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J Am Coll Cardiol, 2006; 48:1579-1587, doi:10.1016/j.jacc.2006.04.101
(Published online 25 September 2006). © 2006 by the American College of Cardiology Foundation |
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,*
* Division of Cardiology, Washington University School of Medicine, St. Louis, Missouri
Division of Rheumatology, Washington University School of Medicine, St. Louis, Missouri
Division of Oncology, Washington University School of Medicine, St. Louis, Missouri
Manuscript received December 5, 2005; revised manuscript received March 29, 2006, accepted April 4, 2006.
* Reprint requests and correspondence: Dr. Daniel Link, Division of Oncology, Washington University School of Medicine, Box 8007, 660 South Euclid Avenue, St. Louis, Missouri 63110 (Email: dlink{at}im.wustl.edu).
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Abstract |
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BACKGROUND: Previous studies have suggested an inverse relationship between levels of circulating EPCs/CACs and the presence of CAD or cardiovascular risk factors, whereas other studies have observed increased numbers of EPCs in the setting of acute ischemia. However, the criteria used to identify specific angiogenic cell subpopulations and methods of evaluating CAD varied in these studies. In the present study, we used rigorous criteria to identify EPCs and CACs in the blood of patients undergoing coronary angiography.
METHODS: The number of EPCs and CACs were measured in the blood of 48 patients undergoing coronary angiography. Patients with acute coronary syndromes were excluded.
RESULTS: Compared with patients without angiographically significant CAD, the number of EPCs was increased (1.11 ± 2.50 vs. 4.01 ± 3.70 colonies/well, p = 0.004) and the number of CACs trended higher (175 ± 137 vs. 250 ± 160 cells per mm2, p = 0.09) among patients with significant CAD. The highest levels of EPCs were isolated from patients subsequently selected for revascularization (5.03 ± 4.10 colonies/well).
CONCLUSIONS: In patients referred for coronary angiography, higher numbers of EPCs, and a trend toward higher numbers of CACs, were associated with the presence of significant CAD, and EPC number correlated with maximum angiographic stenosis severity. Endothelial progenitor cell levels were highest in patients with CAD selected for revascularization.
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The capacity of circulating EPCs to repair vascular damage suggests that they may play a key role in maintaining homeostasis of the endothelium. It follows that the number of circulating EPCs may reflect the "vascular health" of an individual. Nevertheless, the relationship between circulating EPCs and vascular diseases, particularly atherosclerosis, remains controversial and incompletely understood. A limited number of studies have examined the association between the circulating EPC and coronary artery disease (CAD). Both an inverse correlation between the level of circulating EPC and the presence of risk factors for CAD (9,10), and a reduced number of circulating EPCs defined as CD34+ or CD34+KDR+ cells, in patients with angiographically documented CAD compared with healthy controls have been reported (11,12). Others have observed that the number of circulating EPCs is increased in patients with acute myocardial infarction (AMI) and unstable ischemic syndromes (13,14), positively correlates with collateral coronary blood flow in patients with CAD (15), and may increase in response to exercise-induced ischemia (16,17). Thus, the relationship between the level of circulating EPCs and the development of, presence of, and severity of CAD remain unclear.
Previous studies of EPC biology may have been hampered by the lack of uniform criteria to precisely identify EPCs. Classically, EPCs have been recognized based on their growth under culture conditions designed to stimulate endothelial cell growth. However, there is now growing evidence that among circulating cells with angiogenic capacity there are subpopulations with distinct marker phenotypes and potentially distinct roles. Previous studies suggest that at least 2 cell populations capable of mediating angiogenesis can be identified in cultures of blood mononuclear cells (18,19). True EPCs (also termed late outgrowth endothelial progenitors, henceforth termed EPCs) are cells with endothelial progenitor capacity (20). Though expression of CD34, CD133, and KDR defines a cell population enriched for EPCs, they are most reliably measured by their ability to produce large colonies of mature endothelial cells in vitro (21). Several studies have described a second angiogenic cell population in these cultures (18,19). These cells, termed circulating angiogenic cells (CACs) or early EPCs (henceforth termed CACs), are adherent and express certain markers associated with mature monocytes/macrophages but do not express CD34 or CD133. Circulating angiogenic cells secrete large amounts of angiogenic growth factors, including vascular endothelial growth factor (VEGF). Importantly, both EPCs and CACs are able to stimulate neoangiogenesis in animal models of vascular injury (18).
In the present study, we use rigorous criteria to measure EPCs and CACs in the blood of patients referred for cardiac catheterization in whom the presence and severity of CAD was assessed by coronary angiography. Our results show that the number of EPCs and CACs isolated from the blood is increased in patients with angiographically significant CAD.
