The Number of Endothelial Progenitor Cell Colonies in the Blood Is Increased in Patients With Angiographically Significant Coronary Artery Disease

Transcription

1 Journal of the American College of Cardiology Vol. 48, No. 8, by the American College of Cardiology Foundation ISSN /06/$32.00 Published by Elsevier Inc. doi: /j.jacc The Number of Endothelial Progenitor Cell Colonies in the Blood Is Increased in Patients With Angiographically Significant Coronary Artery Disease Hasan Güven, MD,* Rebecca M. Shepherd, MD, Richard G. Bach, MD, FACC,* Benjamin J. Capoccia, BS, Daniel C. Link, MD St. Louis, Missouri Coronary Artery Disease OBJECTIVES BACKGROUND METHODS RESULTS CONCLUSIONS The objective of this study was to determine whether the number of endothelial progenitor cells (EPCs) and circulating angiogenic cells (CACs) in peripheral blood was associated with the presence and severity of coronary artery disease (CAD) in patients undergoing coronary angiography. 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. The number of EPCs and CACs were measured in the blood of 48 patients undergoing coronary angiography. Patients with acute coronary syndromes were excluded. Compared with patients without angiographically significant CAD, the number of EPCs was increased ( vs colonies/well, p 0.004) and the number of CACs trended higher ( vs cells per mm 2,p 0.09) among patients with significant CAD. The highest levels of EPCs were isolated from patients subsequently selected for revascularization ( colonies/well). 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. (J Am Coll Cardiol 2006;48: ) 2006 by the American College of Cardiology Foundation Post-natal neovascularization, or neoangiogenesis, was originally thought to result exclusively from the proliferation and migration of mature endothelial cells from pre-existing blood vessels, until the recent demonstration that a population of leukocytes in the blood could differentiate into endothelial cells in vitro and contribute to neoangiogenesis in vivo (1). Evidence has now accumulated that bonemarrow derived cells with angiogenic capability, loosely See page 1588 From the Divisions of *Cardiology, Rheumatology, and Oncology, Washington University School of Medicine, St. Louis, Missouri. This work was supported by grants from the National Institutes of Health (R01 HL to Dr. Link and T32 HL to Dr. Shepherd). Manuscript received December 5, 2005; revised manuscript received March 29, 2006, accepted April 4, termed endothelial progenitor cells (EPCs), circulate in the blood of adults (2,3). Endothelial progenitor cells possess the ability to home to sites of vascular injury and contribute to neoangiogenesis (4). As a consequence, there has been considerable interest in the therapeutic use of EPCs to stimulate angiogenesis after vascular injury. Early clinical trials suggest that local infusion of cell populations containing EPCs may improve revascularization after myocardial infarction or other vascular syndromes (5 8). 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

