Capacity Of Endothelial Progenitor Cells Research Articles

Age-associated declined function of endothelial progenitor cells and its correlation with plasma IL-18 or IL-23 concentrations in patients with ST-segment elevation myocardial infarction

BackgroundST-segment elevation myocardial infarction (STEMI) persists to be prevalent in the elderly with a dismal prognosis. The capacity of endothelial progenitor cells (EPCs) is reduced with aging. Nevertheless, the influence of aging on the functionality of EPCs in STEMI is not fully understood.MethodThis study enrolled 20 younger STEMI patients and 21 older STEMI patients. We assessed the Thrombolysis in Myocardial Infarction (TIMI) and Global Registry of Acute Coronary Events Risk (GRACE) scores in two groups. Then, we detected EPC migration, proliferation, adhesion, and plasma interleukin (IL)-18 and IL-23 concentrations in two groups. In addition, we analyzed the interconnection between age, EPC function, plasma IL-18 and IL-23 concentrations, and GRACE or TIMI scores in STEMI patients.ResultGRACE and TIMI scores in older STEMI patients were higher than in younger STEMI patients, whereas EPC function declined. GRACE and TIMI scores were found to have an inverse relationship with the EPC function. In older STEMI patients, plasma concentrations of IL-18 and IL-23 increased. Plasma IL-18 and IL-23 concentrations were adversely connected to EPC capacity and positively related to GRACE and TIMI scores. Moreover, age was positively correlated with plasma IL-18 or IL-23 concentrations, as well as GRACE or TIMI scores. However, age was adversely correlated with EPC function.ConclusionIn patients with STEMI, aging results in declined EPC function, which may be associated with inflammatory cytokines. The current investigation may offer new perception about mechanism and therapeutic targets of aging STEMI.

Novel Angiogenesis Role of GLP-1(32-36) to Rescue Diabetic Ischemic Lower Limbs via GLP-1R-Dependent Glycolysis in Mice.

Restoring the capacity of endothelial progenitor cells (EPCs) to promote angiogenesis is the major therapeutic strategy of diabetic peripheral artery disease. The aim of this study was to investigate the effects of GLP-1 (glucagon-like peptide 1; 32-36)-an end product of GLP-1-on angiogenesis of EPCs and T1DM (type 1 diabetes) mice, as well as its interaction with the classical GLP-1R (GLP-1 receptor) pathway and its effect on mitochondrial metabolism. In in vivo experiments, we conducted streptozocin-induced type 1 diabetic mice as a murine model of unilateral hind limb ischemia to examine the therapeutic potential of GLP-1(32-36) on angiogenesis. We also generated Glp1r-/- mice to detect whether GLP-1R is required for angiogenic function of GLP-1(32-36). In in vitro experiments, EPCs isolated from the mouse bone marrow and human umbilical cord blood samples were used to detect GLP-1(32-36)-mediated angiogenic capability under high glucose treatment. We demonstrated that GLP-1(32-36) did not affect insulin secretion but could significantly rescue angiogenic function and blood perfusion in ischemic limb of streptozocin-induced T1DM mice, a function similar to its parental GLP-1. We also found that GLP-1(32-36) promotes angiogenesis in EPCs exposed to high glucose. Specifically, GLP-1(32-36) has a causal role in improving fragile mitochondrial function and metabolism via the GLP-1R-mediated pathway. We further demonstrated that GLP-1(32-36) rescued diabetic ischemic lower limbs by activating the GLP-1R-dependent eNOS (endothelial NO synthase)/cGMP/PKG (protein kinase G) pathway. Our study provides a novel mechanism with which GLP-1(32-36) acts in modulating metabolic reprogramming toward glycolytic flux in partnership with GLP-1R for improved angiogenesis in high glucose-exposed EPCs and T1DM murine models. We propose that GLP-1(32-36) could be used as a monotherapy or add-on therapy with existing treatments for peripheral artery disease. URL: www.ebi.ac.uk/metabolights/; Unique identifier: MTBLS9543.

