Mobilization of Endogenous CD34+/CD133+ Endothelial Progenitor Cells by Enhanced External Counter Pulsation for Treatment of Refractory Angina.

Adult stem cell therapy via intramyocardial injection of autologous CD34+ stem cells has been shown to improve exercise capacity and reduce angina frequency and mortality in patients with refractory angina (RA). However, the cost of such therapy is a limitation to its adoption in clinical practice. Our goal was to determine whether the less costly, less invasive, and widely accessible, FDA-approved alternative treatment for RA patients, known as enhanced external counterpulsation (EECP), mobilizes endogenous CD34+ stem cells and whether such mobilization is associated with the clinical benefits seen with intramyocardial injection. We monitored changes in circulating levels of CD34+/CD133+ and CD34+/KDR+ cells in RA patients undergoing EECP therapy and in a comparator cohort of RA patients undergoing an exercise regimen known as cardiac rehabilitation. Changes in exercise capacity in both cohorts were monitored by measuring treadmill times (TT), double product (DP) scores, and Canadian Cardiovascular Society (CCS) angina scores between pre- and post-treatment treadmill stress tests. Circulating levels of CD34+/CD133+ cells increased in patients undergoing EECP and were significant (ß = -2.38, p = 0.012) predictors of improved exercise capacity in these patients. CD34+/CD133+ cells isolated from RA patients could differentiate into endothelial cells, and their numbers increased during EECP therapy. Our results support the hypothesis that mobilized CD34+/CD133+ cells repair vascular damage and increase collateral circulation in RA patients. They further support clinical interventions that can mobilize adult CD34+ stem cells as therapy for patients with RA and other vascular diseases.

Human Umbilical Cord Blood Endothelial Progenitor Cell-Derived Extracellular Vesicles Control Important Endothelial Cell Functions.

Circulating endothelial progenitor cells (EPCs) play a pivotal role in the repair of diseases in which angiogenesis is required. Although they are a potentially valuable cell therapy tool, their clinical use remains limited due to suboptimal storage conditions and, especially, long-term immune rejection. EPC-derived extracellular vesicles (EPC-EVs) may be an alternative to EPCs given their key role in cell-cell communication and expression of the same parental markers. Here, we investigated the regenerative effects of umbilical cord blood (CB) EPC-EVs on CB-EPCs in vitro. After amplification, EPCs were cultured in a medium containing an EVs-depleted serum (EV-free medium). Then, EVs were isolated from the conditioned medium with tangential flow filtration (TFF). The regenerative effects of EVs on cells were investigated by analyzing cell migration, wound healing, and tube formation. We also analyzed their effects on endothelial cell inflammation and Nitric Oxide (NO) production. We showed that adding different doses of EPC-EVs on EPCs does not alter the basal expression of the endothelial cell markers nor change their proliferative potential and NO production level. Furthermore, we demonstrated that EPC-EVs, when used at a higher dose than the physiological dose, create a mild inflammatory condition that activates EPCs and boosts their regenerative features. Our results reveal for the first time that EPC-EVs, when used at a high dose, enhance EPC regenerative functions without altering their endothelial identity.

TNFα priming through its interaction with TNFR2 enhances endothelial progenitor cell immunosuppressive effect: new hope for their widespread clinical application.