2 1580 JACC Vol. 48, No. 8, 2006 October 17, 2006: Abbreviations and Acronyms AMI acute myocardial infarction CABG coronary artery bypass grafting CAC circulating angiogenic cell CAD coronary artery disease EPC endothelial progenitor cell FITC fluorescein isothiocynate hs-crp high-sensitivity C-reactive protein LDL low-density lipoprotein PBS phosphate buffered saline VEGF vascular endothelial growth factor 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. METHODS Study protocol. The protocol was approved by the Institutional Review Board at Washington University School of Medicine. Forty-eight patients undergoing cardiac catheterization were enrolled in the study. Inclusion criteria included the ability to give informed consent and referral for coronary angiography for the evaluation of CAD. Exclusion criteria included the presence of an AMI, unstable coronary syndrome, unstable angina, prior coronary artery bypass surgery (CABG), prior coronary angioplasty or stenting, pre-menopausal state or pregnancy, presence of diabetic retinopathy, the presence of symptomatic peripheral vascular disease, underlying infection, cancer, immune suppression, end-stage renal or liver disease, or being initiated on a statin within the past 3 months. In 5 patients, technical problems precluded EPC enumeration. The clinical profile of the patients is shown in Table 1. Angiogenic cell culture. After placement of an arterial sheath, 3 to 5 ml of blood was wasted and then 30 ml of whole blood obtained for cell culture. The heparinized specimen was diluted with phosphate buffered saline (PBS), and the specimen was centrifuged across a density gradient (Histopaque, Sigma-Aldrich, St. Louis, Missouri) at 1,700 g for 30 min. The isolated mononuclear cells were suspended in EGM-2 medium (Cambrex Bio Science, Walkersville, Maryland) supplemented with 20% fetal calf serum and plated onto a 6-well cell culture plate coated with Table 1. Baseline Characteristics No Significant CAD* (n 24) Significant CAD* (n 24) p Value Age (yrs) 59 (38 85) 66 (46 79) 0.69 Male gender (%) Diabetes (%) Smoking history (%) Hypertension (%) Hypercholesterolemia (%) Family history of CAD (%) ACE-I use (%) Statin use (%) WBC (cells/ l) 7,935 7, Lymphocytes (cells/ l) 2,055 1, Monocytes (cells/ l) Max coronary stenosis (%) Revascularization (%) *Significant CAD was defined as 1 or more coronary artery occlusions 70%; subsequent revascularization procedures included percutaneous coronary intervention or coronary artery bypass grafting. ACE-I angiotensin-converting enzyme inhibitor; CAD coronary artery disease; WBC white blood cell.

3 JACC Vol. 48, No. 8, 2006 October 17, 2006: % gelatin (Corning Life Sciences, Acton, Massachusetts) at a concentration of 5 million cells/well. The cell media was replenished every 4 days after a gentle washing with phosphate-buffered saline to remove non-adherent cells. The number of CACs was assessed on days 7 to 12 on culture. Twenty fields of view were assessed by random field imaging at 200 magnification with a Nikon inverted microscope with a grid lens by an observer who was blinded to the clinical profile of the patients. Endothelial progenitor cells were assessed on days 12 to 28 of culture by scanning culture plates for discrete colonies. Because a similar amount of blood was analyzed for each sample, the data are reported as the number of CACs per mm 2 or EPCs per well. A similar reporting system has been used in previously published reports (10,13,22,23). Complete EPC and CAC culture data were available for 43 and 48 patients, respectively. Immunofluorescence. Cells were incubated in 1,1= dioctadecyl-3,3,3=,3=-tetramethyindocarbocyanide-labeled acetylated low-density lipoprotein (Dil-Ac-LDL) at 10 g/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 isotypematched 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. RESULTS Measurement of EPCs and CACs. We first established criteria to rigorously identify EPCs and CACs. Standard methods were used to culture freshly isolated blood mononuclear cells under conditions that favor endothelial cell growth (19). Endothelial progenitor cells were identified by the formation of discrete colonies of endothelial cells on days 14 to 28 of culture. Cells in these colonies stained with ulex-lectin and took up acetylated LDL (Fig. 1). These cells expressed high levels of the endothelial antigens CD34, CD31, CD105, and CD144 but were largely negative for the leukocyte antigens CD45 and CD14 (Fig. 2). Circulating angiogenic cells were identified as adherent spindleshaped cells that stained for ulex-lectin and took up acetylated LDL (Fig. 1). Consistent with previous reports (18,19), these cells expressed the leukocyte antigens CD45 and CD14 but were generally negative for the endothelial antigens CD34, CD105 (endoglin), and CD144 (VE cadherin) (Fig. 2). CD31 (platelet/endothelial cell adhesion molecule-1), which is normally expressed at high levels on endothelial cells and weakly on leukocytes, was expressed at a low level on CACs. Herein, we report the number of circulating EPCs and CACs based on the culture-based assays described above. However, it should be noted that culture-based assays, by their nature, provide an indirect measure of the number of circulating EPCs and CACs. In the population studied, the level of EPCs and CACs did not vary significantly according to age, systolic blood pressure, white blood cell count, serum glucose, serum creatinine, the

