Endothelial Progenitor Cells Dysfunction Research Articles

MiR-155 targets PTCH1 to mediate endothelial progenitor cell dysfunction caused by high glucose

Endothelial progenitor cells (EPCs) are involved in diabetes-associated complications, including diabetic foot ulcer (DFU). Recent reports showed that miR-155 downregulation promotes wound healing in diabetic rats and ameliorates endothelial injury induced by high glucose, but its role in DFU is unknown. We found that miR-155 was overexpressed in EPCs from patients with DFU and in high glucose–induced EPCs from healthy people. Reductions in cell viability, migration, tube formation and nitric oxide production, as well as increases in lactated hydrogenase, cell apoptosis, and reactive oxygen species induced by high glucose, were enhanced by miR-155 overexpression and restrained by miR-155 inhibition. Additionally, dual-luciferase reporter assay demonstrated that miR-155 directly targeted the 3′ untranslated region of patched-1 (PTCH1), a receptor of the sonic hedgehog signaling pathway, and downregulated the mRNA and protein expression of PTCH1. qRT-PCR and Western blot results revealed that the PTCH1 was downregulated in EPCs treated with high glucose. Silencing PTCH1 by PTCH1 siRNA alleviated the protective effect of anti-miR-155 on high glucose–induced EPC dysfunction. Our results indicate that miR-155 worsened high glucose–induced EPC function by downregulating PTCH1. These findings suggest that miR-155 may be a potential therapeutic target for DFU.

Methylglyoxal induced advanced glycation end products (AGE)/receptor for AGE (RAGE)-mediated angiogenic impairment in bone marrow-derived endothelial progenitor cells

ABSTRACTEndothelial cells (ECs) maintain the structure and function of blood vessels and are readily exposed to exogenous and endogenous toxic substances in the circulatory system. Bone marrow-derived endothelial progenitor cells (EPCs) circulate in the blood and differentiate to EC, which are known to participate in angiogenesis and regeneration of injured vessels. Dysfunction in EPC contributes to cardiovascular complications in patients with diabetes, but the precise molecular mechanisms underlying diabetic EPC abnormalities are not completely understood. The aim of this study was to investigate the mechanisms underlying diabetic EPC dysfunction using methylglyoxal (MG), an endogenous toxic diabetic metabolite. Data demonstrated that MG decreased cell viability and protein expression of vascular endothelial growth factor receptor (VEGFR)-2 associated with functional impairment of tube formation in EPC. The generation of advanced glycation end (AGE) products was increased in EPC following exposure to MG. Blockage of receptor for AGE (RAGE) by FPS-ZM1, a specific antagonist for RAGE, significantly reversed the decrease of VEGFR-2 protein expression and angiogenic dysfunction in MG-incubated EPC. Taken together, data demonstrated that MG induced angiogenic impairment in EPC via alterations in the AGE/RAGE-VEGFR-2 pathway which may be utilized in the development of potential therapeutic and preventive targets for diabetic vascular complications.

Resveratrol Improves Tube Formation in AGE-Induced Late Endothelial Progenitor Cells by Suppressing Syndecan-4 Shedding.

Dysfunction of endothelial progenitor cells (EPCs) contributes to cardiovascular complications in diabetes, and resveratrol has been shown to improve EPC functions. Syndecan-4 (Synd4), a cell surface heparin sulfate proteoglycan, has been shown to promote neovascularization. Thus, the present study was performed to determine whether resveratrol promoted angiogenesis of EPCs by regulating Synd4. Late EPCs were isolated from human peripheral blood and stimulated with AGEs. Western blot showed that AGEs induced Synd4 shedding in a dose- and time-dependent manner. AGE-induced Synd4 shedding was partly reversed by NAC or resveratrol, along with normalized ROS production. Overexpression of Synd4 or pretreatment of resveratrol reversed AGE-impaired tube formation of EPCs and regulated the Akt/eNOS pathway. Furthermore, resveratrol suppressed Synd4 shedding via the inhibition of oxidative stress and improved tube formation of late EPCs via the regulation of the Synd4/Akt/eNOS pathway.

