Functional Impairment Of Endothelial Progenitor Cells Research Articles

The Impact of Modern Anti-Diabetic Treatment on Endothelial Progenitor Cells.

Diabetes is one of the leading chronic diseases globally with a significant impact on mortality. This condition is associated with chronic microvascular and macrovascular complications caused by vascular damage. Recently, endothelial progenitor cells (EPCs) raised interest due to their regenerative properties. EPCs are mononuclear cells that are derived from different tissues. Circulating EPCs contribute to regenerating the vessel's intima and restoring vascular function. The ability of EPCs to repair vascular damage depends on their number and functionality. Diabetic patients have a decreased circulating EPC count and impaired EPC function. This may at least partially explain the increased risk of diabetic complications, including the increased cardiovascular risk in these patients. Recent studies have confirmed that many currently available drugs with proven cardiovascular benefits have beneficial effects on EPC count and function. Among these drugs are also medications used to treat different types of diabetes. This manuscript aims to critically review currently available evidence about the ways anti-diabetic treatment affects EPC biology and to provide a broader context considering cardiovascular complications. The therapies that will be discussed include lifestyle adjustments, metformin, sulphonylureas, gut glucosidase inhibitors, thiazolidinediones, dipeptidyl peptidase 4 inhibitors, glucagon-like peptide 1 receptor analogs, sodium-glucose transporter 2 inhibitors, and insulin.

Impaired Angiogenic Function of Fetal Endothelial Progenitor Cells via PCDH10 in Gestational Diabetes Mellitus.

Maternal hyperglycemia, induced by gestational diabetes mellitus (GDM), has detrimental effects on fetal vascular development, ultimately increasing the risk of cardiovascular diseases in offspring. The potential underlying mechanisms through which these complications occur are due to functional impairment and epigenetic changes in fetal endothelial progenitor cells (EPCs), which remain less defined. We confirm that intrauterine hyperglycemia leads to the impaired angiogenic function of fetal EPCs, as observed through functional assays of outgrowth endothelial cells (OECs) derived from fetal EPCs of GDM pregnancies (GDM-EPCs). Notably, PCDH10 expression is increased in OECs derived from GDM-EPCs, which is associated with the inhibition of angiogenic function in fetal EPCs. Additionally, increased PCDH10 expression is correlated with the hypomethylation of the PCDH10 promoter. Our findings demonstrate that in utero exposure to GDM can induce angiogenic dysfunction in fetal EPCs through altered gene expression and epigenetic changes, consequently increasing the susceptibility to cardiovascular diseases in the offspring of GDM mothers.

HIF-1α-Dependent Autophagy Deficiency in Bone Marrow Endothelial Progenitor Cells Contributes to Acute Graft-Versus-Host Disease after Allotransplant