4 1582 JACC Vol. 48, No. 8, 2006 October 17, 2006: Figure 1. Phenotype of circulating angiogenic cells (CAC) and endothelial progenitor cells (EPC). Representative photomicrographs of cells on day 7 (CAC) or day 21 (EPC) of culture are shown in the left and right panels, respectively. The top panel shows phase contrast images; the middle panel demonstrates staining for Ulex-lectin; and the lower panel shows DiI-labeled acetylated-low density lipoprotein uptake (LDL-U). Original magnification 100. presence of hypertension, diabetes mellitus, or cigarette smoking (all p NS, data not shown). Patient characteristics. Patients were divided into groups by the presence or absence of significant (ⱖ70% 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 ). 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 ( vs 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. Levels of EPCs were then stratified by dividing patients into groups according to the maximum angiographic stenosis severity: 30%, n 13; ⱖ30% to 70%, n 10; and ⱖ70%, n 20 (Fig. 3C). The number of EPCs was highest among the patients with angiographically severe disease ( colonies per well), lower among patients with intermediate severity CAD ( colonies per well), and significantly lower among patients with normal coronary arteries ( colonies per well, p vs. severe CAD). A significant correlation was observed between the maximum stenosis severity and the number of EPCs (r 0.57, p ) (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 com-

5 JACC Vol. 48, No. 8, 2006 October 17, 2006: Figure 2. Cell surface phenotype of circulating angiogenic cells and endothelial progenitor cells. (A) Representative forward scatter (FSC-H) and side scatter (SSC-H) histograms of cultures on day 7 and 21 of culture. The gates used to analyze cells are shown. (B) Representative histograms of cells analyzed on day 7 (upper panel) and day 21 (lower panel). Isotype controls are shown as green lines. pared with patients who did not have revascularization ( vs colonies/well, respectively, p ) (Fig. 5C). The number of CACs is elevated in patients with angiographically significant CAD. Peripheral blood obtained from the patients without and with significant CAD yielded an average of CACs per mm 2 and CACs per mm 2, respectively (p 0.09) (Fig. 3B). When divided according to degree of CAD (Fig. 3D), the highest number of CACs was observed in the peripheral blood of the 24 patients with at least 1 stenosis 70% ( per mm 2 ), a lower level among the 10 patients with moderate severity disease ( per mm 2 ), and the lowest level among the 14 patients with no stenosis 30% ( per mm 2 ), 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 ( per mm 2,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 ( vs per mm 2, 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). DISCUSSION In this study, we examined the hypothesis that the level of circulating EPCs and CACs was associated with the presence and/or severity of CAD. Because uniform criteria to identify these cells were lacking, an emphasis was placed in this study on distinguishing CACs from EPCs. As shown previously, we demonstrate that CACs are CD45 leukocytes that express the monocytic marker CD14 (18,19). Consistent with previous reports (18,19), these cells secrete VEGF and are able to stimulate angiogenesis in a murine model of hindlimb ischemia (20). In contrast, EPCs are true progenitor cells that form discrete colonies in a culture comprised mainly of mature endothelial cells. In fact,

6 1584 JACC Vol. 48, No. 8, 2006 October 17, 2006: Figure 3. Number of endothelial progenitor cells (EPC) (A and C) and circulating angiogenic cells (CAC) (B and D) isolated from patients without and with significant coronary artery disease (stenosis 70% vs. 70%; A and B), and among patients stratified by maximum coronary stenosis 30%, 30% to 70%, and 70% (C and D), as assessed by coronary angiography. Ingram et al. (20) have shown that EPCs are capable of exponential growth in vitro, achieving at least 100 cell doublings. True EPCs are exceedingly rare in peripheral blood with an estimated frequency in healthy volunteers of 1 EPC per 20 ml of blood (21). This translates to a frequency of approximately 1 EPC per 100 million leukocytes, making EPCs difficult to reliably quantify by flow cytometry without prior sample purification. 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 Figure 4. Correlation of endothelial progenitor cell (EPC) (A) and circulating angiogenic cell (CAC) (B) number with maximum angiographic percent diameter coronary stenosis. Spearman rank correlation was used to assess statistical significance.