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Methods |
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Immunofluorescence. Cells were incubated in 1,1' dioctadecyl-3,3,3',3'-tetramethyindocarbocyanide-labeled acetylated low-density lipoprotein (Dil-Ac-LDL) at 10 µg/ml in EGM2 media at 37°C for 4 h (Biomedical Technologies, Staughton, Massachusetts). Cells were then washed in PBS and fixed in 3% formaldehyde/PBS for 20 min at room temperature. Ulex-lectin staining was performed by incubating cells with fluorescein isothiocynate (FITC)-conjugated Ulex europeaus agglutinin I (ulex-lectin, Sigma, St. Louis, Missouri) for 1 h at 4°C. Cells were washed with PBS before visualization using a Nikon microphot-SA fluorescent microscope.
Flow cytometry. Adherent cells were washed twice with PBS and detached with Cell Dissociation Buffer (Invitrogen Corporation, Carlsbad, California) for 20 min at 37°C. The cells were then resuspended in fluorescence activated cell sorting buffer (0.1% sodium azide, 0.2% bovine serum albumin, 1 mM ethylenediaminetetraacetic acid in PBS) and incubated with Fc block (Miltenyi Biotec, Auburn, California) for 10 min at 4°C followed by incubation with 1 of the following antibodies: phycoerythrin-conjugated CD14, CD45, CD34; FITC-conjugated CD31 (all from BD Biosciences, San Diego, California); CD105 (BD Biosciences) conjugated to Alexa Fluor 488 using monoclonal antibody labeling kit (Molecular Probes, Eugene, Oregon); FITC-conjugated CD-144 (VE-cadherin) (Novus Biologicals, Littleton, Colorado). Fluorescent isotype-matched antibodies were used as negative controls. The cells were analyzed on a FACScan flow cytometer using Cell Quest analysis software (Becton-Dickinson, Franklin Lakes, New Jersey) (24).
Assessment of CAD severity.
Coronary angiography was performed using 4-F or 6-F catheters at a standard 30 frames/s and stored on digital media. Angiograms were independently reviewed by 2 cardiologists who were blinded to the results of EPC and CAC cultures and to the clinical history and outcomes. To underscore clinical relevance, and given the limitations of quantitative coronary angiography for assessing the entire coronary tree (24), stenosis severity was assessed by visual estimation. In accordance with lesion severity previously correlated with reduced coronary flow reserve (25) and with clinical standards recognized in the American College of Cardiology/American Heart Association guidelines for coronary angiography (26), patients with severe disease had at least 1 coronary stenosis of 
70%. Coronary arteries were considered normal when angiograms showed smooth lumens with no stenoses >30%. Moderate disease was considered present when angiograms showed stenosis of >30% to <70%. Patients were selected for revascularization by percutaneous coronary intervention or CABG at the discretion of the referring cardiologist.
High-sensitivity C-reactive protein (hs-CRP). Serum hs-CRP was measured on blood samples collected at the time of coronary angiography using an immunonephelometric assay on a BN II analyzer (Dade Behring, Deerfield, Illinois).
Statistical analysis. Data are expressed as mean values ± SEM unless otherwise indicated. Statistical significance was assessed using chi-square analysis or logistic regression. For comparisons of data with more than 2 categorical groups, the Fisher exact test was applied. The correlation between maximal coronary artery stenosis and EPC or CAC number was assessed using the Spearman rank test. A p value of <0.05 was considered statistically significant.
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Results |
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70% diameter stenosis) CAD by angiography. Baseline characteristics of the 2 groups are shown in Table 1. The average maximum stenosis in the patients without and with significant CAD was 19.2% ± 24% and 87.0% ± 16%, respectively (p < 0.0001). Hypertension and hypercholesterolemia were more frequent among patients with significant CAD. The total white blood cell, neutrophil, lymphocyte, and monocyte counts were similar between the 2 groups (all p > 0.10). The number of EPCs is increased in patients with angiographically significant CAD. When compared with patients with no significant CAD, peripheral blood obtained from the patients with angiographically significant CAD yielded a greater number of circulating EPCs (1.11 ± 2.5 vs. 4.01 ± 3.70 EPC colonies per well, respectively, p = 0.004; Fig. 3A). Logistic regression showed that the odds of having angiographically significant CAD increased by 38% for each unit increase in EPCs (odds ratio 1.38, 95% confidence interval 1.07 to 1.77; p = 0.01). For those patients with greater than 4 EPCs per well (n = 12), the odds ratio increased to 6.43 with a 95% confidence interval of 1.32 to 31.3 (p = 0.02). A significant association of EPC number and angiographically significant CAD was still observed after adjusting for age, gender, cardiac risk factors, left ventricular ejection fraction, angiotensin-converting enzyme inhibitor or statin use.
