intracoronary cell therapy on left ventricular ejection fraction (LVEF), reduction in infarct size, end-systolic volume, and end-diastolic volume. 35

36 Slide Effects of intracoronary cell therapy on LVEF Study EF change % (random) EF change % or sub-category 95% CI 95% CI ASTAMI, (-2.81, 0.01) Bartunek, (-9.14, 2.94) BOOST, (-3.00, -0.60) Jannsens, (-2.68, 0.68) MAGIC-3, (-7.18, 1.23) Meluzin, (-2.94, -1.04) REPAIR-AM, (-1.54, -1.44) Strauer, (-4.06, 2.04) TCT-STAMI, (-1.89, -3.51) Zhan-Quan, (-3.19, -2.83) Total (-1.04, -1.22) Favors cell therapy Favors control Test for heterogenicity, Chi 2 = 33.62, αf = 9 (P = ), P = 73.2% Test for overall effect: Z = 5.35 (P = ) Lipinsky MJ et al. J Am Coll Cardiol. 2007;50: Effects of intracoronary cell therapy on LVEF Pooled data from these ten trials confirmed the statistically significant improvement of LVEF. No major differences were found in LVEF effect size between randomized and nonrandomized studies. Intracoronary cell therapy was also associated with a moderately significant reduction in recurrent AMI and with trends toward reduced death, rehospitalization for heart failure, and repeat revascularization (data not shown). Intracoronary cell therapy following PCI for AMI appears to provide statistically and clinically relevant benefits for cardiac function and remodeling, confirming the benefits of this novel therapy and provides further support for multicenter randomized trials targeted to address the effect of intracoronary cell therapy on overall and event-free long-term survival. 36

37 Slide 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: Autologous CD34 + cells for intractable angina Twenty-four patients with Canadian Cardiovascular Society (CCS) class 3 or 4 angina who were undergoing optimal medical treatment and who were not candidates for mechanical revascularization received granulocyte colony-stimulating factor 5 µg/kg/day for 5 days with leukapheresis on the fifth day. Selection of CD34+ cells was performed with a FDA approved device. Efficacy parameters including angina frequency, nitroglycerine usage, exercise time, and CCS class showed trends that favored CD34+ cell treated patients vs control subjects given placebo. The completed randomized trial of intramyocardial injection of autologous CD34+ cells in patients with intractable angina provided evidence for feasibility, safety, and bioactivity. Evaluation of the bioactivity revealed trends in favor of the cell-treated patient in most of the outcome parameters, as summarized in the next slide. 37

38 Slide Decrease in angina frequency with CD34 + cell therapy Control CD34+ Cell Angina episodes per week (baseline) Months 6 Losordo DW et al. Circulation. 2007;115: Decrease in angina frequency with CD34 + cell therapy At 3 months after injection, angina frequency was increased in the placebo group and decreased in the active treatment group. At 6 months after injection, the frequency of angina was reduced in both placebo and CD34 + cell treated patients. At both time points, the CD34 + stem cell treated patients reported a greater magnitude of reduction of symptoms. 38

39 Slide 39 Skeletal Myoblast Cells Skeletal Myoblast Cells This section will explore therapy with skeletal myoblast cells. 39

40 Slide 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 Skeletal myoblasts Autologous skeletal myoblasts or satellite cells contained in skeletal muscle injected into an area of myocardial infarction differentiated into multinucleated myotubules, not cardiomyocytes. This resulted in improved global cardiac function, with a linear relationship between the extent of improvement of ejection fraction and the number of injected autologous skeletal myoblasts. However, a lack of complete electrical and mechanical integration into surrounding viable cardiac muscle, potentially a cause for arrhythmias, has limited the clinical application of these cells. 40

41 Slide 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. Skeletal myoblasts Myogenic cell grafting in a damaged myocardium is a promising development in the treatment of heart failure and has been shown experimentally to improve heart function after infarction. Myoblasts (or satellite cells), which normally lie in a quiescent state under the basal membrane of mature muscular fibers, have several properties that can be exploited for clinical use: autologous origin, ease of expansion in vitro, exclusive differentiation into muscle-fiber cells, and its high resistance to ischemia. 41

