doi: 10.1161/hh1301.093953
© 2001 American Heart Association, Inc.
UltraRapid Communication |
Number and Migratory Activity of Circulating Endothelial Progenitor Cells Inversely Correlate With Risk Factors for Coronary Artery Disease
From the Division of Molecular Cardiology, Department of Internal Medicine IV (M.V., S.F., A.A., K.A., C.U., A.M.Z., S.D.), and Department of Hematology, Internal Medicine III (H.M.), University of Frankfurt, Germany.
Correspondence to Stefanie Dimmeler, PhD, Molecular Cardiology, Department of Internal Medicine IV, University of Frankfurt, Theodor Stern-Kai 7, 60590 Frankfurt, Germany. E-mail dimmeler{at}em.uni-frankfurt.de
Abstract
Abstract—Recent
studies provide increasing evidence that postnatal neovascularization
involves bone marrow–derived circulating endothelial
progenitor cells (EPCs). The regulation of EPCs in patients with
coronary artery disease (CAD) is unclear at present.
Therefore, we determined the number and functional activity of EPCs in
45 patients with CAD and 15 healthy volunteers. The numbers of isolated
EPCs and circulating CD34/kinase insert domain receptor
(KDR)-positive precursor cells were significantly reduced in
patients with CAD by 
40% and 48%, respectively. To determine the
influence of atherosclerotic risk factors, a risk factor score
including age, sex, hypertension, diabetes, smoking, positive family
history of CAD, and LDL cholesterol levels was used. The
number of risk factors was significantly correlated with a reduction of
EPC levels (R=-0.394,
P=0.002) and CD34-/KDR-positive
cells (R=-0.537,
P<0.001). Analysis of
the individual risk factors demonstrated that smokers had significantly
reduced levels of EPCs
(P<0.001) and
CD34-/KDR-positive cells
(P=0.003). Moreover, a positive
family history of CAD was associated with reduced CD34-/KDR-positive
cells (P=0.011). Most
importantly, EPCs isolated from patients with CAD also revealed an
impaired migratory response, which was inversely correlated with the
number of risk factors
(R=-0.484,
P=0.002). By
multivariate analysis, hypertension was
identified as a major independent predictor for impaired EPC migration
(P=0.043). The present
study demonstrates that patients with CAD revealed reduced levels and
functional impairment of EPCs, which correlated with risk factors for
CAD. Given the important role of EPCs for neovascularization of
ischemic tissue, the decrease of EPC numbers and activity may
contribute to impaired vascularization in patients with CAD. The full
text of this article is available at
http://www.circresaha.org.
Key Words: coronary disease • angiogenesis • endothelium
Recent studies provide increasing evidence that postnatal neovascularization does not exclusively rely on sprouting of preexisting vessels, but involves bone marrow-derived circulating endothelial cells.1 These bone marrow–derived endothelial progenitor cells (EPCs) are considered to originate from hematopoietic stem cells, which are positive for CD34 or the more immature marker protein CD133.1 2 3 4 In animals, CD34-positive leukocytes were shown to home to sites of ischemia, to express endothelial antigens such as kinase insert domain receptor (KDR) (vascular endothelial growth factor [VEGF] receptor-2),2 5 and to make a significant contribution to adult blood vessel formation.5 Importantly, injection of isolated CD34-positive cells or cultivated EPCs enhances neovascularization6 7 8 and accelerates the restoration of blood flow in diabetic mice.9 Moreover, increased neovascularization by bone marrow–derived angioblasts or CD34-positive cells was shown to improve cardiac function.10 11
The regulation of EPC mobilization and differentiation in patients with coronary artery disease (CAD) has not been studied so far. Therefore, we investigated the influence of atherosclerotic risk factors on the number and functional activity of EPCs. The present study demonstrates that patients with CAD exhibit reduced levels and functional impairment of EPCs. The reduction of the levels and migratory capacity of EPCs were inversely correlated with the number of risk factors. Multivariate analysis of the individual risk factors revealed smoking as the major independent predictor for the reduction of EPC levels, whereas the migration of EPCs was mainly influenced by hypertension.
Materials and Methods
Characteristics of Study Patients and Healthy
Control Subjects
Forty-five patients with angiographically documented
CAD were prospectively studied. The patient characteristics are
summarized in
Table 1
. Patients with concomitant inflammatory or
malignant disease were excluded. Patients had stable CAD (n=22), acute
coronary syndrome (n=9), or myocardial infarction (n=14) with
positive troponin test. In patients with myocardial infarction, blood
samples were taken 4.2±0.4 days after the acute ischemic
event. None of the patients had previously been treated with a statin.