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30% to < 70%, n = 10; and 70%, n = 20 (Fig. 3C). The number of EPCs was highest among the patients with angiographically severe disease (4.01 ± 3.70 colonies per well), lower among patients with intermediate severity CAD (1.78 ± 3.1 colonies per well), and significantly lower among patients with normal coronary arteries (0.59 ± 1.8 colonies per well, p = 0.0006 vs. severe CAD). A significant correlation was observed between the maximum stenosis severity and the number of EPCs (r = 0.57, p = 0.00005) (Fig. 4A). When patients were divided according to number of vessels with significant stenosis, there was no significant difference in the number of EPCs isolated between patients with 1-, 2-, or 3-vessel CAD (Fig. 5A). After angiographic assessment, 13 of 43 study patients were selected by their clinician, independent of this study, for a revascularization procedure. These patients had a significantly higher number of EPCs compared with patients who did not have revascularization (5.03 ± 4.10 vs. 1.34 ± 2.34 colonies/well, respectively, p = 0.0006) (Fig. 5C).
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70% (250 ± 160 per mm2), a lower level among the 10 patients with moderate severity disease (190 ± 122 per mm2), and the lowest level among the 14 patients with no stenosis >30% (165 ± 150 per mm2), although these differences did not reach statistical significance. A significant correlation was observed between the maximum stenosis severity and the level of CAC (r = 0.36, p = 0.01) (Fig. 4B), whereas no significant correlation was observed between coronary stenosis severity and the white blood cell, lymphocyte, or monocyte count assessed in the same peripheral blood (data not shown). When patients were divided according to number of diseased vessels, the greatest number of CACs were isolated from the blood of patients with severe 3-vessel CAD (349 ± 182 per mm2, p = 0.08 vs. patients with no stenosis >30%) (Fig. 5B). Patients who were subsequently selected independently to undergo revascularization (n = 16) had a significantly higher number of CACs compared with patients who did not have revascularization (287 ± 182 vs. 175 ± 121 per mm2, respectively, p = 0.01) (Fig. 5D). The number of EPCs or CACs does not correlate with serum hs-CRP. In blood collected from the 48 study patients simultaneously with the samples used to determine EPC or CAC number, the levels of hs-CRP were measured. By linear regression, there was no correlation observed between the serum level of hs-CRP and either EPCs or CACs (Figs. 6A and 6B); the results were similar when the analysis was repeated and patients with significant CAD were excluded (data not shown).
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Discussion |
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Previous studies have examined the relationship between circulating EPCs and CAD or risk factors predisposing to CAD. Hill et al. (9) reported that the number of circulating EPCs, identified as cell colonies on day 9 of culture that stained with VEGFR2, CD31, and BS1-lectin and took up acetylated LDL, correlated inversely with cardiac risk factors. In that study, CAD was not directly assessed. Vasa et al. (10) observed an inverse correlation between the number of circulating EPCs and both cardiac risk factors and angiographically documented CAD. In their analysis, EPC were identified as cells present on day 4 of culture that stained with ulex-lectin and took up acetylated LDL or by flow cytometry to detect CD34+VEGFR2+ cells in the blood. Eizawa et al. (11) reported that the number of circulating CD34+ cells in the blood inversely correlated with the presence of angiographically documented CAD (11). However, the control group with "no CAD" in that study did not undergo coronary angiography. In the largest study to date, Werner et al. (12) observed an inverse association between the level of circulating EPCs and the risk of cardiovascular events among patients with angiographically documented CAD. Endothelial progenitor cells were measured by flow cytometry as CD34+KDR+ cells and, in a subset of patients, using a culture-based method. However, colonies were only scored on day 7 of culture, whereas, as shown by Ingram et al. (21), many endothelial colonies only appear after 10 to 14 days. With respect to the correlations between EPCs and angiographic or clinical end points, all comparisons in this study were performed with CD34+KDR+ cells quantified by flow cytometry. It should also be noted that patients in whom no CAD was detected by angiography were excluded from the study population, and the relationship between EPC number and the severity of angiographic CAD was not specified in this report.