Dapagliflozin restores diabetes-associated decline in vasculogenic capacity of endothelial progenitor cells via activating AMPK-mediated inhibition of inflammation and oxidative stress

Sodium-glucose cotransporter 2 inhibitors (SGLT2i) provide added vascular protection beyond glucose lowering to patients with type 2 diabetes mellitus (T2DM). Endothelial progenitor cells (EPCs) are an important endogenous repair mechanism for diabetic vascular complications. Yet, whether SGLT2i protect vessels in diabetic patients by improving the function of EPCs remains to be elucidated. Here we enrolled Sixty-three T2DM patients and 60 healthy participants and 15 of T2DM group took dapagliflozin for 3 months. Retinal capillary density (RCD) was examined before and after meditation. Moreover, vasculogenic capacity of EPCs cocultured with or without dapagliflozin in vitro and in vivo (hind limb ischemia model) were assessed. Mechanically, genes related to inflammation/oxidative stress, and the AMPK signaling of EPCs were determined. Our results found T2DM demonstrated a declined RCD and a decreased number of circulating EPCs compared with healthy controls. Compared with the EPCs from healthy individuals, vasculogenic capacity of T2DM EPCs was significantly impaired, which could be restored by dapagliflozin meditation or dapagliflozin coculture. Increased expression of inflammation correlative genes and decreased anti-oxidative stress related genes expression were found in EPCs form T2DM, which were accompanied with reduced phosphorylation level of AMPK. Dapagliflozin treatment activated AMPK signaling, decreased the level of inflammation and oxidative stress, and rescued vasculogenic capacity of EPCs from T2DM. Furthermore, AMPK inhibitor pretreatment diminished the enhancement vasculogenic capacity of diabetic EPCs from dapagliflozin treatment. This study demonstrates for the first time that dapagliflozin restores vasculogenic capacity of EPCs via activating AMPK-mediated inhibition of inflammation and oxidative stress in T2DM.

Inhibition of oxidative stress delays senescence and augments functional capacity of endothelial progenitor cells

Ageing is characterised by a progressive loss of vascular endothelial function and integrity. Endothelial progenitor cells (EPCs) play an integral role in endothelial regeneration but are prone to age-dependent changes which may accelerate their senescence and diminish their availability and functionality. Considering these, we firstly investigated the quantity of circulating EPCs in older (73.3 ± 7.2 years) and younger (40.2 ± 14.3 years) healthy volunteers and showed sharp declines in the number of EPCs expressing stemness markers (CD34 + and/or CD133 + ) in older people. These coincided with the decreases in total anti-oxidant capacity (TAC) and concomitant increases in plasma levels of pro-inflammatory cytokine, TNF-α and anti-angiogenic factor, endostatin and thrombospondin-1. The subsequent experimental studies to scrutinise the effect of ageing on molecular and functional properties of outgrowth endothelial cells (OECs), the functional subtype of EPCs, showed that chronological ageing, mimicked by replicative senescence, profoundly impaired proliferation, migration, tubulogenesis, and blood–brain barrier (BBB)-forming capacity of these cells. Similar to those seen in the clinical observational studies, senescent OECs also manifested decreased TAC and increased pro-oxidant NADPH oxidase activity and endostatin level. Suppressing oxidative stress level using structurally and functionally distinct anti-oxidants, namely vitamin C or VAS2870, an NADPH oxidase inhibitor, delayed OEC senescence and restored their tubulogenic and BBB-forming capacities. In conclusion, the enhanced oxidative stress level that develops during physiological ageing may promote EPC senescence and evoke endothelial dysfunction. Effective control of oxidative stress using either compound somewhat delays both phenomena and augments EPC functionality.

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.

Shear stress improves the endothelial progenitor cell function via the CXCR7/ERK pathway axis in the coronary artery disease cases

