ENDOTHELIAL PROGENITOR CELLS AS A NOVEL BIOMARKER OF VASCULAR HEALTH I Jialal, MD, PhD. FRCPath.DABCC Robert E. Stowell Chair in Experimental Pathology Professor of Pathology and Medicine Director, Laboratory for Atherosclerosis and Metabolic Research UC Davis Medical Center Sacramento, CA
Steps in the Progression of the Atherosclerotic Plaque Libby Nature 2002
enos-no
Endothelial Dysfunction Diabetes Hypertension Smoking Dyslipidemia enos Prostacyclin Endothelin-1 CAMS PAI-1
ENDOTHELIAL CELL NO Reduced enos activity, stability, espression and phosphorylation, enos uncoupling ENDOTHELIAL CELL NO ROS Reduced enos activity, stability, espression and phosphorylation, enos uncoupling ROS No Bioavailability Apoptosis and senescene Vascular homeostasis NORMAL ENDOTHELIAL FUNCTION Aging and other cardiovascular risk factors Disrupted Vascular Homeostasis IMPAIRED ENDOTHELIAL FUNCTION Normal numbers and functional activity Circulating Endothelial Progenitor Cell Reduced mobilisation, survival and homing Reduced numbers and impaired functional activity Circulating Endothelial Progenitor Cell Craenenbroeck and Conraads. Microvasc Res. 2010
CARDIOVASCULAR RISK FACTORS Advanced age, hypertension, obesity diabetes, smoking, hyperlipidemia, sedentarity ENDOTHELIAL DYSFUNTION ENDOTHEILIAL PROGENATOR CELLS Reduced circulating numbers Impaired anglogenic capacity Physical Activity Healthy Diet Smoking cessasion Weight Reduction Stress Reduction Craenenbroeck and Conraads. Microvasc Res. 2010
Schematic drawing of conditions influencing the liberation of EPCs from the bone marrow, and its therapeutical application in various cardiovascular diseases Mobius-Winkler et al. Cytometry, 2009
Endothelial Progenitor Cells Werner and Nickenig. JCEM, 2006
EPC Measurement Flow Cytometry Cell Culture: Colony forming units (CFUs) Migration (VEGF, SDF) Adhesion (Fibronectin) Tubule Formation (DiI-EPC and HAEC) In-vivo (Hind limb ischemia)
Schematic drawing of the isolation and characterization of endothelial progenitor cells starting with circulating blood Mobius-Winkler et al. Cytometry, 2009
The two main protocols for culture of putative EPCs. On the left, the 5-day CFU-EC assay, which is based on pre-plating total mononuclear cells (MNC) and re-plating nonattaching cells. On the right, the prolonged culture protocol needed to isolate late ( true ) EPCs (Endothelial Colony Forming Cells, ECFC). Fadini et al. Atherosclerosis, 2008
Mean cell count of diverse progenitor cell phenotypes obtained in a total of 439 subjects using FITC-conjugated anti-cd34, PEconjugated anti-kdr and APC-conjugated anti-cd133 Fadini et al. Atherosclerosis, 2008
Technical Hints for Analyses of EPCs Fadini et al. Atherosclerosis, 2008
Relation between the Number of Endothelial Progenitor Cells and Endothelial Function Hill J et al. N Engl J Med 2003
Association between Cardiovascular Risk Factors and Endothelial- Progenitor-Cell Colony Counts Hill J et al. N Engl J Med 2003
Effect of risk factors on CD34/KDR positive cells Vasa, M. et al. Circ Res 2001
Effect of risk factors on migration of EPCs Vasa, M. et al. Circ Res 2001
Cumulative Event-free Survival in an Analysis of Death from Cardiovascular Causes at 12 Months, According to Levels of Circulating CD34+KDR+ Endothelial Progenitor Cells at the Time of Enrollment Werner N et al. N Engl J Med 2005
Number of Colony Forming Units Endothelial Cells and Outcome P = 0.034 Werner and Nickenig. JCEM, 2006
Median EPC counts by severity of CAD CFU Us 14 12 10 8 6 4 2 0 P <0.0088 0/1 Vessel Multivessel Median Kunz et al. Am Heart J 2006
Multivariable predictors of CAD severity Kunz et al. Am Heart J 2006