42 Slide Skeletal myoblast transplantation in post-mi HF patients Before surgery After surgery Menasché P et al. Lancet. 2001;357: Skeletal myoblast transplantation in post-mi HF patients On M-mode echocardiograms before and after surgery, an increase in left ventricular posterior wall contractility occurred after myoblast transplantation. Left ventricular end-diastolic dimensions did not change after surgery (70 6 mm) compared with dimensions before surgery (68 6 mm). Echocardiographic studies done in the months since the operation showed a left ventricular mean ejection fraction of 30%, compared with 21% before surgery. 42

43 Slide Autologous skeletal myoblast injection for ischemic cardiomyopathy trial (MAGIC) Patients: Moderate to severe LVSD referred for CABG Patients enrolled Placebo 41 Low-Dose Myoblasts 39 High-Dose Myoblast 40 P value Cells: Muscle Bx from thigh Skeletal muscle myoblasts cultured Delivery: Direct injection into scar at surgery Patients injected Change in LVEF (echo)* Change in LVEF (nuc) Change in LVEDD (ml) +9 (-21 to 28) 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 % % 2% -9 (-33 to 25) % 3% -23 (-42 to 0) NS < Cleland JGF et al. Eur J Heart Failure 2007;9: Autologous skeletal myoblast injection for ischemic cardiomyopathy trial (MAGIC) This study used stem cells to regenerate functioning myocardium. The patient s own cells were used to grow skeletal muscle myoblasts. One-hundred twenty patients were enrolled, but only 97 received treatment. The trial has created many questions, such as whether the cell injection would have been more effective delivered into the non-scarred but dysfunctional regions, and the possibility that perhaps there could be longer-term effects. 43

44 Slide 44 Cell Therapy in CV Disease: Newest Evidence Cell Therapy in CV Disease: Newest Evidence This section will explore some of the current directions and findings in cell therapy in CV disease. 44

45 Slide Induction of pluripotent stem cells from human fibroblasts Transcription factors introduced via retroviral transduction Study Takahashi, 2007 Source of fibroblasts Adult dermis Transcription factors Oct3/4, Sox2, Klf4, c-myc Yield (ips colonies/ 10 6 fibroblasts) ~200 Yu, 2007 Postnatal foreskin Oct4, Sox2, NANOG, Lin28 ~95 ips = induced pluripotent stem cells Takahashi K et al. Cell Yu J et al. Science Induction of pluripotent stem cells from human fibroblasts Takahashi and colleagues have demonstrated the generation of ips cells from adult human dermal fibroblasts with 4 factors: Oct3/4, Sox2, Klf4, and c-myc. Yu and colleagues used human newborn foreskin fibroblasts transduced with Oct4, Sox2, NANOG, and Lin28. Human ips cells were similar to human embryonic stem (ES) cells in morphology, proliferation, surface antigens, gene expression, epigenetic status of pluripotent cellspecific genes, and telomerase activity. These cells could differentiate into cell types of the 3 germ layers in vitro and in teratomas. 45

46 Slide 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 *Demonstrated in both studies. Takahashi K et al. Cell Yu J et al. Science Evidence for successful reprogramming of human fibroblasts Takahashi et al was able to generate ips cells from adult human dermal fibroblasts and other human somatic cells, which are comparable to human ES cells in their differentiation potential in vitro and in teratomas. Yu et al chose 4 colonies of which (ips(foreskin) -1 to -4) for continued expansion and analyses. Each of the 4 ips (foreskin) clones had a human ES cell morphology, a normal karyotype and expressed telomerase, cell surface markers, and genes characteristic of human ES cells. These studies have created a path to generate patient and disease-specific pluripotent stem cells. Even with retroviral integration, human ips cells are potentially useful for comprehending disease mechanisms, drug screening, and toxicology. 46

47 Slide 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 Yu J et al. Science Effects of composite transcription factor modulation of human fibroblasts: Conclusions Takahashi showed that ips cells can be generated from adult HDF and other somatic cells by retroviral transduction of the same 4 transcription factors with mouse ips cells, namely Oct3/4, Sox2, Klf4, and c-myc. The human ips cells described in the Yu study meet the defining criteria originally proposed for human ES cells but ips cells are not derived from embryos. As with human ES cells, human ips cells should prove expedient for studying the development and function of human tissues, for discovering and testing new drugs, and for transplantation medicine. 47

48 Slide 48 Cell Therapy in CV Disease: Future Directions Cell Therapy in CV Disease: Future Directions Much exciting work is ongoing in cell therapy in CV disease, and interesting directions are being formed. 48

