115 Endothelial Progenitor Cells: A Novel Laboratory-Based Biomarker of Vascular Health. Ishwarlal Jialal

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1 115 Endothelial Progenitor Cells: A Novel Laboratory-Based Biomarker of Vascular Health Ishwarlal Jialal 211 Annual Meeting Las Vegas, NV AMERICAN SOCIETY FOR CLINICAL PATHOLOGY 33 W. Monroe, Ste. 16 Chicago, IL 663

2 115 Endothelial Progenitor Cells: A Novel Laboratory-Based Biomarker of Vascular Health Heart disease is the leading cause of morbidity and mortality in westernized populations. One of the earliest events in atherogenesis is endothelial dysfunction. There is tremendous interest in a sub?type of progenitor cells, isolated from bone marrow and peripheral blood of adults that have the capacity to circulate, proliferate and differentiate into mature endothelial cells, termed endothelial progenitor cells (EPCs). However, there is much controversy with respect to the definition of EPCs. EPCs are characterized by surface markers, CD34 and VEGFR?2 (KDR) by flow cytometry or by their functionality(migration, Colony Forming units(cfu) and Tubule formation). CD34 KDR is the only EPC phenotype that is reduced with established risk factors and an independent predictor of cardiovascular outcomes. In this session, participants will learn i) isolation (flow cytometry and assessment of EPC function (CFU, tubules, migration etc.) and ii) Overview of EPCs and cardiovascular risk/events. Understand the definition of endothelial progenitor cells and their isolation from circulation. Understand functional assays for EPCs. Understand the role of EPCs in cardiovascular risk. FACULTY: Ishwarlal Jialal Entire Pathology Team New Techniques and Technologies New Techniques & Technologies 1. CME/CMLE Credit Accreditation Statement: The American Society for Clinical Pathology (ASCP) is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education (CME) for physicians. This activity has been planned and implemented in accordance with the Essential Areas and Policies of the Accreditation Council for Continuing Medical Education (ACCME). Credit Designation: The ASCP designates this enduring material for a maximum of 1 AMA PRA Category 1 Credits. Physicians should only claim credit commensurate with the extent of their participation in the activity. ASCP continuing education activities are accepted by California, Florida, and many other states for relicensure of clinical laboratory personnel. ASCP designates these activities for the indicated number of Continuing Medical Laboratory Education (CMLE) credit hours. ASCP CMLE credit hours are acceptable to meet the continuing education requirements for the ASCP Board of Registry Certification Maintenance Program. All ASCP CMLE programs are conducted at intermediate to advanced levels of learning. Continuing medical education (CME) activities offered by ASCP are acceptable for the American Board of Pathology s Maintenance of Certification Program.

Professor of Pathology and Medicine Director,

Research UC Davis Medical Center Sacramento,

3 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 22 enos-no 1

4 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. 21 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. 21 2

Cytometry, 29 Endothelial Progenitor Cells Werner and Nickenig.

5 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, 29 Endothelial Progenitor Cells Werner and Nickenig. JCEM, 26 EPC Measurement Flow Cytometry Cell Culture: Colony forming units (CFUs) Mi ti (VEGF SDF) Migration (VEGF, SDF) Adhesion (Fibronectin) Tubule Formation (DiI-EPC and HAEC) In-vivo (Hind limb ischemia) 3

Schematic drawing of the isolation and characterization of endothelial progenitor cells starting with circulating blood Mobius-Winkler et al.

6 Schematic drawing of the isolation and characterization of endothelial progenitor cells starting with circulating blood Mobius-Winkler et al. Cytometry, 29 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, 28 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, 28 4

7 Technical Hints for Analyses of EPCs Fadini et al. Atherosclerosis, 28 Relation between the Number of Endothelial Progenitor Cells and Endothelial Function Hill J et al. N Engl J Med 23 Association between Cardiovascular Risk Factors and Endothelial- Progenitor-Cell Colony Counts Hill J et al. N Engl J Med 23 5

8 Effect of risk factors on CD34/KDR positive cells Vasa, M. et al. Circ Res 21 Effect of risk factors on migration of EPCs Vasa, M. et al. Circ Res 21 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 25 6

9 Number of Colony Forming Units Endothelial Cells and Outcome P =.34 Werner and Nickenig. JCEM, 26 Median EPC counts by severity of CAD CFU s P <.88 /1 Vessel Multivessel Median Kunz et al. Am Heart J 26 Multivariable predictors of CAD severity Kunz et al. Am Heart J 26 7