Endothelial progenitor cell (EPC) colony-forming units (A) and migration (B) in healthy, sedentary young, middle-aged, and older men Hoetzer, G. L. et al. J Appl Physiol, 2007
Event-free survival according to levels of circulating CD34+KDR+-EPCs defined by ROC curve analysis Schmidt-Locke, C. et al. Circulation 2005
EPC and MACE Crude, Disease Activity-Adjusted, and Risk Factor-Adjusted Relative Risks of a First Major Cardiovascular Event in Patients With Low EPC Count. Variable HR for MACE (95% Cl) Crude relative risk 6.3 (1.8-21.8) 0.003 Disease activity-adjusted relative 4.2 (1.1-16.0) 0.032 risk Risk factor-and disease activityadjusted 3.9 (1.1-14.6) 0.036 MACE indicates major adverse cardiovascular event. P Schmidt-Lucke, C. et al. Circulation 2005
The Northern Manhattan Study (NOMAS) Flow Media ated Dilation(%) 8 7 6 5 4 3 2 1 6.29 ± 3.76 5.26 ± 3.70 P <0.0001 1 2 No MetS (n = 442) MetS (n = 377) Suzuki et al. Am Heart J 2008
Control (n=30) MetS (n=45) P value Age (yrs) 49 ± 11 54 ± 11 0.08 Females/Males 25/6 35/10 0.99 Waist circumference (cm) 94 ± 15 110±14 0.0001 Weight (kg) 85±22 102±19 0.0005 BMI (kg/m2) 30±6 36±6 0.0002 Systolic BP(mmHg) 121 ± 12 130± 13 0.001 Diastolic BP (mmhg) 74 ± 8 82 ± 10 0.0003 Fasting Glucose (mg/dl) 90 ± 7 101± 10 0.0001 Total Cholesterol (mg/dl) 185 ± 33 199 ± 27 0.048 HDL-Cholesterol (mg/dl) 65 ± 13 41 ± 12 0.0001 LDL-Cholesterol (mg/dl) 114 ± 27 129± 20 0.0001 Triglycerides (mg/dl) 75 (62,95) 134 (106,172) 0.0001 Fasting insulin (uiu/ml) 8.6 ±4.9 16.6 ±9.9 0.0001 HOMA-IR 1.7 (0.9, 2.8) 3.3 (2.3, 5.8) 0.0001 hscrp (mg/l) 1.3 (0.5,3) 3.5(1.6,5.7) 0.005 Data are summarized as mean ± SD except for hscrp and triglycerides which are presented as median (25% percentile, 75% percentile) Jialal et al. Atherosclerosis, 2010
Assays of EPC Functionality
Enumeration of EPCs by FACS Surface Expression (MFI) 30 15 CD34+ PC *p<0.001 CD34+/KDR+ EPC *p<0.001 0 Control (n=30) MetS (n=46) Control (n=30) MetS (n=46) Jialal et al. Atherosclerosis, 2010
CFU in Control and Metabolic Syndrome Subjects 80 70 Number of CFUs/5 hpf 60 50 40 30 20 10 0 Control MetS *p<0.001 (n=25) (n=43) Jialal et al. Atherosclerosis, 2010
Vasculogenic capacity of EPCs in Control and MetS 60 50 Tubules (/5 hpf) 40 30 20 10 *p<0.05 0 Control (n=15) MetS (n=15) Jialal et al. Atherosclerosis, 2010
Migration assay in Control and Metabolic Syndrome Subjects 1000 Migratio on (%) 500 0 Control (n=16) MetS (n=36) Jialal et al. Atherosclerosis, 2010
EPC Mobilizing Factors 600 500 **P<0.01 pg/m ml 400 300 200 100 ***P<0.001 Control (n=38) MetS (n=36) 0 G-CSF SCF Jialal et al. (Submitted), 2010
EPC Mobilizing Factors pg/m ml 90 80 70 60 50 40 30 20 10 0 VEGF* *P<0.05 Control (n=38) MetS (n=36) Jialal et al. 2011
EPC Mobilizing Factors EPC Mobilizing Factors 600 500 **P<0.01 400 pg/ml 300 Control (n=38) MetS (n=36) 200 100 **P<0.01 *P<0.05 ***P<0.001 0 SCF-sR VEGF G-CSF SCF Jialal et al. A.J.Cardiol. 2010
EPC Mobilizing Factors 0.9 0.8 0.7 **P<0.01 ng/m ml 0.6 0.5 0.4 0.3 Control (n=38) MetS (n=36) 0.2 0.1 0 MMP-9
Drugs that modulate Endothelial Progenitor cells Statins Insulin Oestrogens ACE-inhibitors PPAR-Gamma-agonists
Acknowledgements Grants: ADA, NIH S. Devaraj C. Duncan-Staley M. Kaur
Cumulative Event-free Survival in Analysis of a First Major Cardiovascular Event (Myocardial Infarction, Hospitalization, Revascularization, or Cardiovascular Death) at 12 Months, According to Levels of Circulating CD34+KDR+ Endothelial Progenitor Cells at the Time of Enrollment Werner N et al. N Engl J Med 2005
Effect of risk factors on number of EPCs Vasa, M. et al. Circ Res 2001
CD133+ Endothelial Progenitor Cells and Outcome Werner and Nickenig. JCEM, 2006
Craenenbroeck and Conraads. Microvasc Res. 2010
Fadini and Avogaro. Diabetes Obes Metab. 2010
Everaert B. et al. Int J Cardiol. 2010