Background: Acute Graft-Versus-Host Disease (aGVHD) constitutes a prevalent component after allogeneic hematopoietic stem cell transplantation(allo-HSCT). Among the components of the Bone Marrow（BM)microenvironment, Endothelial Progenitor Cells (EPCs) hold significant importance in hematopoietic-supporting and immune reconstitution in post-transplantation patients. Consequently, a discernible correlation emerges between the impairment of EPCs and the incidence and severity of GVHD. Our recent study has shown impaired EPC function and heightened reactive oxygen species (ROS) levels in aGVHD patients (2018, BJH). Additionally, reduced levels of hypoxia-inducible factor-1α (HIF-1α) and autophagy were observed in BM EPCs from aGVHD patients in comparison to those without GVHD（non-GVHD）. HIF-1α is closely linked to ROS generation. Therefore， exploring the role of HIF-1α and autophagy in the development of aGVHD after allo-HSCT is significant. Aims： To conduct a comparative analysis of the functions of EPCs and assess the variations in the expression levels of HIF-1α and autophagy-related proteins between aGVHD and non-GVHD patients. Additionally, the study aims to assess whether the low HIF-1α level is responsible for the autophagy deficiency and mitochondrial dysfunction observed in EPCs of aGVHD patients, and to delineate the specific pathways through which this downregulation influences their capacity of hematopoiesis-supporting. Methods: The current prospective clinical case-matched study included 20 patients with aGVHD and 40 patients without GVHD. BM mononuclear cells（BMMNCs） were cultured as former publications reported. EPCs（CD34+, CD133+, CD309+) were flow-sorted for evaluating apoptosis, ROS, and autophagy levels. SiRNA transfection knocked down HIF-1α in human umbilical vein endothelial cells (HUVECs). Endothelial cells(ECs） gene expression levels were determined using Western blot, RT-qPCR. ECs function was assessed by angiogenesis, Transwell cell migration, CCK-8 cell proliferation, and JC-1 mitochondrial staining. ECs were co-cultured with hematopoietic stem cells （HSCs） for seven days, and HSCs were subjected to colony-forming unit assays and other experiments to evaluate hematopoietic stem cell function. ECs NAD+ and NADH levels were assessed by the kit. Results: Downregulation of autophagy signaling pathway in HUVECs following knockdown of HIF-1α, leading to inhibited cell proliferation, migration, and angiogenesis. Similarly, patients affected by aGVHD exhibited reduced HIF-1α expression, impaired autophagy, and cellular function in EPCs compared to non-GVHD patients. HSCs co-culturing with HUVECs with HIF-1α knockdown and EPCs from aGVHD patients resulted in increased apoptosis, elevated levels of ROS, and impaired colony formation ability. However, intervention with HIF-1α activators Roxadustat, which served to upregulate HIF-1α levels in ECs, activated autophagy, and improved cellular function, also enhanced the hematopoiesis-supporting ability. Furthermore, the knockdown of HIF-1α in HUVECs and EPCs of aGVHD patients led to the abnormal activation of SIRT1 and PARP1 and other NAD+-consuming proteins, which subsequently caused a decrease in NAD+ levels and resulted in abnormal mitochondrial membrane potential. The supplementation of NAD+ precursors, specifically β-nicotinamide mononucleotide, effectively increased NAD+ levels in ECs and restored cellular function. Conclusion: The results revealed that the autophagy deficiency was mediated by reduced expression of HIF-1α in EPCs of patients with aGVHD. The deficiency of autophagy also led to abnormal activation of SIRT1 and PARP1, which in turn affected the levels of NAD+ and mitochondrial membrane potential, ultimately impairing cellular function and hematopoiesis-supporting ability. These molecular changes may be identified as potential contributors to the pathogenesis of aGVHD after allo-HSCT. On the other hand, Roxadustat upregulates HIF-1α expression in ECs, which subsequently enhanced the autophagy level and improved mitochondrial function. As a result, cellular function and hematopoiesis-supporting ability were restored. This promising therapeutic approach highlights a novel strategy for preventing and treating of patients with aGVHD after allo-HSCT.

Il ruolo dei progenitori delle cellule endoteliali nel diabete mellito

Diabetes is associated with increased risk of cardiovascular morbidity and mortality. Endothelial progenitor cells (EPCs) are bone marrow-derived cells involved in endothelial health, vasculogenesis and damage repair. Both subjects with type 1 and type 2 diabetes showed a lower number of EPCs and impaired EPC function, suggesting a potential pathogenic role of these cells in both micro and macrovascular complications, as well as in sexual dysfunctions in diabetes. Several glucose-lowering drugs have been reported to potentially improve EPCs levels and bioreactivity. However, larger and longer studies are needed to better explain the mechanisms involved.

Fibroblast growth factor 21 reverses high-fat diet-induced impairment of vascular function via the anti-oxidative pathway in ApoE knockout mice.

Circulating endothelial progenitor cells (EPCs), which function in vascular repair, are the markers of endothelial dysfunction and vascular health. Fibroblast growth factor 21 (FGF21), a liver‐secreted protein, plays a crucial role in glucose homeostasis and lipid metabolism. FGF21 has been reported to attenuate the progression of atherosclerosis, but its impact on EPCs under high oxidative stress conditions remains unclear. In vitro studies showed that the β‐klotho protein was expressed in cultured EPCs and that its expression was upregulated by FGF21 treatment. Hydrogen peroxide (H2O2)‐induced oxidative stress impaired EPC function, including cell viability, migration and tube formation. Pretreatment with FGF21 restored the functions of EPCs after the exposure to H2O2. Administration of N(ω)‐nitro‐L‐arginine methyl ester (L‐NAME), an inhibitor of nitric oxide synthase, inhibited the effects of FGF21 in alleviating oxidative injury by suppressing endothelial nitric oxide synthase (eNOS). In an in vivo study, the administration of FGF21 significantly reduced total cholesterol (TC) and blood glucose levels in apolipoprotein E (ApoE)‐deficient mice that were fed a high‐fat diet (HFD). Endothelial function, as reflected by acetylcholine‐stimulated aortic relaxation, was improved after FGF21 treatment in ApoE‐deficient mice. Analysis of mRNA levels in the aorta indicated that FGF21 increased the mRNA expression of eNOS and upregulated the expression of the antioxidant genes superoxide dismutase (SOD)1 and SOD2 in ApoE‐deficient mice. These data suggest that FGF21 improves EPC functions via the Akt/eNOS/nitric oxide (NO) pathway and reverses endothelial dysfunction under oxidative stress. Therefore, administration of FGF21 may ameliorate a HFD‐induced vascular injury in ApoE‐deficient mice.

GPR4 signaling is essential for the promotion of acid-mediated angiogenic capacity of endothelial progenitor cells by activating STAT3/VEGFA pathway in patients with coronary artery disease

BackgroundPatients with coronary artery disease (CAD) are characterized by a decline in vascular regeneration, which is related to the dysfunction of endothelial progenitor cells (EPCs). G-protein-coupled receptor 4 (GPR4) is a proton-sensing G-protein-coupled receptor (GPCR) that contributes to neovascularization in acidic microenvironments. However, the role of GPR4 in regulating the angiogenic capacity of EPCs from CAD patients in response to acidity generated in ischemic tissue remains completely unclear.MethodsThe angiogenic capacity of EPCs collected from CAD patients and healthy subjects was evaluated in different pH environments. The GPR4 function of regulating EPC-mediated angiogenesis was analyzed both in vitro and in vivo. The downstream mechanisms were further investigated by genetic overexpression and inhibition.ResultsAcidic environment prestimulation significantly enhanced the angiogenic capacity of EPCs from the non-CAD group both in vivo and in vitro, while the same treatment yielded the opposite result in the CAD group. Among the four canonical proton-sensing GPCRs, GPR4 displays the highest expression in EPCs. The expression of GRP4 was markedly lower in EPCs from CAD patients than in EPCs from non-CAD individuals independent of acid stimulation. The siRNA-mediated knockdown of GPR4 with subsequent decreased phosphorylation of STAT3 mimicked the impaired function of EPCs from CAD patients at pH 6.4 but not at pH 7.4. Elevating GPR4 expression restored the neovessel formation mediated by EPCs from CAD patients in an acidic environment by activating STAT3/VEGFA signaling. Moreover, the beneficial impact of GPR4 upregulation on EPC-mediated angiogenic capacity was abrogated by blockade of the STAT3/VEGFA signaling pathway.ConclusionsOur present study demonstrated for the first time that loss of GPR4 is responsible for the decline in proton sensing and angiogenic capacity of EPCs from CAD patients. Augmentation of GPR4 expression promotes the neovessel formation of EPCs by activating STAT3/VEGF signaling. This finding implicates GPR4 as a potential therapeutic target for CAD characterized by impaired neovascularization in ischemic tissues.

Metoprolol rescues endothelial progenitor cell dysfunction in diabetes.

Added risk portended by diabetes in addition to hypertension has been related to an amplification of endothelial dysfunction. β-blockers are widely used for cardiovascular diseases and improve the endothelial function compared with a placebo. However, the effect of β-blockers on the endothelial progenitor cells (EPCs) function in diabetes is still unknown. Five β-blockers (metoprolol, atenolol, propranolol, bisoprolol, and nebivolol) were tested in EPC functional screening. Metoprolol improved EPC function significantly among the five β-blockers and was chosen for the in vivo tests in STZ induced diabetic mice. Reactive hyperemia peripheral arterial tonometry (RH-PAT) measurements were performed using the Endo-PAT2000 device in diabetic patients. Metoprolol, but not other β-blockers, improved EPC function in both tube formation and migration assay. EPC function was significantly decreased in diabetic mice, and metoprolol treatment restored damaged EPC migration capabilities and circulation EPC number. Metoprolol treatment promoted wound healing and stimulated angiogenesis in diabetic mice. Furthermore, metoprolol significantly enhanced eNOS phosphorylation and decreased O2− levels in EPCs of diabetic mice. In clinical trials, the RH-PAT index was significantly higher in metoprolol-treated versus bisoprolol-treated diabetics. Metoprolol could accelerate wound healing in diabetic mice and improve endothelial function in diabetic subjects, which may be mediated in part by improving impaired EPC function.

Dipeptidyl Peptidase-4 Inhibitor Decreases Allograft Vasculopathy Via Regulating the Functions of Endothelial Progenitor Cells in Normoglycemic Rats.

