Circulating Progenitor Cells
and Cardiovascular Disease
Arshed A. Quyyumi, MD
Professor of Medicine
Emory Clinical Cardiovascular Research Institute (ECCRI)
Emory University School of Medicine
Atlanta, GA
Grant support:
National Institutes of Health,
American Heart Association,
Woodruff Fund,
Emory Heart and Vascular Center,
Sanofi Aventis,
Novartis,
Lilly,
Pfizer,
Forest,
Amgen,
Genzyme
Advisory Boards:
Amorcyte/Neostem,
Genway/Firstmark
Soteria
Stemedica
 Circulating progenitor cells
 CD34+ cell populations
 Circulating progenitor cells and gender
 Circulating progenitor cells and ischemic syndromes
 Circulating progenitor cells and CVD outcomes
 CD34+ cells as therapy for vascular diseases
Adult Bone Marrow Stem Cell Plasticity
Neural cells
Epidermal cells
Ectodermal
Progenitor Cells
Endothelial Progenitor
Cells
Blood cells
Mesodermal
Progenitor Cells
Hematopoeitic
cells
Bone Marrow
Stem Cells
Endodermal
Progenitor Cells
Hepatocytes
Resident stem cells:
Heart, skeletal muscle,
Adipose tissue, brain,
Lung etc.
Stromal or
Mesenchymal
MAPC
Osteocytes,
Chondrocytes
Myocytes (Skeletal)
(Cardiac)
4
 Circulating progenitor cells
 CD34+ cell populations
 Circulating progenitor cells and gender
 Circulating progenitor cells and ischemic syndromes
 Circulating progenitor cells and CVD outcomes
 CD34+ cells as therapy for vascular diseases
Flow Cytometry
250K
105
200K
200K
104
<APC-A>: CD133
250K
SSC-A
SSC-A
31.8
150K
150K
100K
100K
0.113
10
3
10
2
35.2
0.585
62.4
1.85
0
50K
50K
0
0
0
50K
100K
150K
FSC-A
200K
Gated on MNCs
CD45med
250K
2
3
0 10
10
10
<FITC-A>: CD34
4
Gated on CD34+
within MNCs
10
5
2
3
0 10
10
10
<PE-A>: VEGF-R2
4
10
Within CD34+ gate,
4 quadrants for
marker CD133 and
VEGF-2R
11
5
Fluorescent activated cell sorting (FACS) analysis
for bone marrow derived progenitor cell
populations
 CD34: Hematopoietic stem cell
 CD133: Immature progenitors
 CD133+/VEGF2R+:
Differentiates between
immature and mature
endothelial PCs
 CD34+/CD133+/VEGF2R+:
Presumed ‘EPC’ enriched
 CXCR4: Epitope that is
associated with homing to
areas of ischemia that express
SDF-1
Peichev M et al Blood 2000; 95:952
Hirschi KK. et al ATVB 2008;28:1584
12
Cell Type: Isolated CD34⁺Cells Most Able to Improve Perfusion,
Prevent Apoptosis and Rescue Hibernating Cardiomyocytes
CD34⁺ Cells Exhibit Increased Potency and Safety for Therapeutic Neovascularization after
AMI Compared with Total Mononuclear Cells in Nude Rats:
PBS = Phosphate-buffered saline
loMNCs = 5x10^5 MNC
hiMNCs = contains 5x10^5 CD34+ cells
within MNCs
CD34+ = 5x10^5 CD34+ cells
Capillary Density (perfusion) is greatest in CD34+ cell cohort, and this
correlates with decreased incidence of fibrosis. Effect increases with dose.