The LDL cholesterol serum levels ranged from 57 to 261
mg/dL at the time of inclusion into the study.
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The age-matched healthy control group (n=9) consisted of 3 women and 6 men with a mean age of 60±5 years without any evidence of CAD by history and physical examination. Moreover, 6 healthy volunteers (5 women and 1 men; mean age 29±2.3 years) were included in the study. Informed consent was obtained from all patients and healthy volunteers; the study protocol was approved by the local Ethics Committee of the University of Frankfurt.
Definition of Risk Factors for CAD
To determine the overall risk factor load of an
individual subject, a risk factor score including age >40 years, male
sex, hypertension, diabetes, smoking, positive family history of CAD,
and hypercholesterolemia was calculated
according to Vita et al.12
Hypertension was defined as a history of hypertension for >1 year that
required the initiation of antihypertensive therapy by the primary
physician. Smoking was defined as patients revealing a history of
smoking for >2 pack-years and current smoking. Positive family history
for CAD was defined as documented evidence of CAD in a parent or
sibling before 60 years of age.
Hypercholesterolemia was defined as fasting LDL
cholesterol levels exceeding 130 mg/dL. Diabetes was
defined as the need for oral antidiabetic drug therapy or insulin
use.
Isolation, Cultivation, and
Characterization of EPCs
Mononuclear cells were isolated by density gradient
centrifugation with Biocoll
(Biochrom, Berlin, Germany) from 20 mL of
peripheral blood, and 4x106
mononuclear cells were plated on 24-well culture dishes coated with
human fibronectin and gelatin (Sigma) in
endothelial basal medium (EBM) (CellSystems)
supplemented with endothelial growth medium SingleQuots and 20%
FCS. After 4 days in culture, nonadherent cells were removed by
thorough washing with PBS and adherent cells underwent cytochemical
analysis. To detect the uptake of
1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine–labeled
acetylated LDL (DiLDL), cells were incubated with DiLDL (2.4
µg/mL) at 37°C for 1 hour. Cells were then fixed with 2%
paraformaldehyde for 10 minutes and incubated with
FITC-labeled Ulex europaeus
agglutinin I (lectin, 10 µg/mL) (Sigma) for 1
hour. Dual-staining cells positive for both lectin and DiLDL were
judged as EPCs and counted per well. Two or three independent
investigators evaluated the number of EPCs per well by counting 3
randomly selected high-power
fields.6 The reproducibility
of the method was tested in healthy volunteers, which revealed
essentially unchanged levels during a 4-week observational period (data
not shown).
To confirm the endothelial phenotype, the expression of endothelial marker proteins was additionally measured by flow cytometry. EPCs were detached with 1 mmol/L EDTA in PBS, and cells were incubated for 15 minutes with mouse anti-human KDR (Sigma), anti–vascular endothelium cadherin (Santa Cruz), or anti–von Willebrand factor (vWF; Becton Dickinson). Phycoerythrin (PE)–conjugated goat anti-mouse F(ab')2 (DAKO) was used as secondary antibody. For DiLDL/von Willebrand double staining, EPCs were incubated with DiLDL as described above, washed with PBS, and then stained with rabbit anti-human vWF (Calbiochem-Novabiochem) followed by swine anti-rabbit FITC antibodies (DAKO). All incubations were performed at 4°C followed by fixation in 2% paraformaldehyde before fluorescence-activated cell sorter analysis. Single- and 2-color flow cytometric analyses were performed using a FACScan flow cytometer (Becton Dickinson).
To further characterize the endothelial phenotype of EPCs, shear stress–induced upregulation of endothelial NO synthase (eNOS), a specific feature of differentiated endothelial cells, was assessed as previously described.13
Flow Cytometry Analysis
A volume of 100 µL peripheral blood was
incubated for 15 minutes in the dark with monoclonal antibodies against
human KDR (Sigma) followed by PE-conjugated
secondary antibody, with the FITC-labeled monoclonal antibodies against
human CD45 (Becton Dickinson), with the
PE-conjugated monoclonal antibody against human CD133 (Milteny), and
with FITC- or PE-conjugated monoclonal antibodies against human CD34
(Becton Dickinson). Isotype-identical antibodies
served as controls (Becton Dickinson). After
incubation, cells were lysed, washed with PBS, and fixed in 2%
paraformaldehyde before analysis. Each
analysis included 60 000 events.