7 JACC Vol. 48, No. 8, 2006 October 17, 2006: Figure 5. Number of endothelial progenitor cells (EPC) (A and C) and circulating angiogenic cells (CAC) (B and D) observed in patients stratified by number of vessels with stenosis 70% (A and B), and by subsequent clinical decision for revascularization (C and D). Revasc revascularization; W number of vessels with stenosis 70%. 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 Figure 6. Correlation of endothelial progenitor cell (EPC) (A) and circulating angiogenic cell (CAC) (B) number with high-sensitivity C-reactive protein (hscrp).

8 1586 JACC Vol. 48, No. 8, 2006 October 17, 2006: 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. Acknowledgments The authors would like to thank the cardiologists of the Washington University School of Medicine as well as the staff of the Barnes-Jewish Hospital Cardiac Catherization

9 JACC Vol. 48, No. 8, 2006 October 17, 2006: Laboratory, St. Louis, Missouri, for their assistance with this study. Reprint requests and correspondence: Dr. Daniel Link, Division of Oncology, Washington University School of Medicine, Box 8007, 660 South Euclid Avenue, St. Louis, Missouri REFERENCES 1. Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science 1997;275: Asahara T, Masuda H, Takahashi T, et al. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Circ Res 1999;85: Shi Q, Rafii S, Wu MH, et al. Evidence for circulating bone marrow-derived endothelial cells. Blood 1998;92: Urbich C, Dimmeler S. Endothelial progenitor cells: characterization and role in vascular biology. Circ Res 2004;95: Assmus B, Schachinger V, Teupe C, et al. Transplantation of Progenitor Cells and Regeneration Enhancement in Acute Myocardial Infarction (TOPCARE-AMI). Circulation 2002;106: Kawamoto A, Tkebuchava T, Yamaguchi J, et al. Intramyocardial transplantation of autologous endothelial progenitor cells for therapeutic neovascularization of myocardial ischemia. Circulation 2003; 107: Strauer BE, Brehm M, Zeus T, et al. Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans. Circulation 2002;106: Tateishi-Yuyama E, Matsubara H, Murohara T, et al. Therapeutic angiogenesis for patients with limb ischaemia by autologous transplantation of bone-marrow cells: a pilot study and a randomised controlled trial. Lancet 2002;360: Hill JM, Zalos G, Halcox JP, et al. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med 2003;348: Vasa M, Fichtlscherer S, Aicher A, et al. Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease. Circ Res 2001;89:E Eizawa T, Ikeda U, Murakami Y, et al. Decrease in circulating endothelial progenitor cells in patients with stable coronary artery disease. Heart 2004;90: Werner N, Kosiol S, Schiegl T, et al. Circulating endothelial progenitor cells and cardiovascular outcomes. N Engl J Med 2005;353: George J, Goldstein E, Abashidze S, et al. Circulating endothelial progenitor cells in patients with unstable angina: association with systemic inflammation. Eur Heart J 2004;25: Massa M, Rosti V, Ferrario M, et al. Increased circulating hematopoietic and endothelial progenitor cells in the early phase of acute myocardial infarction. Blood 2005;105: Lambiase PD, Edwards RJ, Anthopoulos P, et al. Circulating humoral factors and endothelial progenitor cells in patients with differing coronary collateral support. Circulation 2004;109: Adams V, Lenk K, Linke A, et al. Increase of circulating endothelial progenitor cells in patients with coronary artery disease after exerciseinduced ischemia. Arterioscler Thromb Vasc Biol 2004;24: Sandri M, Adams V, Gielen S, et al. Effects of exercise and ischemia on mobilization and functional activation of blood-derived progenitor