The Endothelial Progenitor Cell Dysfunction in Type 2 Diabetes Mellitus: The Link with Heart Failure Developing

The circulating number of Endothelial Progenitor Cells (EPCs) decrease in patients with established heart failure (HF) depending the severity of the disease and may reflect poor prognosis. Developing type 2 diabetes mellitus (T2DM) associates with decreased number and weak function of EPCs that corresponds to impaired vascular repair and endothelial dysfunction. It is well known that T2DM is a risk factor of HF, but the role of EPC dysfunction in T2DM-induced HF is not fully investigated. The short communication is depicted a role of decreased circulating number and lowered function of EPCs in T2DM patients as a predictive biological marker of HF manifestation. It has been suggested that the potential use of EPCs as a novel personified indicator of cardiac dysfunction in diabetics is challenging and requires being under scrutinizes in future

The Impact of Endothelial Progenitor Cell Dysfunction in Heart Failure “Obesity Paradox”

Heart failure (HF) is a major factor in premature death and inability in the patient with established cardiovascular (CV) disease worldwide [1]. Although CV diseases mediate half of the cases of all newly diagnosed HF, diabetes mellitus and other metabolic diseases may sufficiently increase a risk of HF development and progression [2]. On the other hand overweight and obesity strongly associating with the exaggerated level of the CV risk among none-HF individuals do not relate to poor clinical outcomes in HF patients [3,4]. This phenomenon determines the “obesity paradox”, which is being a U-shaped association between body mass index (BMI) and long-term prognosis in HF patients [5,6]. Interestingly, this paradox has found in many people of the HF patients without corresponding to the number of conventional CV risk factors [7]. Although several explanations of causes of “obesity paradox” development are discovered, most studies appear to be limited by the daily use of BMI to classify the severity of obesity [8]. Indeed, standard anthropometric measures of body mass adjusted to height square, hip and waist circumstances with a further calculation of hip-to-waist ratio that appears to be useful for grading overweight, underweight and obesity have implemented into routine clinical practice to easily stratify the patients from the general population.

The Impact of Endothelial Progenitor Cell Dysfunction in Heart Failure “Obesity Paradox”

The Impact of Endothelial Progenitor Cell Dysfunction in Heart Failure “Obesity Paradox”

Puerarin protects endothelial progenitor cells from damage of angiotensin II via activation of ERK1/2‑Nrf2 signaling pathway.

Endothelial progenitor cell (EPC) dysfunction is associated with the formation of carotid atherosclerosis. It has been demonstrated that angiotensin II (AngII) may impair the function of EPCs and puerarin, a natural product, possesses cardiovascular protective effects against oxidative stress and inflammation. Therefore, the present study aimed to investigate the beneficial effects of puerarin in AngII‑induced EPC injury, and to elucidate the underlying mechanisms. Treatment with AngII suppressed EPC proliferation and migration, increased the expression of the senescence marker β‑galactosidase, and the adhesion molecules intracellular adhesion molecule‑1 and vascular cell adhesion molecule‑1. However, the above effects were markedly alleviated by treatment with puerarin in a dose‑dependent manner (1, 10 and 100µM). In addition, AngII significantly increased reactive oxygen species production and the levels of the inflammatory cytokine tumor necrosis factor‑α and interleukin‑6. Notably, these effects were reversed by puerarin. However, it was identified that the impaired EPC functions were due to inhibition of the phosphorylation of extracellular signal‑regulated kinase 1 and 2 (ERK1/2) and the degradation of nuclear factor erythroid 2 like 2 (Nrf2), and treatment with puerarin activated the ERK1/2‑Nrf2 signaling pathway. The results of the present study indicated that puerarin protected AngII‑induced EPC dysfunction via activation of the ERK1/2‑Nrf2 signaling pathway.

Homocysteine inhibits endothelial progenitor cells proliferation via DNMT1-mediated hypomethylation of Cyclin A

Endothelial progenitor cells (EPCs) contribute to neovasculogenesis and reendothelialization of damaged blood vessels to maintain the endothelium. Dysfunction of EPCs is implicated in the pathogenesis of vascular injury induced by homocysteine (Hcy). We aimed to investigate the role of Cyclin A in Hcy-induced EPCs dysfunction and explore its molecular mechanism. In this study, by treatment of EPCs with Hcy, we found that the expression of Cyclin A mRNA and protein were significantly downregulated in a dose-dependent manner. Knockdown of Cyclin A prominently reduced proliferation of EPCs, while over-expression of Cyclin A significantly promoted the cell proliferation, suggesting that Hcy inhibits EPCs proliferation through downregulation of Cyclin A expression. In addition, epigenetic study also demonstrated that Hcy induces DNA hypomethylation of the Cyclin A promoter in EPCs through downregulated expression of DNMT1. Moreover, we found that Hcy treatment of EPCs leads to increased SAM, SAH and MeCP2, while the ratio of SAM/SAH and MBD expression decrease. In summary, our results indicate that Hcy inhibits Cyclin A expression through hypomethylation of Cyclin A and thereby suppress EPCs proliferation. These findings demonstrate a novel mechanism of DNA methylation mediated by DNMT1 in prevention of Hcy associated cardiovascular disease.

Ginsenoside Rb1 prevents homocysteine-induced EPC dysfunction via VEGF/p38MAPK and SDF-1/CXCR4 activation

Hyperhomocystinemia (HHcy) is known as an independent risk factor for cardiovascular disease. Our previous study showed that ginsenoside Rb1, the major active constituent of ginseng, prevents homocysteine (Hcy)-induced endothelial damage. However, the role of ginsenoside Rb1 in Hcy-induced dysfunction in endothelial progenitor cells (EPCs) remains unknown. In the study, we found that ginsenoside Rb1 reversed the Hcy-induced impairment of adhesive and migratory ability in EPCs which were significantly abolished by CXCR4 antagonist AMD3100 and VEGFR2 inhibitor SU5416. Ginsenoside Rb1 significantly reversed Hcy-induced SDF-1 reduction in the supernatant and in the serum. Ginsenoside Rb1 reversed downregulation of SDF-1 and VEGFR2 protein expression, inhibition of p38MAPK phosphorylation induced by Hcy. Re-endothelialization in balloon-injured carotid arteries significantly increased with EPCs transplant, and was even better with Rb1 treatment. This effect was significantly abolished by AMD3100. AMD3100 also decreased the number of CM-DiI labeled EPCs in injured arteries. Here we show for the first time that Rb1 prevents Hcy-induced EPC dysfunction via VEGF/p38MAPK and SDF-1/CXCR4 activation. These findings demonstrate a novel mechanism of the action of Rb1 that may have value in prevention of HHcy associated cardiovascular disease.

Influence of cytokine status on insulin resistance and circulating endothelial progenitor cells in type 2 diabetes mellitus

BackgroundType 2 diabetes mellitus (T2DM), a chronic metabolic disorder caused by insulin resistance (IR) and elevated blood glucose level, may lead to endothelial dysfunction. This can result in the development of various vascular complications, even in clinically controlled glycemic state. AimIt has been experimentally proven that cytokine influences both IR and endothelial progenitor cell (EPC) dysfunction in T2DM patients. The present study evaluated the effect of clinical and metabolic risk factors and cytokine levels on IR and EPC, which are used as critical early biomarkers for estimating the risks associated with T2DM. MethodsThe study involved 58 T2DM patients. They were further subdivided into three groups based on IR score: 32 (55.17%) with normal, 11 (18.97%) with mild-moderate and 15 (25.86%) with severe IR. The relationship of clinical, metabolic and immune mediators with IR and EPCs was verified. ResultsHbA1c% was significantly elevated in severe (P=0.022) and mild-moderate IR groups (P= 0.012) than the normal group. The IR normal group had significantly elevated TNF levels compared to mild-moderate and severe groups. The regression analysis indicated that patients with increased body mass index (BMI) were 19.5% more likely to be significantly associated with severe IR. Association studies demonstrated that IL6 and IL10 values correlated with EPCs. ConclusionIL6 and IL10 were associated with circulating EPCs than IR and other clinical characteristics including glycemic control (glycated hemoglobin). TNF-α was associated with IR, but had no relationship with EPCs. The effect of cytokine status on IR and circulating EPCs in T2DM may indicate the risk of vascular complications.

MeCP2 mediated dysfunction in senescent EPCs.

Aging endothelial progenitor cells (EPCs) exhibit functional impairment in terms of proliferation, migration and survival. SIRT1 plays an important role in improving EPCs function. MeCP2, another important epigenetic regulator, is involved in regulating many life-related activities such as cell growth, death and senescence. Here we aim to explore the effect of MeCP2 on the functional activities of senescent EPCs and the underlying mechanisms. By using western blot and real-time PCR, we found that the expression levels of MeCP2 were up-regulated and SIRT1 were down-regulated with replicative senescence and H2O2-induced senescence. Through transduction with adenoviral vectors, EPCs overexpressing MeCP2 had significantly reduced EPCs function, and silencing MeCP2 improved EPCs function. In addition, the protein and mRNA levels of SIRT1 were decreased with MeCP2 overexpression and increased with MeCP2 knockdown. Through co-transfection of EPCs with MeCP2 and SIRT1, we observed that SIRT1 could reverse the effects of MeCP2 on EPCs. In summary, our work demonstrated that MeCP2 inhibited SIRT1 in senescent EPCs.

Microvesicles Derived from Indoxyl Sulfate Treated Endothelial Cells Induce Endothelial Progenitor Cells Dysfunction.

Cardiovascular disease is a major cause of mortality in chronic kidney disease patients. Indoxyl sulfate (IS) is a typical protein-bound uremic toxin that cannot be effectively cleared by conventional dialysis. Increased IS is associated with the progression of chronic kidney disease and development of cardiovascular disease. After endothelial activation by IS, cells release endothelial microvesicles (EMV) that can induce endothelial dysfunction. We developed an in vitro model of endothelial damage mediated by IS to evaluate the functional effect of EMV on the endothelial repair process developed by endothelial progenitor cells (EPCs). EMV derived from IS-treated endothelial cells were isolated by ultracentrifugation and characterized for miRNAs content. The effects of EMV on healthy EPCs in culture were studied. We observed that IS activates endothelial cells and the generated microvesicles (IsEMV) can modulate the classic endothelial roles of progenitor cells as colony forming units and form new vessels in vitro. Moreover, 23 miRNAs were contained in IsEMV including four (miR-181a-5p, miR-4454, miR-150-5p, and hsa-let-7i-5p) that were upregulated in IsEMV compared with control endothelial microvesicles. Other authors have found that miR-181a-5p, miR-4454, and miR-150-5p are involved in promoting inflammation, apoptosis, and cellular senescence. Interestingly, we observed an increase in NFκB and p53, and a decrease in IκBα in EPCs treated with IsEMV. Our data suggest that IS is capable of inducing endothelial vesiculation with different membrane characteristics, miRNAs and other molecules, which makes maintaining of vascular homeostasis of EPCs not fully functional. These specific characteristics of EMV could be used as novel biomarkers for diagnosis and prognosis of vascular disease.

In vitro assessment of xenogeneic complement and endothelial cell activation using a microvascular flow model

Endothelial progenitor cells (EPCs) contribute to neovasculogenesis and reendothelialization of damaged blood vessels to maintain the endothelium. Dysfunction of EPCs is implicated in the pathogenesis of vascular injury induced by homocysteine (Hcy). We aimed to investigate the role of Cyclin A in Hcy-induced EPCs dysfunction and explore its molecular mechanism. In this study, by treatment of EPCs with Hcy, we found that the expression of Cyclin A mRNA and protein were significantly downregulated in a dose-dependent manner. Knockdown of Cyclin A prominently reduced proliferation of EPCs, while over-expression of Cyclin A significantly promoted the cell proliferation, suggesting that Hcy inhibits EPCs proliferation through downregulation of Cyclin A expression. In addition, epigenetic study also demonstrated that Hcy induces DNA hypomethylation of the Cyclin A promoter in EPCs through downregulated expression of DNMT1. Moreover, we found that Hcy treatment of EPCs leads to increased SAM, SAH and MeCP2, while the ratio of SAM/SAH and MBD expression decrease. In summary, our results indicate that Hcy inhibits Cyclin A expression through hypomethylation of Cyclin A and thereby suppress EPCs proliferation. These findings demonstrate a novel mechanism of DNA methylation mediated by DNMT1 in prevention of Hcy associated cardiovascular disease.

Toll‑like receptor 4 is expressed and functional in late endothelial progenitor cells.

It has been previously demonstrated that lipopolysaccharides (LPS) inhibit the viability, migration, adhesion and invitro angiogenesis of late endothelial progenitor cells (EPCs). However, the mechanisms underlying this LPS‑induced impairment of late EPC functional activity are unknown. The aim of the present study was to investigate whether Toll‑like receptor 4 (TLR4) is expressed and functional on late EPCs, using late EPCs of 3‑5 passages. Cells were deprived of serum for 24h prior to experiments and incubated with 10µg/ml LPS for 24h with or without pretreatment with 2µg/ml TLR4 signaling inhibitor CLI‑095 for 30min. The viability, migration, adhesion and invitro angiogenesis, as well as the expression of silent information regulator1 (SIRT1), in late EPCs were evaluated. Treatment with 10µg/ml LPS decreased the viability, migration and adhesion abilities, and invitro angiogenesis of late EPCs. Pretreatment with the TLR4 signaling inhibitor reversed this LPS‑induced dysfunction of late EPCs. LPS downregulated the expression of SIRT1 protein, however, blocking TLR4 attenuated the effect of LPS on SIRT1 expression. Therefore, the results of the present study indicate that LPS impaired the functional activity of late EPCs via TLR4, which may be associated with decreased SIRT1 expression.