10 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, 27 Event-free survival according to levels of circulating CD34+KDR+-EPCs defined by ROC curve analysis Schmidt-Locke, C. et al. Circulation 25 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 P (95% Cl) Crude relative risk 6.3 ( ).3 Disease activity-adjusted relative 4.2 ( ).32 risk Risk factor-and disease activityadjusted 3.9 ( ).36 MACE indicates major adverse cardiovascular event. Schmidt-Lucke, C. et al. Circulation 25 8

11 The Northern Manhattan Study (NOMAS) Flow Media ated Dilation(%) ± ± 3.7 P < No MetS (n = 442) MetS (n = 377) Suzuki et al. Am Heart J 28 Control (n=3) MetS (n=45) P value Age (yrs) 49 ± ± 11.8 Females/Males 25/6 35/1.99 Waist circumference (cm) 94 ± 15 11±14.1 Weight (kg) 85±22 12±19.5 BMI (kg/m2) 3±6 36±6.2 Systolic BP(mmHg) 121 ± 12 13± 13.1 Diastolic BP (mmhg) 74 ± 8 82 ± 1.3 Fasting Glucose (mg/dl) 9 ± 7 11± 1.1 Total Cholesterol (mg/dl) 185 ± ± HDL-Cholesterol (mg/dl) 65 ± ± 12.1 LDL-Cholesterol (mg/dl) 114 ± ± 2.1 Triglycerides (mg/dl) 75 (62,95) 134 (16,172).1 Fasting insulin (uiu/ml) 8.6 ± ±9.9.1 HOMA-IR 1.7 (.9, 2.8) 3.3 (2.3, 5.8).1 hscrp (mg/l) 1.3 (.5,3) 3.5(1.6,5.7).5 Data are summarized as mean ± SD except for hscrp and triglycerides which are presented as median (25% percentile, 75% percentile) Jialal et al. Atherosclerosis, 21 Assays of EPC Functionality 9

12 Enumeration of EPCs by FACS Expression (MFI) Surface E 3 15 CD34+ PC *p<.1 CD34+/KDR+ EPC *p<.1 Control (n=3) MetS (n=46) Control (n=3) MetS (n=46) Jialal et al. Atherosclerosis, 21 CFU in Control and Metabolic Syndrome Subjects 8 7 CFUs/5 hpf Number of C Control MetS *p<.1 (n=25) (n=43) Jialal et al. Atherosclerosis, 21 Vasculogenic capacity of EPCs in Control and MetS 6 5 hpf) Tubules (/5 h *p<.5 Control (n=15) MetS (n=15) Jialal et al. Atherosclerosis, 21 1

13 Migration assay in Control and Metabolic Syndrome Subjects 1 Migratio on (%) 5 Control (n=16) MetS (n=36) Jialal et al. Atherosclerosis, 21 EPC Mobilizing Factors EPC Mobilizing Factors 6 5 **P<.1 4 pg/ml *P<.5 ***P<.1 **P<.1 SCF-sR VEGF G-CSF SCF Control (n=38) MetS (n=36) Jialal et al. A.J.Cardiol. 21 EPC Mobilizing Factors.9.8 **P< ng/m ml Control (n=38) MetS (n=36).2.1 MMP 9 Jialal et al. (Submitted), 21 11

14 Drugs that modulate Endothelial progenesis Statins Insulin Oestrogens ACE-inhibitors PPAR-Gamma-agonists Acknowledgements Grants: ADA, NIH S. Devaraj C. Duncan Staley M. Kaur EPC Mobilizing Factors 6 5 **P<.1 pg/m ml ***P<.1 Control (n=38) MetS (n=36) G-CSF SCF Jialal et al. (Submitted), 21 12

15 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 25 EPC Mobilizing Factors pg/m ml VEGF* *P<.5 Control (n=38) MetS (n=36) Jialal et al. (Submitted), 21 Effect of risk factors on number of EPCs Vasa, M. et al. Circ Res 21 13

16 CD133+ Endothelial Progenitor Cells and Outcome Werner and Nickenig. JCEM, 26 Craenenbroeck and Conraads. Microvasc Res. 21 Fadini and Avogaro. Diabetes Obes Metab

17 Everaert B. et al. Int J Cardiol

ENDOTHELIAL PROGENITOR CELLS AS A NOVEL BIOMARKER OF VASCULAR HEALTH

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