BackgroundDysfunction in the late Endothelial Progenitor Cells (EPCs) is responsible for endothelial repair in patients with Coronary Artery Disease (CAD), and the shear stress is beneficial for EPCs function. However, the impact of shear stress on the capacity of EPCs in CAD patients has not been elucidated yet. The C-X-C chemokine receptor 7/extracellular signal-regulated kinase (CXCR7)/(ERK) pathways are identified to regulate EPCs function in CAD patients. Here, we hypothesize that shear stress upregulates the CXCR7/ERK pathways, which restore the EPCs function in CAD patients.MethodsThe human Peripheral Blood Mononuclear Cells (PBMCs) were collected from healthy adults and CAD patients and then used for EPCs cultivation. The Lv-siRNA for human CXCR7 was transfected into induced EPCs isolated from the CAD patients. Meanwhile, the EPCs from CAD patients were subjected to shear stress generated by a biomimetic device. Next, the cell viability, migration, tube formation, and apoptosis were detected by CCK-8, Transwell assay, Matrigel, and flow cytometry, respectively. Also, the CXCR7/ERK pathways in human EPCs were analyzed by Western blotting and qRT-PCR.ResultCompared to the EPCs collected from normal adults, the CAD patient-derived EPCs showed reduced in vitro vasculogenic capacity. Also, the level of CXCR7 in CAD patient-derived EPCs was significantly reduced compared to the EPCs of healthy subjects. Meanwhile, the extracellular signal-regulated kinase (ERK), which represents a CXCR7 downstream signaling pathway, had decreased phosphorylation level. The shear stress treatment augmented the CXCR7 expression and also elevated ERK phosphorylation, which is comparable to the up-regulation of CAD patient-derived EPCs function. Further, the small interfering RNA (siRNA)-mediated CXCR7 knockdown diminished the enhanced migration, adhesion, and tube formation capacity of shear stress treated CAD patient-derived EPCs.ConclusionUp-regulation of the CXCR7/ERK pathways by shear stress can be a promising new target in enhancing the vasculogenic ability of CAD patient-derived EPCs.

Aliskiren Improved the Endothelial Repair Capacity of Endothelial Progenitor Cells from Patients with Hypertension via the Tie2/PI3k/Akt/eNOS Signalling Pathway.

Background Studies show that aliskiren exerts favourable effects not only on endothelial progenitor cells (EPCs) but also on endothelial function. However, the mechanism of the favourable effect of aliskiren on EPCs from patients with hypertension is unclear and remains to be further studied. Methods The object of this study was to investigate and assess the in vitro function of EPCs pretreated with aliskiren. After treated with aliskiren, the human EPCs were transplanted into a nude mouse model of carotid artery injury, and the in vivo reendothelialization of injured artery was estimated by staining denuded areas with Evans blue dye via tail vein injection. Results We found that aliskiren increased the in vitro migration, proliferation, and adhesion of EPCs from patients with hypertension in a dose-dependent manner and improved the reendothelialization capability of these EPCs. Furthermore, aliskiren increased the phosphorylation of Tie2, Akt, and eNOS. After the blockade of the Tie2 signalling pathway, the favourable effects of aliskiren on the in vitro function and in vivo reendothelialization capability of EPCs were suppressed. Conclusions This study demonstrates that aliskiren can improve the in vitro function and in vivo reendothelialization capability of EPCs from patients with hypertension via the activation of the Tie2/PI3k/Akt/eNOS signalling pathway. These findings further indicate that aliskiren is an effective pharmacological treatment for cell-based repair in hypertension-related vascular injury.

β2AR-dependent signaling contributes to in-vivo reendothelialization capacity of endothelial progenitor cells by shear stress

Endothelial progenitor cells (EPCs) play a crucial role in the endothelial repair after arterial injury. Shear stress has a beneficial effect on modulating EPC functions. The molecular mechanism underlying the influence of EPCs on the endothelial integrity and shear stress effects on EPC regulation remained unclear. Herein, we investigated the influence of β2 adrenergic receptor (β2AR)-dependent signaling on in-vitro shear stress-mediated function and in-vivo reendothelialization capacity of human EPCs. The human EPCs from healthy population were exposed to in-vitro 5, 10, and 20 dyn/cm shear stress for 15 h, and 10 dyn/cm for 5, 10, and 15 h, respectively. The in-vitro proliferation was assessed by CCK8 and BrDU tests. The migration and adhesion were evaluated by Transwell system and human umbilical vein endothelial cells (HUVECs) incorporation assays. Meanwhile, the angiogenic cytokine stromal derived factor-1 (SDF-1) and vascular endothelial growth factor (VEGF) concentration of supernatant were tested by ELISA. Phosphorylated β2AR, Akt, and eNOS were detected by western blot. In an in-vivo study, mice carotid injury models were produced through denuding the endothelium with a curved flexible wire, and thereafter CM-Dil-labeled EPCs were injected intravenously. After 3 days, cells recruited to the injury sites were detected by fluorescent microscopy, and the in-vivo reendothelialization capacity was assessed by Evans blue dye. Shear stress improved in-vitro functions and in-vivo reendothelialization capacity of human EPCs. In parallel, shear stress up-regulated the phosphorylation of β2AR, Akt, and eNOS, and promoted vascular endothelial growth factor (VEGF) secretion of human EPCs. With ICI118,551 (a β2AR inhibitor) treatment, shear stress-induced Akt and eNOS phosphorylation as well as VEGF secretion were suppressed. After β2AR/PI3K/Akt/eNOS pathway of EPCs was blocked, the effects of shear stress on in-vitro functions and in-vivo reendothelialization capacity of EPCs were inhibited. The present study provided the novel data that shear stress-induced β2AR/Akt/eNOS pathway enhanced reendothelialization capacity of EPCs. Shear stress-induced β2AR-dependent pathway may be a novel and important therapeutic target for endothelial repair.