BACKGROUND: Bone marrow derived endothelial progenitor cells (EPCs) are immature endothelial cells (ECs) involved in neo-angiogenesis and endothelial homeostasis and are considered as a circulating reservoir for endothelial repair. Many studies showed that EPCs from patients with cardiovascular pathologies are impaired and insufficient; hence, allogenic sources of EPCs from adult or cord blood are considered as good choices for cell therapy applications. However, allogenic condition increases the chance of immune rejection, especially by T cells, before exerting the desired regenerative functions. TNFα is one of the main mediators of EPC activation that recognizes two distinct receptors, TNFR1 and TNFR2. We have recently reported that human EPCs are immunosuppressive and this effect was TNFα-TNFR2 dependent. Here, we aimed to investigate if an adequate TNFα pre-conditioning could increase TNFR2 expression and prime EPCs towards more immunoregulatory functions. METHODS: EPCs were pre-treated with several doses of TNFα to find the proper dose to up-regulate TNFR2 while keeping the TNFR1 expression stable. Then, co-cultures of human EPCs and human T cells were performed to assess whether TNFα priming would increase EPC immunosuppressive and immunomodulatory effect. RESULTS: Treating EPCs with 1 ng/ml TNFα significantly up-regulated TNFR2 expression without unrestrained increase of TNFR1 and other endothelial injury markers. Moreover, TNFα priming through its interaction with TNFR2 remarkably enhanced EPC immunosuppressive and anti-inflammatory effects. Conversely, blocking TNFR2 using anti-TNFR2 mAb followed by 1 ng/ml of TNFα treatment led to the TNFα-TNFR1 interaction and polarized EPCs towards pro-inflammatory and immunogenic functions. CONCLUSIONS: We report for the first time the crucial impact of inflammation notably the TNFα-TNFR signaling pathway on EPC immunological function. Our work unveils the pro-inflammatory role of the TNFα-TNFR1 axis and, inversely the anti-inflammatory implication of the TNFα-TNFR2 axis in EPC immunoregulatory functions. Priming EPCs with 1 ng/ml of TNFα prior to their administration could boost them toward a more immunosuppressive phenotype. This could potentially lead to EPCs' longer presence in vivo after their allogenic administration resulting in their better contribution to angiogenesis and vascular regeneration. Video Abstract.

METTL1 limits differentiation and functioning of EPCs derived from human-induced pluripotent stem cells through a MAPK/ERK pathway.

Transplantation of endothelial progenitor cells (EPCs) has high therapeutic potential for ischemia-related ailments like heart attacks and claudication. Due to limited EPC sources, direct reprogramming is a fast-developing way to convert human-induced pluripotent stem cells (hiPSCs) into EPCs fit for transplantation. However, the procedural efficacy was affected by multiple factors, including epigenetic modifications. Recent studies have shown that m7G methylation mediated by Methyltransferase like 1 (METTL1) is required for mouse embryonic stem cells (mESCs) to differentiate normally. Yet, its contributions to EPC differentiation still require elucidation. Here, using immunofluorescence microscopy and Fluorescence-activated Cell Sorting (FACS), we found that the typical EPC markers were significantly increased in METTL1 knockdown (METTL1-KD) hiPSCs-derived EPCs compared to those of control types. In addition, we found that METTL1 knockdown activates the MAPK/ERK signaling pathway during EPCs differentiation from hiPSCs. Furthermore, functional properties of METTL1-KD EPCs were significantly raised above those of control hiPSCs-derived EPCs. Moreover, we proved that METTL1-KD hiPSCs-derived EPCs significantly accelerate vascular smooth muscle cell proliferation and 'phenotype switching' through a co-culture system. To sum up, our results demonstrate that METTL1-KD significantly promotes the differentiation of EPCs along with their in vitro functions, and this effect may be achieved through activation of the MAPK/ERK signaling pathway. This enhances current knowledge of EPC generation from hiPSCs and presents a new therapeutic target of vascular diseases.

The TNF/TNFR2 signaling pathway is a key regulatory factor in endothelial progenitor cell immunosuppressive effect.