Kawamoto et al., Circulation 2006;114;2163-2169
13
Endothelial progenitor cell therapy for acute
myocardial infarction
 Cell 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
BM mononuclear cells
• Uptake of BM mononuclear cells:
1.3 to 2.6% in MI region
CD34+ cells
• Uptake of CD34+ cells: 14-39% in MI
region
Hofmann et al Circ 2005;111:2198-2202
 Circulating progenitor cells
 CD34+ cell populations
 Circulating progenitor cells and gender
 Circulating progenitor cells and ischemic syndromes
 Circulating progenitor cells and CVD outcomes
 CD34+ cells as therapy for vascular diseases
Figure3: Age-related changes in circulating PC populations in men (green) and
women (blue)
Hematopoietic (top) and endothelial-enriched PCs (bottom)
Men: r=-0.16, p=0.011
Women: r=-0.13, p=0.003
Men: r=-0.1, p=0.05
Women: r=-0.04, p=0.4
Men: r=-0.19, p=0.003
Women: r=-0.14, p=0.001
Men: r=-0.1, p=0.09
Women: r=-0.05, p=0.2
18
 Circulating progenitor cells
 CD34+ cell populations
 Circulating progenitor cells and ischemic syndromes
 Circulating progenitor cells and CVD outcomes
 CD34+ cells as therapy for vascular diseases
Circulating progenitor cells in acute coronary
syndromes: comparison with stable CAD
 90 ACS patients (mean age 65±15 yrs, 73% male, 10% STEMI, 76% NSTEMI, and 13%
unstable angina)
 Blood samples were obtained at the time of cardiac catheterization for enumeration of
CPCs as CD45dim cells using flow cytometry.
An age- and gender-matched (1:2) cohort of stable CAD patients were randomly selected
as a control group
Comparison of VEGF2R-expressing CPCs between
stable CAD and ACS categories
*
*
*
*
Mental Stress
 Standardized public
speaking task
 Role playing a difficult Mental
Stress
interpersonal situation
where a close relative
in a nursing home is
being mistreated.
Physical
Stress
Rest
Rest
 Hemodynamic and
electrocardiographic
monitoring
Mental stress
Physical stress
Results
 Effect of mental stress on acute mobilization of progenitor cells
• MS challenge provoked an average 21% increase in the number of circulating
CD34+/VEGF2R+/CXCR4+ cells
 Relationship between progenitor cells and ischemia during mental
stress
 Patients with mental stress ischemia had higher circulating number of
CXCR4-expressing cells at baseline
Circulating Progenitor Cells and Coronary Microvascular Dysfunction: Results
from the NHLBI-Sponsored Women’s Ischemia Syndrome Evaluation - Coronary
Vascular Dysfunction (WISE-CVD) Study
Patients:
•
160 women enrolled in the WISE-CVD
Study with ischemia during stress
testing
•
No obstructive CAD
Protocol:
•
CFR measured as ratio of hyperemic
average peak velocity (APV) in
response to intracoronary adenosine
to baseline APV
•
Lower CFR with adenosine correlated
significantly with higher levels of
CD34+, CD34+/CD133+ and
CD34+/CXCR4+ cells
Circulating Progenitor Cells and Coronary Microvascular Dysfunction: Results
from the NHLBI-Sponsored Women’s Ischemia Syndrome Evaluation - Coronary
Vascular Dysfunction (WISE-CVD) Study
Patients:
•
160 women enrolled in the WISE-CVD
Study with ischemia during stress
testing
•
No obstructive CAD
Protocol:
•
Microvascular endothelial function
measured as the CBF response to
intracoronary acetylcholine (n=48)
•
Lower CBF with acetylcholine
correlated significantly with higher
CD34+/CXCR4+ and CD34+/VEGF+.
Progenitor Cells and ischemic syndromes
• ACS is associated with mobilization of VEGF2R and CXCR4
expressing PCs
• Subjects with microvascular coronary ischemia with reduced
flow reserve and endothelial dysfunction have higher
circulating PC subsets
• PC expressing CXCR4, denoting cells with a capacity to home
to areas of ischemia, are increased in those who develop
ischemia during MS.
• This suggests that even mild ischemia during daily living
may stimulate PC mobilization.
 Circulating progenitor cells
 CD34+ cell populations
 Circulating progenitor cells and sub-clinical vascular
disease
 Circulating progenitor cells and ischemic syndromes
 Circulating progenitor cells and CVD outcomes
 CD34+ cells as therapy for vascular diseases
Risk factors, Vascular Injury, and Regenerative
Capacity
Risk Factors
Endothelial injury
Progenitor Cells
Endothelial dysfunction,
Arterial stiffness
Atherogenesis
Vascular Repair
Relationship Between circulating Progenitor Cell Counts and Long term
CVD outcomes (Death/MI)
•
•
•
•
Patients undergoing coronary angiography: Treated with guideline
based therapies
502 patients in a Discovery cohort ;
403 patients in a Validation cohort.