Migration Assay
Isolated EPCs were detached using 1 mmol/L EDTA
in PBS (pH 7.4), harvested by centrifugation,
resuspended in 500 µL EBM, and counted, and
2x104 EPCs were placed in the upper chamber
of a modified Boyden chamber. The chamber was placed in a 24-well
culture dish containing EBM and human recombinant VEGF (50 ng/mL).
After 24 hours incubation at 37°C, the lower side of the filter was
washed with PBS and fixed with 2% paraformaldeyde. For quantification,
cell nuclei were stained with DAPI. Cells migrating into the lower
chamber were counted manually in 3 random microscopic
fields.14
Statistical Analysis
Data are expressed as mean±SEM. Continuous
variables were tested for normal distribution with the
Kolmogorov-Smirnov test and compared by means of 1-way ANOVA. In case
of nonnormal distribution, nonparametric tests were used
(Mann-Whitney U test or
Kruskal-Wallis ANOVA on ranks).The number of risk factors (risk factor
score) was considered a continuous variable. Categorical
variables were compared by means of the
2 test and the Fisher exact test. Linear
regression analysis and nonparametric bivariate
correlation (Spearman rank correlation coefficient) were used to
compare the number and migratory activity of EPCs with each individual
risk factor as well as with the risk factor score, respectively. The
interaction between risk factors and EPC number and migratory activity
was examined by multivariate analysis using the
multiple stepwise logistic regression model. Statistical significance
was assumed if a null hypothesis could be rejected at
P=0.05. All statistical
analysis was performed using SPSS for
Windows 7.0.
Results
Influence of Risk Factors on EPC
Numbers
To determine the influence of risk factors on EPC
levels, mononuclear cells were isolated from 45 patients with CAD (for
patient characteristics see
Table 1
) and 15 healthy volunteers. Adherent EPCs were
characterized by DiLDL uptake and concomitant lectin binding
(Figure 1A).14 The
endothelial origin was further documented by
demonstrating the expression of KDR (79±7.8%), vWF (74±9%), and
vascular endothelium cadherin (80±8%) by flow
cytometry
(Figure 1B). Moreover, EPCs were shown to be positive for
both DiLDL uptake and vWF
(Figure 1C). Moreover, shear stress–induced eNOS
upregulation, a specific feature of differentiated
endothelial cells, was further demonstrated by
immunoblotting
(Figure 1D).
|
As illustrated in
Figure 2A, the number of EPCs was significantly reduced by
40% compared with age-matched healthy volunteers. The number of
risk factors was significantly correlated with a reduction of EPC
levels
(Figure 2B). Moreover, an inverse correlation between EPC
levels and the number of risk factors was also demonstrated when only
patients with CAD were included into the analysis (n=45,
R=-0.301,
P=0.045).
Univariate analysis of the individual risk factors
(Figures 2C through 2E) revealed that smoking was associated
with significantly lower EPC levels, whereas a minor but nonsignificant
reduction of EPC levels was detected in the presence of hypertension,
diabetes, and a positive family history of CAD
(Figure 2C). Neither age nor LDL cholesterol
levels were correlated with the number of EPCs
(Figures 2D and 2E). By multivariate
analysis, only smoking remained as an independent predictor of
reduced EPC numbers
(Table 2).
|
|
Influence of Risk Factors on Circulating
CD34-/KDR-Positive Progenitor Cells
Circulating EPCs are considered to be
characterized by expression of CD34 and the VEGF receptor KDR.
Therefore, we directly determined the number of CD34/KDR
double-positive cells in the peripheral blood of a subset
of 35 patients with CAD by flow cytometry
(Figure 3). CD34-/KDR-positive cells were significantly
reduced by 48% in patients with CAD compared with 9 age-matched
healthy volunteers
(Figure 4A). The number of risk factors was inversely
correlated with the levels of CD34-/KDR-positive cells
(R=-0.537,
P<0,001;
Figure 4B). Similar results were obtained when only patients
with CAD were included
(R=-0.356,
P=0.036, n=35).