cells in patients with ischemic syndromes: results of 3 randomized studies. Circulation 2005;111: Hur J, Yoon CH, Kim HS, et al. Characterization of two types of endothelial progenitor cells and their different contributions to neovasculogenesis. Arterioscler Thromb Vasc Biol 2004;24: Rehman J, Li J, Orschell CM, March KL. Peripheral blood endothelial progenitor cells are derived from monocyte/macrophages and secrete angiogenic growth factors. Circulation 2003;107: Shepherd RM, Capoccia BJ, Devine S, Ingram D, Link DC. Angiogenic cells can be rapidly mobilized and efficiently harvested from the blood following treatment with AMD3100. Blood In press. 21. Ingram DA, Mead LE, Tanaka H, et al. Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood. Blood 2004;104: Asahara T, Kawamoto A. Endothelial progenitor cells for postnatal vasculogenesis. Am J Physiol Cell Physiol 2004;287:C Laufs U, Werner N, Link A, et al. Physical training increases endothelial progenitor cells, inhibits neointima formation, and enhances angiogenesis. Circulation 2004;109: Walter DH, Rittig K, Bahlmann FH, et al. Statin therapy accelerates reendothelialization: a novel effect involving mobilization and incorporation of bone marrow-derived endothelial progenitor cells. Circulation 2002;105: Mack WJ, Azen SP, Dunn M, Hodis HN. A comparison of quantitative computerized and human panel coronary endpoint measures: implications for the design of angiographic trials. Control Clin Trials 1997;18: Gould KL, Lipscomb K. Effects of coronary stenoses on coronary flow reserve resistance. Am J Cardiol 1994;34: Scanlon PJ, Faxon DP, Audet AM, et al. ACC/AHA guidelines for coronary angiography. A report of the American College of Cardiology/ American Heart Association Task Force on Practice Guidelines (Committee on Coronary Angiography). Developed in collaboration with the Society for Cardiac Angiography and Interventions. J Am Coll Cardiol 1999;33: Folland ED, Vogel RA, Hartigan P, et al. Relation between coronary artery stenosis assessed by visual, caliper, and computer methods and exercise capacity in patients with single-vessel coronary artery disease. The Veterans Affairs ACME Investigators. Circulation 1994;89: Cho HJ, Kim HS, Lee MM, et al. Mobilized endothelial progenitor cells by granulocyte-macrophage colony-stimulating factor accelerate reendothelialization and reduce vascular inflammation after intravascular radiation. Circulation 2003;108: Hu J, Takatoku M, Sellers SE, et al. Analysis of origin and optimization of expansion and transduction of circulating peripheral blood endothelial progenitor cells in the rhesus macaque model. Hum Gene Ther 2002;13: Kong D, Melo LG, Gnecchi M, et al. Cytokine-induced mobilization of circulating endothelial progenitor cells enhances repair of injured arteries. Circulation 2004;110: Ridker PM, Cushman M, Stampfer MJ, Tracy RP, Hennekens CH. Inflammation, aspirin, and the risk of cardiovascular disease in apparently healthy men. N Engl J Med 1997;336: Ridker PM, Hennekens CH, Buring JE, Rifai N. C-reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women. N Engl J Med 2000;342: Sata M, Nagai R. Inflammation, angiogenesis, and endothelial progenitor cells: how do endothelial progenitor cells find their place? J Mol Cell Cardiol 2004;36: Ingram DA, Mead LE, Moore DB, Woodard W, Fenoglio A, Yoder MC. Vessel wall-derived endothelial cells rapidly proliferate because they contain a complete hierarchy of endothelial progenitor cells. Blood 2005;105: Lee KW, Lip GY, Tayebjee M, Foster W, Blann AD. Circulating endothelial cells, von Willebrand factor, interleukin-6, and prognosis in patients with acute coronary syndromes. Blood 2005; 105: