Evidence for genetic regulation of endothelial progenitor cells and their role as biological markers of atherosclerotic susceptibility.

AIMS: Endothelial progenitor cells (EPCs) are found in the peripheral circulation and are capable of endothelial repair and neovascularization. EPC number and function are reduced in subjects with cardiovascular risk factors or proven coronary artery disease (CAD). We hypothesized that EPC number and/or function may be genetically regulated and may vary in healthy adult offspring depending on parental history of CAD. METHODS AND RESULTS: We studied 102 subjects comprising 24 healthy parent-healthy offspring pairs and 27 CAD parent-healthy offspring pairs. We measured the number of circulating CD34(+)VEGFR-2(+) and AC133(+)VEGFR-2(+) EPCs, the number of EPCs grown in culture, and the migration capacity of cultured EPCs towards vascular endothelial growth factor. There was significant correlation in the number of cultured EPCs between healthy parents and their offspring (R = 0.492, P = 0.015) and CAD parents and their offspring (R = 0.751, P < 0.001). Offspring of subjects with CAD had significantly higher numbers of circulating CD34(+)VEGFR-2(+) and AC133(+)VEGFR-2(+) cells (P = 0.018 and P < 0.001, respectively). There was no difference in migration capacity between groups. CONCLUSION: Our results suggest that EPC number is, at least in part, genetically regulated. Circulating EPCs may represent biological markers of occult vascular damage in offspring with hereditary risk of CAD.     

Erythropoietin is a potent physiologic stimulus for endothelial progenitor cell mobilization

Increasing evidence suggests that postnatal neovascularization involves the recruitment of circulating endothelial progenitor cells (EPCs). Hematopoietic and endothelial cell lineages share common progenitors. Cytokines formerly thought to be specific for the hematopoietic system have only recently been shown to affect several functions in endothelial cells. Accordingly, we investigated the stimulatory potential of erythropoietin (Epo) on EPC mobilization and neovascularization. The bone marrow of Epo-treated mice showed a significant increase in number and proliferation of stem and progenitor cells as well as in colony-forming units. The number of isolated EPCs and CD34+/flk-1+ precursor cells was significantly increased in spleen and peripheral blood of Epo-treated mice compared with phosphate-buffered saline-treated mice. In in vivo models of postnatal neovascularization, Epo significantly increased inflammation- and ischemia-induced neovascularization. The physiologic relevance of these findings was investigated in patients with coronary heart disease. In a multivariate regression model, serum levels of Epo and vascular endothelial growth factor were significantly associated with the number of stem and progenitor cells in the bone marrow as well as with the number and function of circulating EPCs. In conclusion, the present study suggests that Epo stimulates postnatal neovascularization at least in part by enhancing EPC mobilization from the bone marrow. Epo appears to physiologically regulate EPC mobilization in patients with ischemic heart disease. Thus, Epo serum levels may help in identifying patients with impaired EPC recruitment capacity.     

The role of endothelial progenitor cells in restenosis and stent thrombosis after percutaneous coronary intervention

Background After percutaneous coronary intervention (PCI), some patients may suffer from restenosis and stent thrombosis. Many studies suggest that endothelial progenitor cell (EPC) has an important role in preventing restenosis and stent thrombosis. A novel stent which can attract EPC has been designed to provide a better outcome for these problems. Method The data of the present review was obtained by searching PUBMED and other databases (1994-2011) using the key terms of "endothelial progenitor cell", "reendothelialization", "restenosis", "stent thrombosis", and "percutaneous coronary intervention". Result Rapid reendothelialization is essential in preventing restenosis and stent thrombosis. EPC can differentiate into endothelial cell and accelerate the reendothelialization. After numerous preclinical and clinical researches , the correlation between circulating EPCs to restenosis still remains poorly understood. However , many studies have shown the important role of EPC in diminishing the risk of thrombosis following stent implantation. Some pharmacological agents have been reported can increase the number and / or functions of EPC. Recently , CD34+ antibody coated stent has been developed to attract EPC to the healing endothelium , and has showed favorable results. Conclusion EPC has important role in rapid reendothelialization after vascular injury. EPC can prevent stent thrombosis after PCI , however the effects of EPC in preventing restenosis need further investigations. The capturing CD34+ stent is safe and significantly decreases stent thrombosis.     

Haematopoietic and endothelial progenitor cells are deficient in quiescent systemic lupus erythematosus

BACKGROUND: Systemic lupus erythematosus (SLE) is associated with a high prevalence of cardiovascular disease. Circulating endothelial progenitor cells (EPCs) contribute to vascular regeneration and repair, thereby protecting against atherosclerotic disease. EPCs are derived from CD34+ haematopoietic stem cells (HSCs), which have an increased propensity for apoptosis in the bone marrow of patients with SLE. AIM: To determine whether circulating HSCs and EPCs are reduced in SLE, contributing to an increased cardiovascular risk. METHODS: Progenitor cells were sampled from 15 female patients with SLE in prolonged clinical remission from their disease and 15 matched healthy controls. HSC and CD34+KDR+ EPCs were quantified by flow cytometry. Annexin V staining was used to identify apoptotic cells. RESULTS: Patients with SLE had reduced levels of circulating CD34+ HSCs and CD34+KDR+ EPCs, associated with increased HSC apoptosis. Compared with controls, the fraction of HSCs that could be identified as EPCs was higher in patients with SLE, consistent with a primary defect of HSCs. EPC outgrowth from mononuclear cells, which depends mainly on CD34- cells, was unaffected. CONCLUSIONS: Patients with SLE have lower levels of circulating HSCs and EPCs, even during clinical remission. The data suggest that increased HSC apoptosis is the underlying cause for this depletion. These observations indicate that progenitor cell-mediated endogenous vascular repair is impaired in SLE, which may contribute to the accelerated development of atherosclerosis.     

The association between circulating endothelial progenitor cells and coronary collateral formation

Aim

We investigated the relationship between coronary collateral formation and circulating endothelial progenitor cells (EPC) in patients undergoing coronary angiography.

Methods and results

Circulating CD133⁺/34⁺ and CD34⁺/KDR⁺ EPCs were determined in 68 patients (normal coronary vessels in 24 patients and coronary artery disease (CAD) in 44 patients) (age: 58.7 ± 10.1, 64.7% male). Circulating EPCs were higher among patients with normal coronary vessels compared to patients with CAD for CD133⁺/34⁺ (p < 0.05) and CD34⁺/KDR⁺ cells (p < 0.05). The number of EPCs were significantly greater in patients with good coronary collateral formation (p < 0.05). EPC count was independent predictor for coronary collateral formation after adjustment for other cardiovascular risk factors and extent of CAD (p = 0.037).

Conclusion

In patients with severe coronary stenosis, those with increased circulating EPCs had better collateral formation compared to those with lower EPC counts. Our findings implicate that in addition to presence of critical stenosis, intact response of bone marrow is necessary for collateral formation in CAD.     

How to accelerate the endothelialization of stents

Coronary artery stenting is currently the most frequently performed percutaneous coronary intervention for the treatment of coronary artery disease. The endothelium is a single layer of endothelial cells lining the vascular wall and plays an integral part in maintaining vascular homeostasis. Stenting however causes significant injury to the vascular wall and endothelium, resulting in inflammation, repair and the development of neointimal hyperplasia. The ability of the endothelium to repair itself depends on both the migration of surrounding mature endothelial cells, and the attraction and adhesion of circulating endothelial progenitor cells (EPCs) to the injured region, which then differentiate into endothelial-like cells. Current therapies with drug-eluting stents interrupt the natural response to damage. Accelerating the reendothelialization of the damaged arterial segment following stent implantation is an attractive form of therapy as it is seen as hastening the natural process of repair. It potentially has the benefit of reducing the amount of neointimal hyperplasia and stent thrombosis. Studies have been performed to identify agents that augment the mobilisation and recruitment of EPCs to the injured area (statins, exercise, estrogen and cytokines). Other studies have looked at seeding stents with endothelial cells or EPCs. The most current approach is to coat anti-CD34 antibodies on a stent surface to attract circulating EPCs to the stent which then differentiate into endothelial-like cells. This approach is currently being tested in safety and feasibility clinical studies.     

Reduced numbers of circulating endothelial progenitor cells in patients with coronary artery disease associated with long-term statin treatment

While statin treatment may transiently mobilize endothelial progenitor cells (EPCs), the dose-dependent effects of a continuous statin therapy on EPCs in patients with chronic coronary artery disease (CAD) have not been analyzed. In 209 patients with angiographically documented CAD, 144 of which received 10-40 mg/day of statins for >8 weeks, the EPC number was determined by flow cytometry directly (CD34(+)/KDR(+), n=58) and after in vitro-culture (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-labeled Ac-LDL (DiI-Ac-LDL(+))/lectin(+), n=209). EPC function was assessed by the formation of colony forming units (CFUs). Univariate analysis revealed that the dose of continuous statin therapy inversely correlated with the EPC number. Treatment with 40 mg/day significantly reduced EPC counts. Multivariate analysis unveiled the statin dose and extent of CAD as independent predictors of reduced EPC numbers. Conversely, obesity predicted increased counts, while CFU development was not detectable in all patients and augmented in females and smokers but not in statin-treated patients. Compared with matched controls, statin-treated patients showed significantly reduced absolute and relative EPC counts. In a prospective analysis, initiation of statin therapy significantly diminished the number of circulating and isolated EPCs after 3 but not after 1 month(s). Thus, the statin dose during chronic and continuous treatment independently predicts reduced numbers of circulating as well as isolated EPCs in patients with CAD.     

非阻塞性冠状动脉粥样硬化患者内皮功能不全的影响因素

目的探讨非阻塞性冠状动脉粥样硬化患者内皮功能不全的影响因素。方法选取2013年6月至2015年6月首都医科大学宣武医院心脏内科收治的因胸痛疑诊冠心病、经冠状动脉造影检查证实为非阻塞性冠状动脉粥样硬化症(冠状动脉狭窄<50%)的患者110例。行肱动脉血流介导内皮依赖性血管舒张功能(FMD)检测,用流式细胞仪检测外周血CD133+/KDR+、CD34+/KDR+以及CD34+/CD133+/KDR+内皮祖细胞(EPCs)。根据FMD分为对照组(FMD>10%)和内皮功能不全组(FMD≤10%)。采用SPSS 19.0软件进行统计分析。应用logistic回归模型分析影响内皮功能不全的因素。结果2组患者CD133+/CDR+、CD34+/KDR+或CD34+/CD133+/KDR+循环EPCs数量比较,差异无统计学意义(P>0.05)。多因素logistic回归分析显示,年龄是影响内皮功能不全的独立危险因素(95%CI 1.004~1.104,P=0.033)。结论非阻塞性冠状动脉粥样硬化患者内皮功能不全的独立影响因素是年龄,而非循环EPCs数量。     

Functional impairment of hematopoietic progenitor cells in patients with coronary heart disease

The circulating form of endothelial progenitors cells (EPCs) are derived from bone marrow (BM)-derived hematopoietic stem cells (HSCs). Enhanced mobilization of EPCs was shown to be linked to cardiac diseases. This study investigated whether reduced EPC levels in advanced coronary heart disease (CHD) are secondary to a functional exhaustion of HSCs in the BM or to reduced mobilization. Number and functional properties of EPCs were assessed in 15 healthy controls, and 40 patients with CHD. The colony-forming unit (CFU) capacity of BM-derived mononuclear cells and the CD34+ HSC number were examined in four healthy volunteers, and 15 CHD patients. EPC number was reduced in CHD patients (P < 0.01 vs. controls). Moreover, the migratory capacity was significantly impaired in EPCs of CHD patients (P < 0.05 vs. controls). On multivariate analysis, CHD was an independent predictor of functional EPC impairment. CFUs were reduced in CHD patients (59.6 +/- 21.2 vs. 75.4 +/- 25.8 in controls, P < 0.05). CHD was also predictor of impaired CFU capacity. In this small clinical study, CHD is associated with selective impairment of HSC function in the BM and in the peripheral blood, which may contribute to impairment of cardiac function.     

Endothelial progenitor cells: role in endothelial function recovery and potential therapeutic application

Endothelial dysfunction and cell loss lead to atherosclerosis and its outcomes. Several recent studies suggested that endothelial progenitor cells (EPCs) play a key role in reendothelialization of injured vessels and in endogenous neovascularization of ischemic tissues. Modulation of EPCs number and functional activity or EPCs transplantation are a promising outlook for treatment of cardiovascular disease. In the present review, assessment of endothelial function, mechanisms of its impairment and role of EPCs in their correction are elucidated.     

Endurance training increases the number of endothelial progenitor cells in patients with cardiovascular risk and coronary artery disease

OBJECTIVE: As regular physical exercise improves endothelial dysfunction and promotes cardiovascular health, we investigated the effect of training on angiogenesis by measuring the number of circulating endothelial progenitor cells (EPC), the level of EPC-mobilizing growth factors and tested vascular function by flow-mediated dilation (FMD) in patients with coronary artery disease (CAD) and cardiovascular risk factors (CVRF). In addition, degradation products of the NO pathway (NOx) were determined. METHODS AND RESULTS: Twenty patients with documented CAD and/or CVRF joined a 12-week supervised running training. Circulating EPCs--defined by the surface markers CD34, KDR and CD133--were measured at baseline and after exercise training by flow cytometry. We found a significant increase in circulating EPCs (2.9+/-0.4-fold increase; P < .0001), which was positively correlated with both, the change of FMD (r = .81, P < .001) and the increase of NOx synthesis (r = .83, P < .001). Plasma VEGF and erythropoietin did not change in response to exercise. However, we observed a positive correlation between the number of EPCs and erythropoietin at baseline (r = .70, P < .01) and after training (r = .73, P < .01). CONCLUSIONS: Regular exercise training augments the number of circulating EPCs in patients with CVRF and CAD and is associated with improved vascular function and NO synthesis.     

Nifedipine improves endothelial function: role of endothelial progenitor cells

Nifedipine has been shown to improve endothelial function. Recent studies have indicated that endothelial function is correlated with the number of circulating endothelial progenitor cells (EPCs), but it is unclear whether nifedipine affects the number and function of EPCs. The aims of this study were to determine the effects of nifedipine on the number and function of EPCs and to investigate the relationship between improvement of endothelial function and EPC numbers in patients with hypertension. Stage 1 hypertensive men (n=37) were randomly divided into the nifedipine group and the control untreated group. The nifedipine group was administered slow-release nifedipine (20 mg) once daily. At baseline and after 4 weeks, flow-mediated dilation, blood pressure, biochemical data, and number of circulating CD34+CD133+ progenitor cells and EPCs were measured. The direct effects of nifedipine on EPC number and function were assessed in vitro. In the nifedipine group, flow-mediated dilation and the numbers of circulating CD34+CD133+ progenitor cells and EPCs were increased, along with a decrease of serum malondialdehyde low-density lipoprotein. The improvement of flow-mediated dilation by nifedipine was correlated with the increase of circulating CD34+CD133+ progenitor cells. Nifedipine also improved angiogenesis-related functions of EPCs (differentiation, migration, and resistance to oxidative stress) in vitro. Thus, nifedipine improved endothelial function and EPC function in stage 1 hypertensive subjects. The latter action may be mediated by reduction of oxidative stress and suppression of EPC apoptosis. These results demonstrate that nifedipine preserves endothelial integrity in patients with hypertension, at least partly, by enhancing EPC numbers and activity.     

Circulating Endothelial Progenitor Cells as Markers for Severity of Ischemic Chronic Heart Failure

IntroductionDespite a high potential of endothelial progenitor cells (EPCs) for diagnostic purposes, the EPC role in developing ischemic chronic heart failure (CHF) has not been determined obviously.The objective of this study was to assess the counts of CD45⁺CD34⁺, CD45⁻CD34⁺, CD14⁺CD309⁺, and CD14⁺CD309⁺Tie2⁺ phenotyped circulating EPCs of various subpopulations in patients with ischemic CHF.Methods and ResultsThe study involved 153 patients (86 male), aged 48–62 years, with angiographically proven coronary artery disease (CAD) and 25 healthy volunteers. CHF was diagnosed in 109 patients (71.2%). Mononuclear cell populations were phenotyped by flow cytofluorimetry. Cardiovascular risk factors, such as type 2 diabetes mellitus, hyperlipidemia, arterial hypertension, and adherence to smoking, may have a negative effect on circulating EPC counts in CAD patients regardless of the presence of CHF. The depletion of the CD14⁺CD309⁺- and CD14⁺CD309⁺Tie2⁺-phenotyped circulating EPC counts is associated with the severity of left ventricular dysfunction, whereas the CD45⁺CD34⁺- and CD45⁻CD34⁺-mononuclear cell counts are more representative of the severity of atherosclerotic coronary artery lesions.ConclusionThe authors found that New York Heart Association functional class of CHF, left ventricular ejection fraction <42%, the N-terminal pro–B-type natriuretic peptide level >554 pg/mL, and Е/Еm ratio >15 U had the highest predictive value for the depletion of the EPC count in CAD patients.     

Endothelial progenitor cell capture stent: safety and effectiveness

The endothelial progenitor cell (EPC) capture stent is an innovative device that makes use of the ability of bone marrow-derived EPCs to migrate to injured arterial segments to facilitate healing. The EPC antibody surface, consisting of a covalently coupled polysaccharide intermediate coating with anti-human CD34 antibodies, is attached to a stainless steel stent. Upon stent placement, the anti-human CD34 antibodies will attract circulating EPCs, which are expected to develop into mature functional endothelium. This accelerated healing strategy aims to lower the risk of restenosis and stent thrombosis, as well as obviate prolonged dual antiplatelet therapy. Since the first-in-man study in 2003, a number of small-to-medium size registry and postmarketing studies that confirmed the good safety profile of the EPC capture stent have been published. However, due to lack of large-scale randomized trials, its effectiveness, compared with bare metal stents and drug-eluting stents, cannot be ascertained. Based on restudy angiographic data, instent late loss was approximately 0.7-0.9 mm, which compares unfavorably with that of drug-eluting stents. In order to improve the effectiveness of the EPC capture stent in reducing restenosis-while maintaining its pro-healing property-a bioengineered sirolimus-eluting stent known as the Combo stent was recently designed to combine the EPC capture technology with the abluminal elution of sirolimus. Data from animal studies have been encouraging. The first-in-man study of the Combo stent has been completed and results were presented. (J Interven Cardiol 2012;25:493-500).     

Angiotensin II impairs endothelial progenitor cell number and function in vitro and in vivo: implications for vascular regeneration

Endothelial progenitor cells (EPCs) contribute to endothelial regeneration. Angiotensin II (Ang II) through Ang II type 1 receptor (AT(1)-R) activation plays an important role in vascular damage. The effect of Ang II on EPCs and the involved molecular mechanisms are incompletely understood. Stimulation with Ang II decreased the number of cultured human early outgrowth EPCs, which express both AT(1)-R and Ang II type 2 receptor, mediated through AT(1)-R activation and induction of oxidative stress. Ang II redox-dependently induced EPC apoptosis through increased apoptosis signal-regulating kinase 1, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase phosphorylation; decreased Bcl-2 and increased Bax expression; and activation of caspase 3 but had no effect on the low cell proliferation. In addition, Ang II impaired colony-forming and migratory capacities of early outgrowth EPCs. Ang II infusion diminished numbers and functional capacities of EPCs in wild-type (WT) but not AT(1)a-R knockout mice (AT(1)a(-/-)). Reendothelialization after focal carotid endothelial injury was decreased during Ang II infusion. Salvage of reendothelialization by intravenous application of spleen-derived progenitor cells into Ang II-treated WT mice was pronounced with AT(1)a(-/-) cells compared with WT cells, and transfusion of Ang II-pretreated WT cells into WT mice without Ang II infusion was associated with less reendothelialization. Transplantation of AT(1)a(-/-) bone marrow reduced atherosclerosis development in cholesterol-fed apolipoprotein E-deficient mice compared with transplantation of apolipoprotein E-deficient or WT bone marrow. Randomized treatment of patients with stable coronary artery disease with the AT(1)-R blocker telmisartan significantly increased the number of circulating CD34/KDR-positive EPCs. Ang II through AT(1)-R activation, oxidative stress, and redox-sensitive apoptosis signal-regulating kinase 1-dependent proapoptotic pathways impairs EPCs in vitro and in vivo, resulting in diminished vascular regeneration.     

Circulating endothelial progenitor cells in patients with dysfunctional versus normally functioning congenitally bicuspid aortic valves

Patients with bicuspid aortic valve (BAV) may gradually develop significant valve dysfunction, whereas others remain free of dysfunction. Factors that determine the prognosis of BAV remain unclear. Because endothelial progenitor cells (EPCs) have a role in the repair of endothelial surfaces after injury, we hypothesized that EPCs may also be involved in preventing BAV degeneration. Accordingly, we compared EPC level and function in patients with BAV with versus without valve dysfunction. The study group included 22 patients with BAV and significant valve dysfunction (at least moderate aortic regurgitation and/or at least moderate aortic stenosis). The control group included 28 patients with BAV without valve dysfunction. All patients had 1 blood sample taken. Proportion of peripheral mononuclear cells expressing vascular endothelial growth factor receptor 2, CD133 and CD34 was evaluated by flow cytometry. EPC colony-forming units (CFUs) were grown from peripheral mononuclear cells, characterized, and counted after 7 days of culture. The 2 groups had similar clinical characteristics except for higher prevalence of hypertension in the dysfunctional valve group. Number of EPC CFUs was smaller in the dysfunctional valve group (32 CFUs/plate, 15 to 42.5, vs 48 CFUs/plate, 30 to 62.5, respectively, p = 0.01), and the migratory capacity of the cells in this group was decreased. In addition, the proportion of cells coexpressing vascular endothelial growth factor receptor 2, CD133, and CD34 tended to be smaller in the dysfunctional valve group. In conclusion, patients with BAV and significant valve dysfunction appear to have circulating EPCs with impaired functional properties. These findings require validation by further studies.     

Synergistic effect of combined intramyocardial CD34+ cells and VEGF2 gene therapy after MI

Previous studies have shown that local angiogenic gene therapy acts, in part, by recruiting endothelial progenitor cells (EPCs) to ischemic tissue. Recent data indicate that patients with the most severe vascular disease may have insufficient or deficient EPCs and the poorest response to angiogenic therapy. Accordingly, we hypothesized that combining human CD34(+) cell implantation with local vascular endothelial growth factor 2 (phVEGF2) gene therapy might overcome these deficiencies. The addition of VEGF2 to EPC cultures resulted in significant and dose-dependent decreases in EPC apoptosis. Phosphorylated Akt (p-Akt) was increased in VEGF2-treated EPCs. In vivo, myocardial infarction (MI) was induced by ligation of the left anterior descending coronary artery in 34 immunodeficient rats. The animals were then randomized to one of four treatment groups: cell therapy alone with human CD34(+) cells; VEGF2 gene therapy alone; combination therapy with CD34(+) cells plus phVEGF2; or CD34(-) cells and 50 microg empty plasmid. Four weeks after MI, animals treated with combination therapy showed improved fractional shortening, increased capillary density, and reduced infarct size compared with the other three groups. Combination therapy was also associated with an increased number of circulating EPCs 1 week after MI. Combined subtherapeutic doses of cell and gene therapy result in a significant therapeutic effect compared to monotherapy. This approach may overcome therapeutic failures (e.g. inability of certain patients to mobilize sufficient EPCs) and may also offer safety advantages by allowing lower dosing strategies.     

冠心病患者循环内皮祖细胞与一氧化氮的关系及临床意义

目的探讨冠心病患者循环内皮祖细胞(EPCs)与NO的关系及临床意义。方法78例入选对象均经选择性冠状动脉造影明确有无冠状动脉狭窄,将其分为冠心病组42例,对照组36例。采集入选者外周血测定NO浓度,同时进行循环EPCs分离培养,14天后显微镜下计数细胞克隆形成单位评估循环EPCs水平。结果冠心病组NO水平明显低于对照组(P<0.001),双支、3支冠状动脉病变组较单支病变组NO水平明显降低(P<0.001)。冠心病组循环EPCs水平均明显低于对照组(P<0.01)。NO浓度与循环EPCs水平呈明显正相关(r=0.867,P<0.0001)。结论NO可能在EPCs动员、归巢过程中发挥重要作用,与冠心病发生及临床表现相关。     

Circulating progenitor cells can be reliably identified on the basis of aldehyde dehydrogenase activity.