Total Pooled 905; age 63yrs; 65% male
•
PCs were enumerated by flow cytometry as CD45med+ blood
mononuclear cells expressing CD34, CD133, VEGFR2 and/or CXCR4
•
Followed patients in each cohort for a mean of 2.7 & 1.2 years, for the
primary endpoint of death or myocardial infarction (MI).
92 death/MI (10%)
Higher counts of CD34+ and CD34+/CD133+ cells correlated with:
younger age (p<0.001 both),
male gender (p=0.04 and p<0.011)
higher GFR (p<0.001 both)
Higher CD34+/CD133+ also with greater BMI (p<0.001).
37
CD34+
Relationship Between circulating Progenitor Cell Counts and Event Free
Survival (Major Events - Death/MI)
C statistic improved from 0.713 to 0.752, p=0.024 for CD34/133+38
Relationship Between circulating Progenitor Cell Counts and Long term
CVD outcomes (Death/MI)
•
•
•
•
•
•
Patients undergoing coronary
angiography
502 patients in a Discovery
cohort ;
403 patients in a Validation
cohort.
Total Pooled 905; age 63yrs;
65% male
PCs were enumerated by flow
cytometry as CD45med+ blood
mononuclear cells expressing
CD34, CD133, VEGFR2 and/or
CXCR4
Followed patients in each
cohort for a mean of 2.7 & 1.2
years, for the primary
endpoint of death or
myocardial infarction (MI).
92 death/MI
Cell Population
CD34+
CD34+/133+
Outcome Low v High ROC
Death/MI 2.76 (1.65-4.61)
Death 2.60 (1.45-4.66)
CV Death 2.35 (1.28-4.35)
Death/MI 2.98 (1.68-5.30)
Death 2.66 (1.40-5.03)
CV Death 2.49 (1.28-4.86)
CD34+/VEGF+
Death/MI 1.01(0.60-1.69)
Death 1.18 (0.65-2.14)
CD34+/133+/VEGF
CV Death 1.34 (0.71-2.50)
Death/MI 0.97 (0.59-1.59)
Death 0.83 (0.47-1.47)
CD34+/CXCR4+
CV Death 0.83(0.45-1.50)
Death/MI 2.50 (1.19-5.23)
Death 2.15 (1.00-4.62)
CV death 1.82(0.82-3.96)
39
Relationship Between circulating Progenitor Cell Counts and Event Free
Survival (Major Events - Death/MI)
40
Relationship Between circulating Progenitor Cell Counts and Long term
CVD outcomes (Death/MI)
Conclusion: Low levels of circulating PCs, defined as co-expression of
CD34 and CD133 and CXCR4 epitopes are robustly associated with risk
of future death/MI in patients with CAD.
Implications;
(1) CD34+/CD133+ cells that are enriched for bone marrow-derived
hematopoietic and endothelial progenitors, may represent an index of
global regenerative potential
(2) PCs protect or regenerate damaged endothelium by local or systemic
paracrine effects,
(3) The predictive value of PCs as risk markers was equal or greater than
conventional risk factors such as smoking.
41
 Circulating progenitor cells
 CD34+ cell populations
 Circulating progenitor cells and gender
 Circulating progenitor cells and ischemic syndromes
 Circulating progenitor cells and CVD outcomes
 CD34+ cells as therapy for vascular diseases
Atherosclerosis
Incidence
Risk
PCs
Repair
Age or Risk Factor Years
PCs
Repair
Risk
Acknowledgements
Emory Clinical Cardiovascular Research institute
 Arshed Quyyumi
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Viola Vaccarino
Riyaz Patel
Danny Eapen
Nima Ghassemzadeh
Ronnie Ramadan
Ibhar Al Mheid
Girum Mekonnen
Joseph Poole
Robert Neuman
Pankaj Manocha
Hatem Kassem
Alanna Morris
Ayaz Rahman
Saurabh Dhawan
Salman Sher
Ying Liu
Nino Kavtaradze
Elizabeth Rocco
Sherri Mcdonald
 Cath Lab Attendings and Staff
 Fellows/ Research Volunteers

Emir Veledar

A Maziar Zafari
Laurence Sperling
Edmund Waller
Qunna Li
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DeCode Genetics
Dean Jones PhD (Metabolomics)
Charles Searles (miRNA)
Greg Gibson PhD (Transcriptomics)