MicroRNA-326-5p enhances therapeutic potential of endothelial progenitor cells for myocardial infarction

BackgroundOur study sought to investigate the therapeutic effects and mechanisms of miR-326-5p-overexpressing endothelial progenitor cells (EPCs) on acute myocardial infarction (AMI).MethodsMouse EPCs were isolated, purified, and identified by flow cytometry and uptake of DiI-ac-LDL. The target gene of miR-326-5p was predicted using target prediction algorithms and verified by dual-luciferase reporter assay, RT-qPCR, and Western blot. After EPCs were transfected with the agomir or antagomir of miR-326-5p, tube formation assay and Matrigel plug angiogenesis assay were conducted in four groups (NC, miR-326-5p agomir, miR-326-5p antagomir, and miR-326-5p agomir+Wnt1 agonist). In addition, a mouse model of MI was established and treated with the injection of miR-326-5p-EPCs, miR-326-5p-EPCs+ Wnt1 agonist, EPCs-NC, or PBS/control into the peri-infarcted myocardium. Subsequently, cardiac function was monitored by echocardiography at 7 and 28 days postoperatively. Finally, the infarcted hearts were collected at 28 days, and the size of myocardial infarction was measured by Masson’s trichrome staining and the neovascularization in the peri-infarcted area was examined through immunofluorescence staining.ResultsLuciferase reporter assay indicated that Wnt1 was a direct target of miR-326-5p. Using RT-qPCR and Western blot analysis, we further demonstrated that the expression level of Wnt1 was negatively correlated with miR-326-5p expression in EPCs. Both in vitro study of tube formation assay and in vivo investigation of subcutaneous Matrigel plug assay revealed that the miR-326-5p agomir could significantly enhance the angiogenic capacity of EPCs, and this effect was partially inhibited by Wnt1 agonist. Meanwhile, miR-326-5p antagomir could obviously reduce the the angiogenic capacity of EPCs in vivo compared with that in the NC group. Moreover, the transplantation of miR-326-5p-overexpressing EPCs in the ischemic hearts of mice significantly enhanced the angiogenesis in the peri-infarcted zone and improved the cardiac function. However, the enhanced capacity of angiogenesis of miR-326-5p-overexpressing EPCs was remarkably neutralized by Wnt1 agonist, accompanied by the decreased improvement in cardiac function.ConclusionmiR-326-5p significantly enhanced the angiogenic capacity of EPCs. Transplantation of miR-326-5p-overexpressing EPCs improved cardiac function for AMI therapy, which can be a novel strategy for enhancing therapeutic angiogenesis in ischemic heart diseases.

Metformin inhibits angiogenesis of endothelial progenitor cells via miR-221-mediated p27 expression and autophagy.

Aim: To explore the underlying mechanisms of metformin on the angiogenic capacity of endothelial progenitor cells (EPCs). Results: EPC growth and miR-221 expression decreased concentration-dependence with metformin, and a negative correlation was observed between miR-221 expression and metformin concentration (p <0.001). miR-221 overexpression using a mimic decreased the metformin-mediated angiogenic effects in EPCs (p <0.01). Metformin increased p27 and LC3II expression and AMP-activated protein kinase (AMPK) phosphorylation, and decreased p62 expression, while miR-221 overexpression reversed the effects of metformin. Additionally, AMPK inhibition by compound C reversed the increase in p27 and LC3II levels and AMPK phosphorylation or miR-221 siRNA treatment. Conclusion: Metformin inhibits the angiogenic capacity of EPCs. The underlying mechanism involves AMPK-mediated autophagy pathway activity and increases miR-221-mediated p27 expression.

Beneficial Impact of Moderate to Vigorous Physical Activity Program on Circulating Number and Functional Capacity of Endothelial Progenitor Cells in Children: The Crucial Role of Nitric Oxide and VEGF-A.

Endothelial progenitor cells (EPCs) appear to interact with physical training. This study aimed to provide a comprehensive assessment of the relationship of moderate to vigorous physical activity (MVPA) with both angiogenic factors and EPC function in healthy children. Forty children (22 boys and 18 girls) aged 7 to 11 years participated in a 10-week MVPA program (duration: 45min; intensity: 75%-85% of heart rate reserve; frequency: 4sessions/wk). The anthropometric data, biochemical profile, EPCs number, EPCs colony-forming units, and vascular endothelial growth factor-A (VEGF-A) and nitric oxide (NO) plasma levels were evaluated before and after the MVPA program. After a 10-week MVPA program, a significant increase was detected in circulating/functional capacity of EPCs, NO, and VEGF-A levels, associated with improvement of waist circumference and estimated maximum rate of oxygen consumption (VO2max). A strong positive correlation was found between delta of EPCs number and variation of both NO level (r = .677, P < .001) and VEGF-A level (r = .588, P < .001). Furthermore, a significant correlation between NO level variation and delta of VEGF-A level was observed (r = .708, P < .001). Our findings suggest that lifestyle intervention implemented by MVPA program can contribute meaningfully to improve circulating/functional capacity of EPCs in healthy children, possibly due to the increase of plasma NO and VEGF-A levels.

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.

Detrimental Impact of Low Birth Weight on Circulating Number and Functional Capacity of Endothelial Progenitor Cells in Healthy Children: Role of Angiogenic Factors

To provide a comprehensive assessment of the relationship of birth weight with both endothelial progenitor cell function and angiogenic factors in children. Anthropometric measures, biochemical profile, endothelial progenitor cell number, endothelial progenitor cell colony-forming units, vascular endothelial growth factor-A, and nitric oxide plasma levels of 58 children aged 7-11 years were determined. A positive correlation was observed between birth weight and circulating endothelial progenitor cell number (r= 0.461; P= .001), endothelial progenitor cell colony-forming units (r= 0.512; P < .001), vascular endothelial growth factor-A (r= 0.407; P= .002), and nitric oxide (r= 0.547; P < .001) levels, whereas the adjustment for prematurity, family history of cardiovascular disease, and systolic blood pressure levels did not modify these associations. Low birth weight was associated with a decrease in the circulating/functional capacity of endothelial progenitor cells among healthy children, independent of traditional cardiovascular risk factors. This detrimental impact was accompanied by lower circulating levels of angiogenic factors.

Platelet‑derived growth factorD promotes the angiogenic capacity of endothelial progenitor cells.

Neovascularization and re-endothelialization rely on endothelial progenitor cells (EPCs). However, the recruitment and angiogenic roles of EPCs are subject to regulation through the vascular microenvironment, which remains largely unknown. Platelet-derived growth factor D (PDGF-D) is a new member of the PDGF family that binds the PDGFR-β homodimer. However, it remains unknown whether and how it affects the angiogenic capacity of EPCs and participates in tube-like formation. EPCs were derived from rat bone marrow cells, and the gain-of-function approach was used to study the effects of PDGF-D on the biological activities of EPCs. EPCs that stably express PDGF-D were generated by lentiviral-mediated transduction, and the expression levels were evaluated by western blotting and reverse transcription, followed by real-time quantitative polymerase chain reaction (RT-qPCR). The biological activities of EPCs evaluated in the present study included proliferation, adhesion, migration, tube formation and senescence. Furthermore, the downstream signaling of PDGF-D was explored by western blot analysis. The results revealed that the lentiviral-mediated expression of PDGF-D in the microenvironment promoted the migration, proliferation, adhesion and tube formation of EPCs. In addition, PDGF-D suppressed the senescence of EPCs. Mechanistically, PDGF-D induced the phosphorylation of several signaling molecules, including STAT3, AKT, ERK1/2, mTOR and GSK-3β, suggesting that PDGF-D enhanced the angiogenic function of EPCs through PDGF receptor-dependent and -independent signaling pathways. In conclusion, PDGF-D promotes the angiogenic capacity of EPCs, including proliferation, migration, adhesion and tube formation, and thereby contributes to angiogenesis.

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).

Microparticles harbouring Sonic hedgehog morphogen improve the vasculogenesis capacity of endothelial progenitor cells derived from myocardial infarction patients.

Endothelial progenitor cells (EPC) play a role in endothelium integrity maintenance and regeneration. Decreased numbers of EPC or their impaired function correlates with an increase in cardiovascular events. Thus, EPC are important predictors of cardiovascular mortality and morbidity. Microparticles carrying Sonic hedgehog (Shh) morphogen (MPShh+) trigger pro-angiogenic responses, both in endothelial cells and in ischaemic rodent models. Here, we propose that MPShh+ regulates EPC function, thus enhancing vasculogenesis, and correcting the defects in dysfunctional EPC obtained from acute myocardial infarction (AMI) patients. The mechanisms underlying Shh pathway function and nitric oxide (NO) production in EPC were evaluated. MPShh+ increased both the in vitro and in vivo vasculogenic capacity of EPC isolated from adult human peripheral blood samples. MPShh+ treatment significantly increased the expression of Shh signalling pathway genes (PTCH1, SMO, and GLI1) and masters of pro-angiogenic genes (NOS3, VEGFA, KDR, and KLF2) in EPC. Moreover, MPShh+ increased both the protein expression and activity of eNOS, resulting in increased NO production. Most importantly, MPShh+ improved the vasculogenic capacity of EPC from AMI patients to levels similar to that of EPC from healthy patients. All these effects were due to the activation of Shh pathway. MPShh+ increase both the vasculogenesis of EPC and their capacity to produce NO, including EPC from patients who have recently suffered an AMI. This study emphasizes MPShh+ and EPC as potential therapeutic tools for improving vascular regeneration as a treatment for cardiovascular ischaemic disease.

Shear stress-mediated upregulation of GTP cyclohydrolase/tetrahydrobiopterin pathway ameliorates hypertension-related decline in reendothelialization capacity of endothelial progenitor cells.

Guanosine triphosphate cyclohydrolase/tetrahydrobiopterin (GTPCH)/(BH4) pathway has been proved to regulate the function of endothelial progenitor cells (EPCs) in deoxycorticosterone acetate-salt hypertensive mice, indicating that GTPCH/BH4 pathway may be an important repair target for hypertension-related endothelial injury. Shear stress is an important nonpharmacologic strategy to modulate the function of EPCs. Here, we investigated the effects of laminar shear stress on the GTPCH/BH4 pathway and endothelial repair capacity of circulating EPCs in hypertension. Laminar shear stress was loaded on the human EPCs from hypertensive patients and normotensive patients. The in-vitro function, in-vivo reendothelialization capacity and GTPCH/BH4 pathway of human EPCs were evaluated. Both in-vitro function and reendothelialization capacity of EPCs were lower in hypertensive patients than that in normotensive patients. The GTPCH/BH4 pathway of EPCs was downregulated in hypertensive patients. Shear stress increased in-vitro function and reendothelialization capacity of EPCs from hypertensive patients and normotensive patients. Furthermore, shear stress upregulated the expression of GTPCH I and levels of BH4, nitric oxide, and cGMP of EPCs, and reduced thrombospondin-1 expression. With treatment of GTPCH knockdown or nitroarginine methyl ester inhibition, shear stress-induced increased levels of BH4, nitric oxide and cGMP of EPCs was suppressed. When GTPCH/BH4 pathway of EPCs was blocked, the effects of shear stress on in-vitro function and reendothelialization capacity of EPCs were inhibited. The study demonstrates for the first time that shear stress-induced upregulation of the GTPCH/BH4 pathway ameliorates hypertension-related decline in endothelial repair capacity of EPCs. These findings provide novel nonpharmacologic therapeutic approach for hypertension-related endothelial repair.

The Effect of Atorvastatin on the Viability of Ischemic Skin Flaps in Diabetic Rats.