Chronic rejection induces the occurrence of orthotopic allograft transplantation (OAT) vasculopathy, which results in failure of the donor organ. Numerous studies have demonstrated that in addition to regulating blood sugar homeostasis, dipeptidyl peptidase-4 (DPP-4) inhibitors can also provide efficacious therapeutic and protective effects against cardiovascular diseases. However, their effects on OAT-induced vasculopathy remain unknown. Thus, the aim of this study was to investigate the direct effects of sitagliptin on OAT vasculopathy in vivo and in vitro. The PVG/Seac rat thoracic aorta graft to ACI/NKyo rat abdominal aorta model was used to explore the effects of sitagliptin on vasculopathy. Human endothelial progenitor cells (EPCs) were used to investigate the possible underlying mechanisms. We demonstrated that sitagliptin decreases vasculopathy in OAT ACI/NKyo rats. Treatment with sitagliptin decreased BNP and HMGB1 levels, increased GLP-1 activity and stromal cell-derived factor 1α (SDF-1α) expression, elevated the number of circulating EPCs, and improved the differentiation possibility of mononuclear cells to EPCs ex vivo. However, in vitro studies showed that recombinant B-type natriuretic peptide (BNP) and high mobility group box1 (HMGB1) impaired EPC function, whereas these phenomena were reversed by glucagon-like peptide 1 (GLP-1) receptor agonist treatment. We suggest that the mechanisms underlying sitagliptin-mediated inhibition of OAT vasculopathy probably occur through a direct increase in GLP-1 activity. In addition to the GLP-1-dependent pathway, sitagliptin may regulate SDF-1α levels and EPC function to reduce OAT-induced vascular injury. This study may provide new prevention and treatment strategies for DPP-4 inhibitors in chronic rejection-induced vasculopathy.

Mitochondrial dysfunction-mediated decline in angiogenic capacity of endothelial progenitor cells is associated with capillary rarefaction in patients with hypertension via downregulation of CXCR4/JAK2/SIRT5 signaling.

BackgroundHypertensive patients exhibit decline in capillary density and endothelial progenitor cells (EPCs). However, whether capillary rarefaction in hypertension is associated with defect angiogenesis of EPCs remains unknown. We hypothesized that impaired mitochondrial function of late EPCs in hypertension is associated with the structural lack of capillary microcirculation via deficient CXCR4/JAK2/SIRT5 signaling.MethodsWe performed capillary microcirculation detection in hypertensive patients and healthy subjects. Angiogenic capacity and mitochondrial function of circulating EPCs were evaluated. The underlying mechanisms were further investigated by genetic inhibition and overexpression.FindingsCapillary density of nail fold and eye fundus were significantly reduced in hypertensive patients, which was paralleled to decreased in vitro late EPC function and in vivo angiogenic capacity. Meanwhile the decline of EPC function in hypertension was accompanied by impaired mitochondrial ultrastructure, diminished mitochondrial membrane potential, reduced oxygen consumption, increased ROS generation and NADH level. Rotenone induced inhibition of oxygen consumption rate, excessive ROS generation and loss of MMP, which markedly decreased the in vitro functions of EPCs. Furthermore, SIRT5 expression of EPCs in hypertension was markedly reduced, which was correlated to mitochondrial dysfunction. CXCR4 gene transfer enhanced SIRT5 expression, improved mitochondrial functions and augmented angiogenic capacity of EPCs. The beneficial impacts of SIRT5 up-regulation on late EPC-mediated angiogenesis can be abrogated by blockade of CXCR4/JAK2/SIRT5 signaling pathway.InterpretationMitochondrial dysfunction-mediated fall in angiogenic capacity due to deficient CXCR4/JAK2/SIRT5 signaling of late EPCs is probably responsible for the capillary rarefaction in hypertension. Our findings provide insight into the potential of EPC mitochondria as a novel target for the treatment of hypertension-related loss of microvascular density.FundsNational Nature Science Foundation of China, 973Program, the Nature Science Foundation of Guangdong.

Mitochondrial Dysfunction-Mediated Decline in Angiogenic Capacity of Circulating Endothelial Progenitor Cells is Associated with Capillary Rarefaction in Patients with Hypertension

Hypertensive patients exhibit decline in capillary density and endothelial progenitor cells (EPCs). However, whether capillary rarefaction in hypertension is associated with defect angiogenesis of EPCs remains unknown. We hypothesized that impaired mitochondrial function of late EPCs in hypertension is associated with the structural lack of capillary microcirculation via deficient CXCR4/JAK2/SIRT5 signaling. Capillary density of nail fold was significantly reduced in hypertensive patients, which was paralleled to decreased in vitro late EPC function and in vivo angiogenic capacity. Meanwhile the decline of EPC function in hypertension was accompanied by impaired mitochondrial ultrastructure, diminished mitochondrial membrane potential, reduced oxygen consumption, increased ROS generation and NADH level. Furthermore, SIRT5 expression of EPCs in hypertension was markedly reduced, which was correlated to mitochondrial dysfunction. CXCR4 gene transfer enhanced SIRT5 expression, improved mitochondrial functions and augmented angiogenic capacity of EPCs. The beneficial impacts of SIRT5 up-regulation on late EPC-mediated angiogenesis can be abrogated by blockade of CXCR4/JAK2/SIRT5 signaling pathway. Mitochondrial dysfunction-mediated fall in angiogenic capacity due to deficient CXCR4/JAK2/SIRT5 signaling of late EPCs is probably responsible for the capillary rarefaction in hypertension. Our findings provide insight into the potential of EPC mitochondria as a novel target for the treatment of hypertension-related loss of microvascular density. Funding: This work was supported by the National Nature Science Foundation (31530023, 81500205, 81671379) of China, 973 Program (2013CB531200), the Nature Science Foundation (2016A030310184, 2016A020215056) of Guangdong and Science and Technology Planning Project (2017A020215085) of Guangdong. Declaration of Interest: None. Ethical Approval: The study protocol was approved by the Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University (Guangzhou, China).

Association between Type I interferon and depletion and dysfunction of endothelial progenitor cells in C57BL/6 mice deficient in both apolipoprotein E and Fas ligand

Patients with systemic lupus erythematosus (SLE) have a tremendously increased risk for cardiovascular disease (CVD), which could not be accounted in entirety by traditional Framingham risk factors. To study whether the accelerated atherosclerosis in SLE patients is mediated by type I interferon (IFN-I) through the regulation of endothelial progenitor cells (EPCs), we created a line of C57BL/6 mice with deficiency in both apolipoprotein E (ApoE−/−) and fas ligand (FasL−/−, gld.). As expected, the resultant gld. ApoE−/− mice exhibited both aggravated lupus-like disease and atherosclerosis under normal diet. Increased expression of IFN-I-stimulated genes (ISGs) was closely associated with depletion and dysfunction of EPCs, as well as with accelerated atherosclerotic lesion in gld. ApoE−/− mice. While only IFN-α instead of other interventions, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IRS423 and IRS661, impaired EPC function in vitro. Mechanistically, activation or inhibition of the TLR7/9 signaling could modulate EPC number and function in vivo. Decreased proliferation rate and increased apoptotic rate of EPCs induced by IFN-α might contribute to the results to a certain extent. Thus, our data suggest that excessive expression of IFN-I through the activation of TLR7/9 signaling may induce accelerated atherosclerosis in lupus through the depletion or dysfunction of EPCs, suggesting that targeting IFN-I might have potential therapeutic effects on both lupus disease and premature atherosclerosis in SLE patients.

Inhibition of macrophage inflammatory protein-1β improves endothelial progenitor cell function and ischemia-induced angiogenesis in diabetes.

Systemic inflammation might contribute to the impairment of neovasculogenesis and endothelial progenitor cell (EPC) function in clinical diabetes mellitus (DM). Macrophage inflammatory protein-1β (MIP-1β) is an inflammatory chemokine that may be up-regulated in clinical DM. Its role in diabetic vasculopathy was not clarified. This study aimed to investigate the role of MIP-1β in human EPCs and in neovasculogenesis in different diabetic animal models with hindlimb ischemia. EPCs chamber assay and in vitro tube formation assay were used to estimate the degree of EPC migration and tube formation abilities. Leprdb/JNarl mice, C57BL/6 mice fed a high-fat diet, and streptozotocin-induced diabetic mice were used as different diabetic animal models. Laser Doppler imaging and flow cytometry were used to evaluate the degree of neovasculogenesis and the circulating levels of EPCs, respectively. MIP-1β impaired human EPC function for angiogenesis in vitro. Plasma MIP-1β levels were up-regulated in type 2 DM patients. MIP-1β inhibition enhanced the function and the C-X-C chemokine receptor type 4 expression of EPCs from type 2 diabetic patients, and improved EPC homing for ischemia-induced neovasculogenesis in different types of diabetic animals. MIP-1β directly impaired human EPC function. Inhibition of MIP-1β improved in vitro EPC function, and enhanced in vivo EPC homing and ischemia-induced neovasculogenesis, suggesting the critical role of MIP-1β for vasculopathy in the presence of DM.