Endothelial progenitor cell dysfunction in acute exacerbation of chronic obstructive pulmonary disease.

Endothelial progenitor cells (EPCs) are decreased in cardiac dysfunction morbidity associated with acute exacerbation of chronic obstructive pulmonary disease (AECOPD). Therefore, the present study aimed to assess the role of EPCs in AECOPD. Patients with AECOPD (n=27) or stable COPD (n=26) were enrolled. Systemic inflammatory markers (high-sensitivity C-reactive protein) were measured. In addition, EPCs were counted, isolated and cultured, and their proliferative, migratory, adhesive and tube-forming capabilities were determined, in cells from patients with AECOPD and stable COPD. EPC number was lower in patients with AECOPD (5.1±2.6×103/ml) compared with patients with stable COPD (6.0±3.2×103/ml). Migration assay indicated that the early-EPCs isolated from patients with AECOPD were significantly less mobile than EPCs derived from stable COPD subjects, at a stromal-cell derived factor-1α concentration of 100 ng/ml (3,550/30,000 vs. 7,853/30,000, P<0.05). C-X-C chemokine receptor-4 positivity was significantly reduced in AECOPD patients (16.1±9.9 vs. 56.33±6.3%, P<0.05). Furthermore, fewer early-EPC clusters were formed by EPCs derived from AECOPD, compared with those derived from stable COPD (8.2±0.86 vs. 14.4±1.36, P=0.027). Stable COPD late-EPCs were markedly deficient in intact tubule formation, however AECOPD late-EPCs formed no tubules. The number of AECOPD- and stable COPD-derived late-EPCs adhering to Matrigel-induced tubules was 36.8±1.85 and 20.6±1.36 (P<0.05) respectively, and the cluster of differentiation 31 positivity in late-EPCs was 79.69±1.3 and 29.1±2.47%, in AECOPD and stable COPD patients, respectively (P<0.001). The findings demonstrated that early-EPCs are decreased and dysfunctional in AECOPD patients, which may contribute to the altered vascular endothelium in this patient population.

Curcumin induces therapeutic angiogenesis in a diabetic mouse hindlimb ischemia model via modulating the function of endothelial progenitor cells

BackgroundNeovascularization is impaired in diabetes mellitus, which leads to the development of peripheral arterial disease and is mainly attributed to the dysfunction of endothelial progenitor cells (EPCs). Previous studies proved the promotional effect of curcumin on neovascularization in wound healing of diabetes. Thus, we hypothesize that curcumin could promote neovascularization at sites of hindlimb ischemia in diabetes and might take effect via modulating the function of EPCs.MethodsStreptozotocin-induced type 1 diabetic mice and nondiabetic mice both received unilateral hindlimb ischemic surgery. Curcumin was then administrated to the mice by lavage for 14 days consecutively. Laser Doppler perfusion imaging was conducted to demonstrate the blood flow reperfusion. Capillary density was measured in the ischemic gastrocnemius muscle. In addition, angiogenesis, migration, proliferation abilities, and senescence were determined in EPCs isolated from diabetic and nondiabetic mice. Quantitative PCR was then used to determine the mRNA expression of vascular endothelial growth factor (VEGF) and angiopoetin-1 (Ang-1) in EPCs.ResultsCurcumin application to type 1 diabetic mice significantly improved blood reperfusion and increased the capillary density in ischemic hindlimbs. The in-vitro study also revealed that the angiogenesis, migration, and proliferation abilities of EPCs and the number of senescent EPCs were reversed by curcumin application. Quantitative PCR confirmed the overexpression of VEGF-A and Ang-1 in EPCs after curcumin treatment.ConclusionCurcumin could enhance neovascularization via promoting the function of EPCs in a diabetic mouse hindlimb ischemia model.