Univariate analysis identified smoking and a
positive family history of CAD as significant predictors of reduced
CD34/KDR levels
(Figure 4C). In addition, increased age and elevated LDL
cholesterol serum levels significantly correlated with
lower numbers of CD34-/KDR-positive cells
(Figures 4D and 4E). In contrast, the number of
CD34-/KDR-positive cells did not significantly differ when patients
were stratified according to sex, hypertension, and diabetes
(Figure 4C). Multivariate analysis
revealed that smoking is the most important independent predictor of
reduced circulating CD34-/KDR-positive cells, whereas a positive family
history of CAD, age, and LDL cholesterol levels did not
reach statistical significance
(Table 2).
|
|
Risk Factors Do Not Influence CD34- or
CD133-Positive Hematopoietic Precursor Cell Numbers
To investigate whether the overall number of
hematopoietic precursor cells is affected in patients with CAD, the
total number of CD34-positive cells was determined. However,
CD34-positive leukocytes were similar in patients with CAD compared
with age-matched volunteers (0.056±0.005% versus 0.05±0.007%
CD34/CD45-positive cells, respectively). Moreover, the number of the
more immature hematopoietic cells expressing CD133 was not different in
patients with CAD (0.085±0.009% versus 0.087±0.01% in healthy
volunteers). A potential influence of risk factors on CD34- or
CD133-positive cells was further evaluated by correlation with the risk
factors for CAD. However, neither CD34 nor CD133 or CD34/CD133
double-positive cells were correlated with the number of risk factors
(R=-0.279,
P=0.55;
R=-0.120,
P=0.406; and
R=-0.124,
P=0.392,
respectively).
Effect of Risk Factors on Migratory
Capacity of EPCs
To assess the functional activity of EPCs, migration of
isolated EPC in response to VEGF was determined in 28 patients with CAD
using a modified Boyden chamber. As illustrated in
Figure 5A, the migratory capacity of EPCs isolated from
patients with CAD was significantly impaired compared with healthy
age-matched volunteers. The number of risk factors for CAD
significantly correlated with a reduction of the migratory capacity of
EPCs
(Figure 5B). To investigate whether the impaired migratory
activity of EPCs to VEGF might be due to a downregulation of the VEGF
receptor, the expression of KDR was analyzed in isolated EPCs
by flow cytometry (see
Figure 1B). However, the expression of the KDR receptor on
the EPCs was not correlated with the numbers of risk factors
(R=0.117,
P=0.667; n=16).
|
Analysis of the individual risk factors revealed
that EPC migration was inhibited in patients with hypertension
(Figure 5C). Moreover, a significant negative correlation was
detectable with respect to age
(R=-0.515,
P=0.001) and LDL
cholesterol levels
(R=-0.452,
P=0.005)
(Figures 4D and 4E), whereas individuals with a positive
family history of CAD exhibited a minor but nonsignificant reduction of
their EPC migration
(Figure 5C). In contrast, gender, smoking, and diabetes did
not have a significant effect on EPC migration
(Figure 5C). When we included all risk factors in a
multivariate analysis, only hypertension was
associated with a significant reduction of EPC migration
(Table 2).
Discussion
The results of the present study demonstrate that atherosclerotic risk factors inversely correlate with the number of differentiated EPCs and CD34-/KDR-positive circulating progenitor cells. Moreover, the functional activity of isolated EPCs as measured by their migratory capacity was impaired in relation to the number of risk factors. Analysis of the individual risk factors indicated that smoking is a major factor, which contributes to reduced numbers of circulating EPCs. In contrast, the migratory capacity appears to be mainly influenced by hypertension, but independent of smoking. Serum LDL cholesterol levels, age, and a positive family history of CAD were additionally shown to determine the number of circulating CD34-/KDR-positive cells and EPC migration. However, no influence of LDL cholesterol levels, age, or a positive family history of CAD was detected when the EPCs were counted after isolation and cultivation. One may speculate that the ex vivo cultivation procedure ameliorates the influence of the risk factors on the number of EPCs, whereas the direct measurement of CD34-/KDR-positive cells might more closely resemble the in vivo conditions.
