115 Endothelial Progenitor Cells: A Novel Laboratory-Based...
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115 Endothelial Progenitor Cells: A Novel Laboratory-Based Biomarker of Vascular Health Ishwarlal Jialal 2011 Annual Meeting – Las Vegas, NV AMERICAN SOCIETY FOR CLINICAL PATHOLOGY 33 W. Monroe, Ste. 1600 Chicago, IL 60603
Transcript of 115 Endothelial Progenitor Cells: A Novel Laboratory-Based...
115 Endothelial Progenitor Cells: A Novel Laboratory-Based Biomarker of Vascular Health
Ishwarlal Jialal
2011 Annual Meeting – Las Vegas, NV
AMERICAN SOCIETY FOR CLINICAL PATHOLOGY 33 W. Monroe, Ste. 1600
Chicago, IL 60603
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 1.0 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.
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ENDOTHELIAL PROGENITOR CELLS AS A NOVEL BIOMARKER OF
VASCULAR HEALTH
I Jialal, MD, PhD . FRCPath.DABCC, ,
Robert E. Stowell Chair in Experimental PathologyProfessor of Pathology and Medicine
Director, Laboratory for Atherosclerosis and Metabolic ResearchUC Davis Medical Center
Sacramento, CA
Steps in the Progression of the Atherosclerotic Plaque
Libby Nature 2002
eNOS-NO
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Diabetes Hypertension Smoking Dyslipidemia
Endothelial Dysfunction
↓ eNOS
↓ Prostacyclin
↑ Endothelin-1
↑ CAMS
↑PAI-1
ENDOTHELIAL CELL
NO
ROSNo Bioavailability
Apoptosis and senescene
ENDOTHELIAL CELL
NO
ROSReduced eNOS activity, stability, espression and phosphorylation, eNOS uncoupling
Reduced eNOS activity, stability, espression and phosphorylation, eNOS uncoupling
Normal numbers and functional activity
Circulating Endothelial Progenitor Cell
Reduced numbers and impaired functional activity
Circulating Endothelial Progenitor Cell
Vascular homeostasis
NORMAL ENDOTHELIAL FUNCTION
Disrupted Vascular Homeostasis
IMPAIRED ENDOTHELIAL FUNCTIONAging and other cardiovascular risk factors
Reduced mobilisation, survival and homing
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
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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)Mi ti (VEGF SDF)– Migration (VEGF, SDF)
– Adhesion (Fibronectin)– Tubule Formation (DiI-EPC and HAEC)– In-vivo (Hind limb ischemia)
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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 non-
attaching 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, PE-
conjugated anti-KDR and APC-conjugated anti-CD133
Fadini et al. Atherosclerosis, 2008
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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
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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
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Number of Colony Forming Units – Endothelial Cells and Outcome
Werner and Nickenig. JCEM, 2006
P = 0.034
Median EPC counts by severity of CAD
8
10
12
14
s
P <0.0088
Kunz et al. Am Heart J 2006
0
2
4
6
0/1 Vessel Multivessel
CFU Median
Multivariable predictors of CAD severity
Kunz et al. Am Heart J 2006
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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 MACECrude, 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)
P
Crude relative risk 6.3 (1.8-21.8) 0.003Disease activity-adjusted relative 4.2 (1.1-16.0) 0.032
Schmidt-Lucke, C. et al. Circulation 2005
y jrisk
( )
Risk factor-and disease activity-adjusted
3.9 (1.1-14.6) 0.036
MACE indicates major adverse cardiovascular event.
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45678
ated
Dila
tion(
%)
6.29 ± 3.76
5.26 ± 3.70
P <0.0001
The Northern Manhattan Study (NOMAS)
1234
1 2
Flow
Med
ia
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 0.0001HOMA-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) 0.005Data 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
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Enumeration of EPCs by FACS
*p<0.001
15
30
Expr
essi
on (M
FI)
*p<0.001
CD34+ PC
CD34+/KDR+ EPC
Jialal et al. Atherosclerosis, 2010
(n=30) (n=46)Control MetS
0
15
Surf
ace
E
Control MetS(n=30) (n=46)
CFU in Control and Metabolic Syndrome Subjects
40
50
60
70
80
CFU
s/5
hpf
Jialal et al. Atherosclerosis, 2010
*p<0.001
(n=25) (n=43)
Control MetS0
10
20
30
Num
ber o
f C
Vasculogenic capacity of EPCs in Control and MetS
40
50
60
hpf)
Jialal et al. Atherosclerosis, 2010
(n=15) (n=15)
*p<0.05
Control MetS0
10
20
30
Tubu
les
(/5 h
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Migration assay in Control and Metabolic Syndrome Subjects
500
1000
on (%
)
(n=16) (n=36)
Jialal et al. Atherosclerosis, 2010
Control MetS0
500
Mig
ratio
EPC Mobilizing Factors
EPC Mobilizing Factors
400
500
600**P<0.01
Jialal et al. A.J.Cardiol. 2010
0
100
200
300
SCF-sR VEGF G-CSF SCF
pg/m
L
Control (n=38)MetS (n=36)
**P<0.01
*P<0.05***P<0.001
EPC Mobilizing Factors
0 4
0.5
0.6
0.7
0.8
0.9
Control (n=38)
**P<0.01
mL
Jialal et al. (Submitted), 2010
0
0.1
0.2
0.3
0.4
MMP‐9
( )
MetS (n=36)ng/m
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Drugs that modulate Endothelial progenesis
• Statins• Insulin • Oestrogens• Oestrogens• ACE-inhibitors • PPAR-Gamma-agonists
Acknowledgements
• Grants: ADA, NIH
• S. Devaraj
• C. Duncan‐Staley
• M. Kaur
EPC Mobilizing Factors
400
500
600
Control (n=38)mL
**P<0.01
Jialal et al. (Submitted), 2010
0
100
200
300
G-CSF SCF
Control (n=38)MetS (n=36)pg
/m
***P<0.001
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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
40
50
60
70
80
90
Control (n=38)
EPC Mobilizing Factors
*P<0.05
mL
0
10
20
30
40
VEGF*
MetS (n=36)
Jialal et al. (Submitted), 2010
pg/m
Effect of risk factors on number of EPCs
Vasa, M. et al. Circ Res 2001
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CD133+ Endothelial Progenitor Cells and Outcome
Werner and Nickenig. JCEM, 2006
Craenenbroeck and Conraads. Microvasc Res. 2010
Fadini and Avogaro. Diabetes Obes Metab. 2010
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Everaert B. et al. Int J Cardiol. 2010