Taurine and magnesium supplementation enhances the function of endothelial progenitor cells through antioxidation in healthy men and spontaneously hypertensive rats.

Endothelial damage is repaired by endothelial progenitor cells (EPCs), which are pivotal in preventing cardiovascular diseases and prolonging lifespan. The WHO Cardiovascular Diseases and Alimentary Comparison Study demonstrated that dietary taurine and magnesium (Mg) intake suppresses cardiovascular diseases. We herein evaluate the effects of taurine and Mg supplementation on EPC function and oxidative stress in healthy men and spontaneously hypertensive rats (SHRs). Healthy men received taurine (3 g per day) or Mg (340 mg per day) for 2 weeks. SHRs and Wistar-Kyoto (WKY) rats were housed with high-salt drinking water (1% NaCl). The SHRs received 3% taurine solution and/or a high-Mg (600 mg per 100 g) diet for 4 weeks. Their peripheral blood mononuclear cells were separated to quantify EPC colony formation. Oxidative stress markers in their peripheral blood were evaluated using a free radical analytical system and a thiobarbituric acid reactive substance (TBARS) assay. Taurine and Mg supplementation significantly increased EPC colony numbers and significantly decreased free radical levels and TBARS scores in healthy men. Taurine and Mg supplementation significantly increased EPC colony numbers and significantly decreased TBARS scores and free radical levels in SHRs. Nicotinamide adenine dinucleotide phosphate oxidase component mRNA expression was significantly higher in the renal cortex of salt-loaded SHRs than in WKY rats, in which it was suppressed by taurine and Mg supplementation. Taurine and Mg supplementation increased EPC colony formation in healthy men and improved impaired EPC function in SHRs through antioxidation, indicating that the dietary intake of taurine and Mg may prolong lifespan by preventing the progression of cardiovascular diseases.

Retraction. Fenofibrate improves the impaired endothelial progenitor cell function through inhibiting eNOS uncoupling in diabetic mice.

The above article, published online on 24th December 2014 as an Accepted Article in Wiley Online Library (http://onlinelibrary.wiley.com/doi/10.1111/bph.13054/abstract), has been retracted by agreement between the authors, the journal Editor in Chief, Professor J. McGrath and John Wiley & Sons Limited. The retraction has been agreed owing to duplication of some images. In this paper, there is a figure duplication in Figure 8A (‘Control’ is from an identical tissue slice as ‘STZ+FF’). There are also partially overlapping figures in supplemental figures S3 and S5.The authors explain this as follows: ‘Our technician took fluorescence images and saved them in one folder using the Arabic numerals. Then we use[d] Photoshop to arrange the images. We chose the STZ+FF group image from the Control group by mistake.’ReferenceTing Zhao, Ya‐Ping Deng, Min Ni, Ji‐Kuai Chen, Zhang‐Peng Li, Dong‐Jie Li and Fu‐Ming Shen (2014), . Br J Pharmacol, doi: 10.1111/bph.13054

Impaired function of endothelial progenitor cells in children with primary systemic vasculitis.

IntroductionPreviously, we demonstrated that children with active systemic vasculitis (SV) have higher circulating CD34 + CD133 + KDR+ endothelial progenitor cells (EPC); the function of these EPCs, and their relationship with disease activity in vasculitis remains largely unexplored. We hypothesized that although EPC numbers are higher, EPC function is impaired in active SV of the young. The aims of this study were therefore to: 1. investigate the relationship between disease activity and EPC function in children with SV; and 2. study the influence of systemic inflammation on EPC function by investigating the effects of hyperthermia and TNF-α on EPC function.MethodsWe performed a cross-sectional study of unselected children with SV with different levels of disease activity attending a single center (Great Ormond Street Hospital, London) between October 2008 and December 2014. EPCs were isolated from peripheral blood of children with SV, and healthy child controls. EPC function was assessed by their potential to form colonies (EPC-CFU), and ability to form clusters and incorporate into human umbilical vein endothelial cell (HUVEC) vascular structures in matrigel. The effects of hyperthermia and TNF-α on EPC function were also studied.ResultsTwenty children, median age 12-years (5–16.5; nine males) were studied. EPC-CFU and the number of EPC clusters formed on matrigel were significantly reduced in children with active vasculitis compared with healthy controls (p = 0.02 for EPC-CFU; p = 0.01 for EPC cluster formation). Those with active vasculitis had lower EPC-CFU and EPC cluster formation than those with inactive disease, although non-significantly so. In addition, EPC incorporation into matrigel HUVEC networks was lower in children with SV compared with healthy children, irrespective of disease activity. Ex-vivo pre-treatment of EPC with hyperthermia impaired EPC function; TNF-α down-regulated EPC expression of CD18/CD11b and resulted in decreased incorporation into HUVEC networks.ConclusionsWhilst our previous work showed that circulating CD34 + EPC numbers are well preserved, this study revealed that EPC function is significantly impaired in children with vasculitis. It is possible that the chronic inflammatory milieu associated with vasculitis may impair EPC function, and thus contribute to an unfavourable balance between endothelial injury and repair. The mechanism of this remains to be established, however.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0810-3) contains supplementary material, which is available to authorized users.