OBJECTIVES: Our objective was to develop and assess a novel endogenous progenitor cell (EPC) assay based on aldehyde dehydrogenase (ALDH) activity, and to define the relationship of ALDH-bright (ALDH(br)) cells with previously defined EPCs, patient age, and extent of coronary artery disease. BACKGROUND: Accurate assessment of circulating EPCs is of significant interest, yet current assays have limitations. Progenitor cells display high levels of ALDH activity. An assay based on ALDH activity may offer a simple means for enumerating EPCs. METHODS: We simultaneously determined the numbers of EPCs based on ALDH activity and cell surface expression of CD133, CD34, and vascular endothelial growth factor receptor-2 in 110 patients undergoing cardiac catheterization. We assessed the reproducibility of these estimates, correlation among EPC assays, and the association of ALDH(br) numbers with age and disease severity. RESULTS: Aldehyde dehydrogenase-bright cells were easily identified in nonmobilized peripheral blood with median and mean frequencies of 0.041% and 0.074%, respectively. Aldehyde dehydrogenase-bright cells expressed CD34 or CD133 cell surface markers (57.0% and 27.1%, respectively), correlated closely with CD133+CD34+ cells (r = 0.72; p < 0.001), and differentiated into endothelial cells with greater efficiency than CD133+CD34+ cells. Aldehyde dehydrogenase-bright cell numbers were inversely associated with patient age and coronary disease severity. CONCLUSIONS: Aldehyde dehydrogenase activity represents a novel simplified method for quantifying EPCs. The correlation of ALDH(br) cells with clinical factors and outcomes warrants further study.     

Suppression of endothelial progenitor cells in human coronary artery disease by the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine.

OBJECTIVES: We tested the hypothesis that asymmetric dimethylarginine (ADMA) may be an endogenous inhibitor of endothelial progenitor cells (EPCs). BACKGROUND: Endothelial progenitor cells play a pivotal role in regeneration of injured endothelium, thereby limiting the formation of atherosclerotic lesions. Reduced numbers of EPCs may affect progression of coronary artery disease. Regulation of EPC mobilization and function is mediated in part by nitric oxide (NO). Endogenous inhibitors of NO synthases, such as ADMA, contribute to endothelial dysfunction and injury. METHODS: We used flow cytometry and in vitro assays to investigate the relationship between EPC number and function with ADMA plasma levels in patients with stable angina. RESULTS: The plasma concentration of ADMA was related to the severity of coronary artery disease and correlated inversely with the number of circulating CD34+/CD133+ progenitor cells (r = -0.69; p < 0.0001) and endothelial colony forming units (CFUs) (r = -0.75; p < 0.0001). Adjusting for all patient characteristics, we confirmed these findings in multivariate regression analyses. In vitro differentiation of EPCs was repressed by ADMA in a concentration-dependent manner. Compared with untreated cells, ADMA reduced EPC incorporation into endothelial tube-like structures to 27 +/- 11% (p < 0.001). Asymmetric dimethylarginine repressed the formation of CFUs from cultured peripheral blood mononuclear cells to 35 +/- 7% (p < 0.001). Asymmetric dimethylarginine decreased endothelial nitric oxide synthase activity in EPCs to 64 +/- 6% (p < 0.05) when compared with controls. Co-incubation with the hydroxymethyl glutaryl coenzyme A reductase inhibitor rosuvastatin abolished the detrimental effects of ADMA. CONCLUSIONS: Asymmetric dimethylarginine is an endogenous inhibitor of mobilization, differentiation, and function of EPCs. This contributes to the cardiovascular risk in patients with high ADMA levels and may explain low numbers and function of EPCs in patients with coronary artery disease.