Endothelial progenitor cells play a critical role in neovascularization. However, the mobilization, recruitment, and functional capacity of endothelial progenitor cells are significantly impaired in diabetes. Statins have been shown to augment the number and improve the function of endothelial progenitor cells. This study investigated the effects of statins on the viability of ischemic skin flaps in diabetic rats. Twenty normal and 40 diabetic Sprague-Dawley rats were included in this study. Atorvastatin (10 mg/kg/day) was administered orally in 20 diabetic rats at 2 weeks before flap surgery for 21 consecutive days. Other rats received equal vehicle. Two weeks after first gavage, a 3 × 10-cm skin flap was established on the backs of rats. The necrotic area of each skin flap was measured at 7 days postoperatively. Capillary density and endothelial progenitor cells recruited to the flaps were analyzed using immunofluorescence staining. Circulating endothelial progenitor cell number was determined by flow cytometry. In vitro migration and tube formation experiments were used to analyze the function of endothelial progenitor cells. Atorvastatin treatment increased flap survival rate and capillary density. In addition, more endothelial progenitor cells were identified in peripheral blood and skin flaps in diabetic rats receiving atorvastatin. Atorvastatin treatment also restored the impaired function of diabetic endothelial progenitor cells in migration and tube formation. Atorvastatin notably promoted neovascularization and enhanced the viability of ischemic skin flaps in diabetic rats, which may be mediated at least partially by augmenting the number and restoring the functional capacity of endothelial progenitor cells.

Abstract 223: Brain-derived Neurotrophic Factor Promotes Regenerative Functions of Human Endothelial Progenitor Cells - Roles of miRNAs

Our previous study has demonstrated that brain-derived neurotrophic factor (BDNF) enhances anti-oxidant capacity of endothelial progenitor cells (EPCs). Since BDNF is produced in cerebrovascular endothelial cells, and functions as a vasoprotective molecule, we hypothesized that BDNF might enhance regenerative capacity of EPCs. Early EPCs (circulating angiogenic cells) were derived from healthy male subjects (48±1.5 years old, n=10). EPCs were treated with BDNF 100 ng/ml for 7 hours, and miRNA transcriptome was examined using Affymetrix miRNA array (version 3.0). BDNF significantly changed 31 miRNAs (n=4, P&lt;0.05, fold-change cut off=1.5). Notably, miR-181c level was increased by 1.9 fold (P&lt;0.05) after BDNF treatment. Since miR-181c is known to down-regulate TNF-α expression, BDNF may protect EPCs by promoting anti-inflammatory phenotype. We also found that BDNF decreased miR-433 level by 1.6 fold (P&lt;0.05). We further evaluated miR-433 function in human early EPCs. Knock-down of miR-433 in EPCs using siRNA significantly increased expression of guanylate binding protein 2 (GBP2) (n=6, P&lt;0.05), an established direct target for miR-433. In contrast, treatment with miR-433 mimic significantly reduced GBP2 protein expression in EPCs (n=4, P&lt;0.05). Since GBP2 has been shown to play an important role in the growth and colony formation of hematopoietic progenitors, BDNF/miR-433/GBP2 signaling may enhance angiogenic capacity of EPCs. In addition, neither miR-433-siRNA nor miR433 mimic significantly changed protein expressions of other targets such as growth factor receptor-binding protein2, cAMP response element-binding protein-1, cyclin-dependent kinase 6, and γ glutamyl cysteine synthetase (n=4-6, P&gt;0.05). In summary, changes in miRNA transcriptome induced by treatment with BDNF may promote anti-inflammatory and angiogenic phenotype of EPCs. These effects of BDNF may have important implications for therapeutic enhancement of regenerative capacity of EPCs.

Resveratrol prevents endothelial progenitor cells from senescence and reduces the oxidative reaction via PPAR‑γ/HO‑1 pathways.

Increasing evidence suggests endothelial progenitor cells (EPCs) improve neovascularization and endothelium regeneration. Resveratrol (RSV) is a natural polyphenolic compound, which has been demonstrated to exert multiple protective effects on the cardiovascular system, including inhibition of platelet adhesion and aggregation, reduction of myocardial ischemia-reperfusion injury, and suppression of neointimal hyperplasia of injured vascular tissue. The present study investigated the role of RSV on levels of oxidative stress and senescence of EPCs, and the effects of RSV on vascular-promoting and/or vascular-healing capacity of EPCs. It was demonstrated that EPCs could promote the repair of endothelium of the injured artery. RSV reduced the oxidative reaction of EPCs and inhibited EPC senescence, and these effects may occur via the peroxisome proliferator-activated receptor-γ/heme oxygenase-1 signaling pathways.