The biphasic effects of oxidized-low density lipoprotein on the vasculogenic function of endothelial progenitor cells.

Late-outgrowth endothelial progenitor cells (EPCs) are stress-resistant and responsible for reparative functions in the cardiovascular system. Oxidized-LDL (oxLDL) plays a critical role in cardiovascular disease pathogenesis. However, it is largely unknown what the impacts of oxLDL are on late-outgrowth EPCs. This study aimed to investigate the concentration-related effects of oxLDL on EPC functions and related angiogenesis, in vitro and in vivo. In this study, early and late-outgrowth EPCs were generated from circulating human mononuclear cells. oxLDL may regulate EPC vasculogenic function via the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1). Lower concentrations (5 μg/mL) of oxLDL can potentiate EPC tube formation in vitro and in vivo by activating eNOS mechanisms, which are mediated by p38 MAPK- and SAPK/JNK-related pathways. Higher concentrations of oxLDL (10-50 μg/mL) impaired EPC function via the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase pathways and consequent inhibition of eNOS activity, which could be reversed by anti-oxidants (diphenylene iodonium and apocynin) and gp91phox siRNA. In conclusion, oxLDL has concentration-dependent biphasic effects on human late-outgrowth EPC tube formation in vitro and in vivo.

A Dysregulated MicroRNA-26a/EphA2 Axis Impairs Endothelial Progenitor Cell Function via the p38 MAPK/VEGF Pathway

Background: Dysfunction of circulating endothelial progenitor cells (EPCs) is associated with the onset of cardiovascular disorders. Circulating microRNAs (miRNAs) have been recognized as novel biomarkers and potential therapeutic targets. Here, we examined the role of miR-26a overexpression in atherosclerosis and explored the underlying mechanisms. Methods: EPCs were obtained from patients with atherosclerosis and healthy controls. Bone marrow (BM)-derived EPCs were exposed to hypoxia to mimic the atherosclerotic environment and miR-26a, EphA2 and p38 MAPK levels were measured by qRT-PCR and western blotting, and VEGF levels were determined by enzyme linked immunosorbent assay. Cell viability was assessed using the MTT assay, and luciferase activity assays confirmed EphA2 as a target of miR-26a. Results: MiR-26a was overexpressed in patients with atherosclerosis and associated with EPC dysfunction. EphA2 was identified as a direct target of miR-26a. Overexpression of miR-26a downregulated EphA2 and impaired EPC function, whereas knockdown of miR-26a upregulated EphA2 and reversed hypoxia-induced EPC dysfunction. MiR-26a overexpression or knockdown modulated the activity of p38 MAPK and the levels of VEGF in EPCs. Conclusions: The role of miR-26a in atherosclerosis is mediated by its target EphA2 via a mechanism involving the p38 MAPK/VEGF pathway.

Endothelial progenitor cells in diabetic patients with myocardial infarction – Can statins improve their function?