Given that several experimental studies indicate a significant contribution of EPCs for adult neovascularization,6 7 8 the reduction in the number of EPCs and their functional impairment might contribute to reduced vascularization in patients with CAD. Indeed, previous studies indicated that atherosclerotic risk factors can impair the formation of new blood vessels in response to tissue ischemia. Thus, increases in age or hypercholesterolemia are associated with reduced angiogenesis.15 16 Moreover, clinical data suggest a relation between impaired coronary blood flow regulation and risk factors for CAD.17 Strikingly, the risk factors identified in the present study to affect the number and functional activity of EPCs are well established to also impair the function of mature endothelial cells. Age,17 hypertension,18 smoking,19 cholesterol levels,12 17 and a positive family of CAD,20 as well as the overall number of risk factors,12 have all been shown to be associated with impaired endothelium-mediated vasodilator function of the coronary circulation. Therefore, one may speculate that the impairment of circulating EPCs may contribute to an insufficient regeneration of the endothelium, which may lead to endothelial dysfunction. In support of this hypothesis may be recent experimental data demonstrating that the age-associated decrease of endothelial function correlates with enhanced endothelial cell apoptosis.21 However, because the role of EPCs for regeneration of the endothelium in mature vessels is not yet clarified, further studies are necessary to address this hypothesis.
The mechanisms by which risk factors for CAD reduce EPC numbers remain to be determined. Because the overall population of CD34 or CD133 hematopoietic progenitor cells was not altered, the present data suggest a specific effect of risk factors on the CD34-/KDR-positive subpopulation of hematopoietic cells, which are considered as endothelial precursor cells. There are several possible scenarios by which atherosclerotic risk factors could reduce the number of circulating EPCs. One explanation might be increased apoptosis of premature progenitor cells. Indeed, CD34-positive EPCs were shown to be very sensitive to apoptosis induction.22 Moreover, risk factors for CAD such as smoking are known to increase oxidative stress, a well-established stimulus for apoptotic cell death.23 24 Alternatively, risk factors may interfere with the signaling pathways regulating EPC differentiation or mobilization. It is known that cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) or VEGF can mobilize EPCs.25 26 However, in the present study, the number of risk factors did not correlate with systemic VEGF or GM-CSF serum levels (data not shown).
In addition to the reduction of EPC numbers, EPCs isolated from patients with CAD exhibited a decreased migration in response to VEGF, indicating a functional impairment. The reduced response to VEGF might be due to a downregulation of the VEGF receptor KDR, which mediates the VEGF effects in endothelial cells.27 However, at least the expression of the KDR receptor was not reduced in EPCs derived from patients with CAD compared with those from healthy volunteers (data not shown) and was not correlated to the number of risk factors, suggesting that defects in the downstream signaling pathways might be responsible for the impairment of cell migration. Of the individual risk factors investigated, hypertension emerged as the most important independent predictor of a dramatically reduced EPC migration. These data are consistent with the observation of structural alterations of the microvascular bed in hypertension.28 Moreover, hypertension was shown to be associated with a profound downregulation of tissue hepatocyte growth factor, which is an essential endothelial growth factor.29 30 Thus, a paracrine effect induced by increased angiotensin II levels in hypertensive patients could potentially be involved in the regulation of EPC migration. Additionally, age and LDL cholesterol levels were shown to negatively correlate with EPC migration. This is in accordance with in vitro studies in differentiated endothelial cells, which demonstrate that aging and oxidized LDL can inhibit VEGF-induced endothelial cell migration31 (S.D., unpublished data, 2001). Thereby, oxidized LDL blocked VEGF-induced Akt activation and NO production, which are essential for endothelial cell migration.32 33 Interestingly, although the total number of risk factors for CAD was the best predictor for both reduced levels and the migratory capacity of circulating EPCs, individual risk factors seem to differentially affect the number and the migratory capacity of EPCs. These data suggest that different mechanisms contribute to the impairment in EPC migration compared with the reduced levels of circulating EPCs.
Taken together, the present study demonstrates that EPC numbers and migratory capacity are impaired in patients with CAD, and this impairment relates to the number of risk factors for CAD. The direct effect of risk factors in persons without apparent CAD requires further investigations. However, given the important role of EPCs for neovascularization of ischemic tissue, these data not only give potential insight into the pathophysiological mechanisms contributing to impaired blood vessel formation in patients with CAD but may further provide the basis to more closely define the molecular pathways, which determine the fate of EPCs in patients at risk for CAD, to identify suitable therapeutic targets.
Acknowledgments
This study was supported by the Deutsche Forschungsgemeinschaft (SFB 335, Projects B6 [to S.D.] and C5 to [A.M.Z.]). We thank Christiane Mildner-Rihm and Marga Müller-Adorgan for excellent technical help.
Footnotes
Original received April 23, 2001; revision received June 1, 2001; accepted June 1, 2001.
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