In the present study, we observed that the number of EPCs positively correlates with the presence and severity of CAD, with the highest number of EPCs detected in patients with angiographically significant CAD selected for a revascularization procedure. Likewise, a trend towards higher CAC number and severity of CAD was observed. Though the results of our study may superficially appear to be at odds with the above studies, there are fundamental differences in the methodologies that likely account for the different results. Importantly, the types of EPCs measured in these studies may well be distinct. For example, in the study by Werner et al. (12), EPCs were primarily measured by flow cytometry as CD34+KDR+ cells. In contrast, we applied the rigorous criteria outlined by Ingram et al. (21) to identify EPCs as cells that formed colonies of mature endothelial cells after 7 to 21 days in culture with angiogenic growth factors. That these 2 methodologies measure distinct cell types is best illustrated by their frequency in the blood. Whereas the frequency of CD34+KDR+ cells in the blood, as reported by Werner et al. (12), was 12 to 1,039 cells/ml, the frequency of true EPCs in our study was 0.05 to 1 per ml of blood. Collectively, these data are consistent with the emerging concept that several blood cell populations with angiogenic activity exist (e.g., true EPC and CD34+KDR+ cells) that have distinct biological properties and are regulated by distinct mechanisms.
There is previous evidence suggesting that coronary ischemia may be associated with an increase in the level of circulating EPCs. Massa et al. (14) showed that the number of CD34+CD133+VEGR2+ cells in the blood was increased in patients with AMI compared with healthy controls or patients with stable CAD. In addition, using a culture-based system to identify EPCs, George et al. (13) reported that circulating EPC number was increased in patients with unstable angina compared with patients with stable angina. Interestingly, clinical stabilization of angina in patients with unstable angina in that cohort resulted in a 2-fold decrease in circulating EPC number. Finally, more recent studies have directly observed that exercise-induced ischemia is associated with an increase in numbers of circulating EPC (16,17). In the present study, we show that EPC number was significantly elevated in patients with at least 1 angiographic stenosis of 70%. Although the classification of significant CAD based on this criteria is somewhat arbitrary, it has been previously correlated with reduced coronary flow reserve and is a widely accepted threshold with clinical relevance as a surrogate for hemodynamic significance (26–28). In our series of generally symptomatic patients, the majority classified with significant CAD by study criteria underwent surgical or percutaneous revascularization, whereas all of the patients independently classified without significant CAD were managed conservatively. It is notable, therefore, that in our study, the highest number of EPCs was observed in patients who underwent a revascularization procedure. Collectively, these data suggest that coronary ischemia may be a proximal determinant of EPC number in the blood.
Although currently speculative, there are several mechanistic possibilities that might account for the increase in EPCs in patients with coronary ischemia. First, it has been established that EPCs can be mobilized from the bone marrow to blood in response to a variety of inflammatory cytokines, including granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor (29–31), and coronary ischemia has been associated with systemic inflammation (32,33). Thus, the increased level of EPCs in patients with coronary ischemia might reflect EPC mobilization from the bone marrow in response to inflammatory cytokine signals. Indeed, previous studies have suggested that the serum level of CRP, a marker of systemic inflammation that has been correlated with mortality from CAD (32,33), may positively correlate with the number of circulating EPCs (13,34). However, in the present study, no significant correlation between EPC number in the blood and serum CRP was observed. A second possibility is suggested by the recent report that EPCs reside within the vascular endothelium (35). It is possible that ischemic damage to the endothelium may result in the release of EPCs into the blood, and this release of EPCs from the vasculature may be sufficient to significantly elevate circulating EPC number. Consistent with this hypothesis, Lee et al. (36) reported that mature endothelial cells were increased in the circulation in patients with acute coronary syndromes.
Study limitations.
As previously described, recent evidence suggests the presence of at least 2 populations of angiogenic cells in the circulation, EPCs and CACs. However, it is possible that other angiogenic cell populations may exist that might alter the interpretation of these data. Moreover, there is suggestive evidence that EPCs as measured in this study represent a heterogenous population of endothelial progenitors with differing proliferative capacity (21). It is possible that stratifying EPCs based on their proliferative status may provide additional insights. In addition, the number of subjects in our study was relatively small, and some group comparisons may have lacked power to detect significant differences for select variables. The classification of significant CAD based on visual estimation of the angiographic percent stenosis of coronary artery lesions at 70% is admittedly arbitrary. However, within the range of angiographically significant CAD including lesions of 70%, this criteria of stenosis severity has been correlated with physiologic significance and has relevance to commonly applied angiographic standards and is widely accepted clinical practice (26–28).
Conclusions. The results of the current study demonstrate that among patients referred for coronary angiography, the number of EPCs in the blood is increased in association with angiographically significant CAD, with a trend toward higher CACs. Given these observed associations, the signals stimulating angiogenic cell proliferation or release, the role of these cells in the progression or stabilization of vascular disease, and their impact on clinical outcome remain important topics for further study.
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