The effect of statins on endothelial progenitor cells (EPCs) function derived from diabetic patients (DMpts) with acute myocardial infarction (AMI) is unknown. In this study we assess the response of early and late EPCs from diabetic versus non-diabetic patients (NDMpts) with AMI to statins. EPCs were obtained from 10 diabetic and 10 age-matched non-diabetic male patients with AMI. For each patient, cultures of early and late EPCs were performed under 4 different conditions: normal glucose concentration (control); high glucose concentration; normal glucose concentration with atorvastatin supplementation and normal glucose concentration with pravastatin supplementation. To compare the effect of these treatments on EPC function in DMpts versus NDMpts, we performed in vitro: EPC colony-forming units (CFU) assay; cell cycle analysis; viability assessment and expression of the surface markers CXCR4, CD133, CD34 and KDR. Under control conditions, CFU numbers were reduced in DMpts-derived EPCs when compared to those of NDMpts (1.4±0.8 vs 2.6±1.2 CFU/well, P=0.021). When early EPCs from DMpts were cultured in the presence of statins, CFU capacity was restored, surmounting that of NDMpts under control conditions. Statins significantly improved viability of early EPCs and delayed the onset of late EPCs senescence, even in cells from DMpts. In addition, statins induced approximately a 2-fold increase in the proportion of late EPCs in S-phase of the cell cycle (P<0.05). Statins have a beneficial effect on both early and late EPCs from DMpts with AMI. Despite the functional impairment of EPCs from DMpts, they exhibit similar responsiveness to statins as equivalent cells from NDMpts.

Hypermethylation of DDAH2 promoter contributes to the dysfunction of endothelial progenitor cells in coronary artery disease patients.

BackgroundCirculating endothelial progenitor cells (EPCs) may be a biomarker for vascular function and cardiovascular risk in patients with coronary artery disease (CAD). Dimethylarginine dimethylaminohydrolase 2 (DDAH2) regulates the function of EPCs. This study aimed to examine whether hypermethylation of DDAH2 promoter contributes to impaired function of EPCs in CAD patients.MethodsPeripheral blood mono-nuclear cells from 25 CAD patients and 15 healthy volunteers were collected and differentiated into EPCs. EPCs were tested for their adhesive capability. DDAH2 mRNA expression was analyzed by real-time PCR, and the methylation of DDAH2 promoter was detected by bisulfite genomic sequencing.ResultsDDAH2 promoter in EPCs from CAD patients was hypermethylated and the methylation level was negatively correlated to DDAH2 mRNA level and adhesion function of EPCs. Homocysteine impaired the adhesion function of EPCs, accompanied by lower DDAH2 expression and higher methylation level of DDAH2 promoter, compared to controls. These effects of homocysteine were reversed by pretreatment with Aza, an inhibitor of DNA methyltransferase.ConclusionHypermethylation in DDAH2 promoter is positively correlated to the dysfunction of EPCs in CAD patients. Homocysteine disrupts EPCs function via inducing the hypermethylation of DDAH2 promoter, suggesting a key role of epigenetic mechanism in the progression of atherosclerosis.

Influence of Dietary Fat Intake on Endothelial Progenitor Cell Function (LB678)

High dietary fat intake has been linked to promoting cardiovascular disease and increasing the risk of heart attack. Bone‐marrow derived circulating endothelial progenitor cells (EPCs) play an important role in repairing and maintaining the health of the cardiovascular system. Circulating EPCs home to sites of vascular damage to initiate a repair response. Declines in circulating EPC function has been linked to greater risk of cardiovascular disease and vascular events. It is currently unknown whether habitual consumption of a diet high in fat is associated with impairments in EPC function. Cells with phenotypic EPC characteristics were isolated from peripheral blood samples collected from 26 middle‐aged and older adults: 13 who habitually consumed a diet high in fat (蠅35% of total calories); 13 who habitually consumed a diet lower in fat (&lt;35% of total calories). All subjects were sedentary and free of cardiometabolic disease. Diet was assessed using 4‐day food records and analyzed using a nutritional software. EPC functional characteristics were assessed by the following: colony formation (in vitro colony forming assay); migration (modified Boyden chamber); release of angiogenic growth factors (ELISA); and apoptosis (active intracellular caspase‐3). There were no significant differences between the high and lower fat groups in EPC colony formation (11±3 vs 8±2) and migration (1297±114 vs 1035±124 RFUs). Basal and phytohemagglutinin (PHA; 10 µg/mL) stimulated release of vascular endothelial growth factor (21.5±2.1 to 84.1±14.3 vs 22.9±1.4 to 95.6±14.1 ng/mL) and granulocyte colony stimulating factor (34.0±6.3 to 1246.9±283.6 vs 38.0±6.6 to 873.7±143.1 ng/mL). Moreover, there was no difference in unstimulated (0.3±0.1 vs 0.3±0.1 ng/mL) and stimulated (2.6±0.3 and 2.5±0.3 ng/mL) active caspase‐3 concentrations between the high and lower fat groups. In conclusion, regular consumption of a diet high in fat (蠅35% of calories) does not appear to adversely influence circulating EPC function in middle‐aged and older adults. Impaired EPC function may not contribute to the increased cardiovascular risk associated with a high fat diet.