49 Slide 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: Cardiac renewal: Is the goal in sight? Even though there is evidence for adult neurogenesis and neural stem cells, the concept of myocyte regeneration has not been fully embraced by the medical community and remains controversial. Better understanding of the origin and behavior of cycling myocytes to maximize myocardial growth and cardiac repair will help. New data suggest that cardiac stem cells (CSCs) exist and are the source of tissue regeneration. The identification, localization, and purification of CSCs, and the understanding of CSC biology, are all essential issues that must be resolved. This may lead to the discovery of mediators of CSC migration, proliferation, and differentiation that, in turn, might just result in the mending of the broken heart. 49

50 Slide Stem cell therapy: More questions than answers? Routes of delivery Surgical vs percutaneous Timing of delivery Acute vs chronic Cell type and marker Myoblast vs BM CD 34/ AC133/ SP? Target patient population Best criteria Dose Dose response Cell origin Embryonic vs adult BM vs peripheral Culture expanded vs fresh Courtesy of Timothy Henry, MD. Stem cell therapy: More questions than answers? Stem cell therapy is in its infancy, and much of the science is still unknown, still controversial, and can be an uncertain journey. 50

51 Slide CCTRN: Addressing gaps in the knowledge base NHLBI Cardiovascular Cell Therapy Research Network, established January 2007 University of Florida MHI U of Minn Network DCC University of Texas Vanderbilt University DCC = Data Coordinating Center MHI = Minneapolis Heart Institute Texas Heart Institute Cleveland Clinic Nat Clin Pract Cardiovasc Med. 2007;4:403. CCTRN: Addressing gaps in the knowledge base A clinical research network has been established by the NHLBI with a central data coordinating center and a number of clinical centers. This network, the Cardiovascular Cell Therapy Research Network (CCTRN), is intended to promote the development and conduct of clinical protocols

52 Slide CCTRN: Goal and initial phase I/II clinical trials Goal: Accelerate research into use of cell-based therapies in management of CV diseases TIME Effect of timing of post-mi BMC administration on measures of LV function Late TIME Effect of late (2-3 weeks) post-mi BMC administration on measures of LV function FOCUS Effect of transmyocardial BMC administration on measures of LV function in patients with chronic IHD Nat Clin Pract Cardiovasc Med. 2007;4:403. CCTRN: Goal and initial phase I/II clinical trials The Steering Committee of the National Heart, Lung, and Blood Institute, CCTRN has stated that the acceptance of the current limitations of the knowledge of cell-based therapies should not halt efforts to harness this knowledge and to ascertain how it can be used in patients with unmet clinical needs. As soon as possible, with the support of the NHLBI and the FDA, and because of the clinical safety observed in early human trials, we must find the best and safest stem cell type or types with which to treat our patients patients with extensive coronary heart disease, heart failure, refractory angina, or claudication for whom there are no other therapeutic options, those who have had cerebrovascular accidents, and those with congenital heart disease. 52

53 Slide CCTRN: Timeline of trials Year 01 Year 02 Year 03 Year 04 Year 05 Protocol 1 Development PRC DSMB Review Protocol 2 Development Protocol 1 PRC DSMB Review Protocol 2 Protocol 3 Development PRC DSMB Review Protocol 3 Courtesy Timothy Henry, MD. CCTRN: Timeline of trials Clinical protocols will be Phase 1 and 2 clinical studies of safety and efficacy. As shown, it is anticipated that multiple protocols will be conducted over the course of the 5-year funding period. In Year 1, the steering committee will develop a protocol that will be submitted to the Protocol Review Committee. If approved, the protocol will then be reviewed by the DSMB. Once approved by the DSMB, each clinical center may submit the protocol for IRB review. Once IRB approval has been obtained, the protocol can be initiated. 53

54 Slide Summary Current clinical efforts have focused in three overlapping areas: Repair of myocardium after MI Reconstitution of myocardium in setting of chronic HF Therapeutic angiogenesis Promising early clinical experience with BMC, endothelial cells, and skeletal myoblasts Numerous questions regarding techniques and mechanism of benefit remain Ongoing trials by the NHLBI CCTRN and other research groups should provide insight Summary In summary, progress has been made in a number of clinical goals, utilizing a variety of cell types. However, numerous questions remain regarding which cells to use, how best to deliver them, and the underlying mechanism of action. Indeed, it is possible that multiple mechanisms may be involved. The establishment of the NHLBC cell therapy network is an important step in addressing these issues and lead the way in further developing this field. 54