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Circulating Progenitor Cells and Cardiovascular DiseaseArshed A. Quyyumi, MD Professor of Medicine Emory Clinical Cardiovascular Research Institute (ECCRI)Emory University School of MedicineAtlanta, GAGrant support: National Institutes of Health, American Heart Association, Woodruff Fund, Emory Heart and Vascular Center,Sanofi Aventis, Novartis, Lilly, Pfizer, Forest,Amgen, GenzymeAdvisory Boards:Amorcyte/Neostem, Genway/FirstmarkSoteriaStemedica

Circulating Progenitor Cells and Cardiovascular Disease2Circulating progenitor cellsCD34+ cell populationsCirculating progenitor cells and genderCirculating progenitor cells and ischemic syndromesCirculating progenitor cells and CVD outcomesCD34+ cells as therapy for vascular diseasesCirculating Progenitor Cells and Cardiovascular Disease4Adult Bone Marrow Stem Cell PlasticityEndodermalProgenitor CellsBone MarrowStem CellsEctodermalProgenitor CellsMesodermalProgenitor CellsNeural cellsEpidermal cellsHepatocytesHematopoeitic cellsEndothelial Progenitor CellsMyocytes (Skeletal) (Cardiac)Osteocytes, ChondrocytesStromal orMesenchymal MAPCBlood cellsResident stem cells:Heart, skeletal muscle,Adipose tissue, brain,Lung etc.5Endothelial Progenitor Cell assaysProgenitor CellsCulture Surface Epitopes

CD45med, CD34, CD133, VEGF2R, CXCR4

Direct Culture(CAC)EarlyOutgrowth(CFU-Hill)4 days 15 days 5-7 days0.0001% of circulatingMononuclear cellsEndothelial ColonyForming Cells (ECFC)Endothelial Progenitor Cell Colony Formation and Cardiovascular Risk Profile45 healthy men aged >21 years (mean 50.3 1.7) without cardiovascular diseasesEPC-CFUFramingham Risk Score-5j05101520010203040506070r = .393P = .008EPC-CFU = endothelial progenitor cell colony-forming units.Hill JM, et al. N Engl J Med. 2003;13:593-600.

Hill, Quyyumi, Finkel N Engl J Med 2003;13:593 6To determine whether a cumulative risk factor profile influenced EPC counts, we calculated the Framingham risk factor score for each patient and found a significant inverse correlation between the calculated risk score and EPCs, with higher scores associated with diminished endothelial progenitor cell counts7Functional assays for endothelial progenitor cellsSenescence:

Hill, Quyyumi, Finkel N Engl J Med 2003;13:593 Hill, Quyyumi, Finkel N Engl J Med 2003;13:593 8Relation between endothelium-dependent function and EPCs

EPC-CFUFlow mediated dilation FMD Hill, Quyyumi, Finkel N Engl J Med 2003;13:593 45 Healthy Males, >21 years (mean age 50.31.7) without cardiovascular diseases

RestReactive HyperemiaFlow-mediated vasodilation (FMD)

EPC-CFU

8Because the vascular endothelium integrates the injury from established and as yet unknown risk factors, we next assessed the relationship between endothelial progenitor cell colony counts and flow mediated brachial reactivity, a composite measure of endothelial integrity. As noted here, there was a strong correlation between endothelial progenitor cell colony count and flow mediated brachial reactivity. When the measured FMD was divided into tertiles (Click) subjects with the highest flow mediated brachial reactivity had endothelial progenitor cell colony counts approximately 3-fold higher than those in the lowest tertile .Risk factors, Vascular Injury, and Regenerative Capacity

Risk FactorsEndothelial injuryEndothelial dysfunction, Arterial stiffnessAtherogenesisVascular RepairProgenitor Cells9Russell Ross classic paradigm argues that seemingly disparate risk factors such as hypertension, increased serum cholesterol, or diabetes function through a final common pathway that culminates in endothelial cell injury. This concept has now been expanded to include both endothelial dysfunction as well as endothelial damage. Consistent with this interpretation are a number of recent studies demonstrating that endothelial function is a predictor of future cardiovascular events. Cardiovascular risk factors thus contribute to atherogenesis by inducing endothelial cell injury and dysfunction.

Cardiologists are now familiarizing themselves with the new biology of bone marrow stem cells. Endothelial progenitor cells or EPCS are one such group of cells which many groups have been investigating for their potential role in angiogenesis. However given their relatively high frequency in the circulation and the limited degree of neovascularization occurring in healthy adults, EPCs may well have an important role beyond angiogenesis. We have become interested in EPCs and the concept that endothelial injury may be opposed by a limited ability of the vessel wall to undergo repair.

Circulating progenitor cellsCD34+ cell populationsCirculating progenitor cells and genderCirculating progenitor cells and ischemic syndromesCirculating progenitor cells and CVD outcomesCD34+ cells as therapy for vascular diseasesCirculating Progenitor Cells and Cardiovascular Disease11

Gated on CD34+ within MNCsWithin CD34+ gate, 4 quadrants for marker CD133 and VEGF-2RGated on MNCsCD45medFlow Cytometry11Forwrad and side scatter red line is gate, bottom left is lymphocyte, granulocytes is big circlepink circle cd34+ - percent is 0.113 look inot this population, gate for cd133 and vegf on left is neg and right is +12Fluorescent activated cell sorting (FACS) analysis for bone marrow derived progenitor cell populationsCD34: Hematopoietic stem cellCD133: Immature progenitorsCD133+/VEGF2R+: Differentiates between immature and mature endothelial PCsCD34+/CD133+/VEGF2R+: Presumed EPC enrichedCXCR4: Epitope that is associated with homing to areas of ischemia that express SDF-1Peichev M et al Blood 2000; 95:952Hirschi KK. et al ATVB 2008;28:1584

Cell Type: Isolated CD34Cells Most Able to Improve Perfusion, Prevent Apoptosis and Rescue Hibernating Cardiomyocytes13

Kawamoto et al., Circulation 2006;114;2163-2169PBS = Phosphate-buffered salineloMNCs = 5x10^5 MNChiMNCs = contains 5x10^5 CD34+ cells within MNCsCD34+ = 5x10^5 CD34+ cellsCapillary Density (perfusion) is greatest in CD34+ cell cohort, and this correlates with decreased incidence of fibrosis. Effect increases with dose.CD34 Cells Exhibit Increased Potency and Safety for Therapeutic Neovascularization after AMI Compared with Total Mononuclear Cells in Nude Rats:Endothelial progenitor cell therapy for acute myocardial infarctionCell types: bone marrow mononuclear cells, CD34+, etc.CD34+ cells constitute 0.1 to 0.2% of bone marrow mononuclear cells

FDG labeled bone marrow mononuclear cells and injected intracoronary in post MI patients

Uptake of BM mononuclear cells: 1.3 to 2.6% in MI region

Uptake of CD34+ cells: 14-39% in MI region

BM mononuclear cellsCD34+ cellsHofmann et al Circ 2005;111:2198-220215Cell Type: Circulating CD34Cell Levels and Migratory Capacity Correlate with Cardiac FunctionCirculating CD34+ cell quantity 1 year post MI significantly correlates to LVEF, wall motion score index, end diastolic volume and end systolic volume. The number of circulating CD34+, CXCR4+, CD117+ and c-met+ cells mobilized early (