Role of microparticles as microrna messengers in reestablishing of atherosclerosis-associated endothelial progenitor cell dysfunction

Role of microparticles as microrna messengers in reestablishing of atherosclerosis-associated endothelial progenitor cell dysfunction

High glucose–induced endothelial progenitor cell dysfunction

Vascular complications contribute significantly to morbidity and mortality of diabetes mellitus. The primary cause of vascular complications in diabetes mellitus is hyperglycaemia, associated with endothelial dysfunction and impaired neovascularization. Circulating endothelial progenitor cells was shown to play important roles in vascular repair and promoting neovascularization. In this review, we will demonstrate the individual effect of high glucose on endothelial progenitor cells. Endothelial progenitor cells isolated from healthy subjects exposed to high glucose conditions or endothelial progenitor cells isolated from diabetic patients exhibit reduced number of endothelial cell colony forming units, impaired abilities of differentiation, proliferation, adhesion and migration, tubulization, secretion, mobilization and homing, whereas enhanced senescence. Increased production of reactive oxygen species by the mitochondria seems to play a crucial role in high glucose-induced endothelial progenitor cells deficit. Later, we will review the agents that might be used to alleviate dysfunction of endothelial progenitor cells induced by high glucose. The conclusions are that the relationship between hyperglycaemia and endothelial progenitor cells dysfunction is only beginning to be recognized, and future studies should pay more attention to the haemodynamic environment of endothelial progenitor cells and ageing factors to discover novel treatment agents.

Cigarette Smoke Extract Changes Expression of Endothelial Nitric Oxide Synthase (eNOS) and p16(INK4a) and is Related to Endothelial Progenitor Cell Dysfunction.

BackgroundEndothelial dysfunction is an important pathophysiologic feature in many smoke-related diseases. Endothelial progenitor cells (EPCs) are the precursors of endothelial cells and play a fundamental role in the maintenance of endothelial integrity and function. Endothelial nitric oxide synthase (eNOS) is the dominant NOS isoform in the vasculature and plays a central role in the maintenance of endothelial homeostasis. p16(INK4a) is a cyclin-dependent kinase inhibitor and could be regarded as a major dominant senescence gene. The present study aimed to determine whether the expression of eNOS and p16(INK4a) in EPCs is related to EPCs function and the possible epigenetic mechanism, if any.Material/MethodsWe investigated EPCs capacity for proliferation, adhesion, and secretion, and the expression of eNOS and p16(INK4a) in EPCs which were altered by cigarette smoke extract (CSE) in vitro. Furthermore, Decitabine (Dec), an agent of demethylation, was used to examine whether it could alter the changes induced by CSE.ResultsThe present study demonstrated that EPCs altered by CSE in vitro displayed decreased capacities of proliferation, adhesion, and secretion, which was accompanied by decreased eNOS expression and increased p16(INK4a) expression in EPCs. Furthermore, Dec could alleviate the changes in the expression of eNOS and p16(INK4a), and protect against the EPCs dysfunction caused by CSE.ConclusionsThe decreased eNOS expression and increased p16(INK4a) expression was associated with dysfunction of EPCs caused by CSE. The mechanism of methylation, one of the most common epigenetic mechanism, may be involved in the EPCs dysfunction caused by CSE.

Urea-induced ROS accelerate senescence in endothelial progenitor cells

Background and aimsThe pathogenic events responsible for the reduction of endothelial progenitor cell (EPC) number and function seen in patients with chronic renal failure (CRF) are poorly understood. Here we investigate the hypothesis that increased concentrations of urea associated with CRF increase ROS production directly in EPCs, causing abnormalities associated with coronary artery disease risk. MethodsHuman EPCs were isolated from peripheral blood mononuclear cells of healthy donors and cultured in the presence or absence of 20 mmol/L urea. ResultsUrea at concentrations seen in CRF induced ROS production in cultured EPCs. Urea-induced ROS reduced the number of endothelial cell colony forming units, uptake and binding of Dil-Ac-LDL and lectin-1, and the ability to differentiate into CD31- and vascular endothelial growth factor receptor 2–positive cells. Moreover, urea-induced ROS generation accelerated the onset of EPC senescence, leading to a senescence-associated secretory phenotype (SASP). Normalization of mitochondrial ROS production prevented each of these effects of urea. ConclusionsThese data suggest that urea itself causes both reduced EPC number and increased EPC dysfunction, thereby contributing to the pathogenesis of cardiovascular disease in CRF patients.