BACKGROUND: Endothelial progenitor cells (EPCs) are non-differentiated endothelial cells (ECs) present in blood circulation that are involved in neo-vascularization and correction of damaged endothelial sites. Since EPCs from patients with vascular disorders are impaired and inefficient, allogenic sources from adult or cord blood are considered as good alternatives. However, due to the reaction of immune system against allogenic cells which usually lead to their elimination, we focused on the exact role of EPCs on immune cells, particularly, T cells which are the most important cells applied in immune rejection. TNFα is one of the main activators of EPCs that recognizes two distinct receptors. TNFR1 is expressed ubiquitously and its interaction with TNFα leads to differentiation and apoptosis, whereas, TNFR2 is expressed predominantly on ECs, immune cells and neural cells and is involved in cell survival and proliferation. Interestingly, it has been shown that different immunosuppressive cells express TNFR2 and this is directly related to their immunosuppressive efficiency. However, little is known about immunological profile and function of TNFR2 in EPCs. METHODS: Using different in-vitro combinations, we performed co-cultures of ECs and T cells to investigate the immunological effect of EPCs on T cells. We interrupted in the TNFα/TNFR2 axis either by blocking the receptor using TNFR2 antagonist or blocking the ligand using T cells derived from TNFα KO mice. RESULTS: We demonstrated that EPCs are able to suppress T cell proliferation and modulate them towards less pro-inflammatory and active phenotypes. Moreover, we showed that TNFα/TNFR2 immune-checkpoint pathway is critical in EPC immunomodulatory effect. CONCLUSIONS: Our results reveal for the first time a mechanism that EPCs use to suppress immune cells, therefore, enabling them to form new immunosuppressive vessels. Furthermore, we have shown the importance of TNFα/TNFR2 axis in EPCs as an immune checkpoint pathway. We believe that targeting TNFR2 is especially crucial in cancer immune therapy since it controls two crucial aspects of tumor microenvironment: 1) Immunosuppression and 2) Angiogenesis. Video Abstract. (MP4 46355 kb).

Chemerin enhances the adhesion and migration of human endothelial progenitor cells and increases lipid accumulation in mice with atherosclerosis.

BACKGROUND: The role of adipokines in the development of atherosclerosis (AS) has received increasing attention in recent years. This study aimed to explore the effects of chemerin on the functions of human endothelial progenitor cells (EPCs) and to investigate its role in lipid accumulation in ApoE-knockout (ApoE-/-) mice. METHODS: EPCs were cultured and treated with chemerin together with the specific p38 mitogen-activated protein kinase (MAPK) inhibitor SB 203580 in a time- and dose-dependent manner. Changes in migration, adhesion, proliferation and the apoptosis rate of EPCs were detected. ApoE-/- mice with high-fat diet-induced AS were treated with chemerin with or without SB 203580. Weights were recorded, lipid indicators were detected, and tissues sections were stained. RESULTS: The data showed that chemerin enhanced the adhesion and migration abilities of EPCs, and reduced the apoptosis ratio and that this effect might be mediated through the p38 MAPK pathway. Additionally, chemerin increased the instability of plaques. Compared with the control group and the inhibitor group, ApoE-/- mice treated with chemerin protein had more serious arterial stenosis, higher lipid contents in plaques and decreased collagen. Lipid accumulation in the liver and kidney and inflammation in the hepatic portal area were enhanced by treatment with chemerin, and the size of adipocytes also increased after chemerin treatment. In conclusion, chemerin can enhance the adhesion and migration abilities of human EPCs and reduce the apoptosis ratio. In animals, chemerin can increase lipid accumulation in atherosclerotic plaques and exacerbate plaques instability. At the same time, chemerin can cause abnormal lipid accumulation in the livers and kidneys of model animals. After specifically blocking the p38 MAPK pathway, the effect of chemerin was reduced. CONCLUSIONS: In conclusion, this study showed that chemerin enhances the adhesion and migration abilities of EPCs and increases the instability of plaques and abnormal lipid accumulation in ApoE-/- mice. Furthermore, these effects might be mediated through the p38 MAPK pathway.

Silica-coated magnetic nanoparticles labeled endothelial progenitor cells alleviate ischemic myocardial injury and improve long-term cardiac function with magnetic field guidance in rats with myocardial infarction.

Low retention of endothelial progenitor cells (EPCs) in the infarct area has been suggested to be responsible for the poor clinical efficacy of EPC therapy for myocardial infarction (MI). This study aimed to evaluate whether magnetized EPCs guided through an external magnetic field could augment the aggregation of EPCs in an ischemia area, thereby enhancing therapeutic efficacy. EPCs from male rats were isolated and labeled with silica-coated magnetic iron oxide nanoparticles to form magnetized EPCs. Then, the proliferation, migration, vascularization, and cytophenotypic markers of magnetized EPCs were analyzed. Afterward, the magnetized EPCs (1 × 106 ) were transplanted into a female rat model of MI via the tail vein at 7 days after MI with or without the guidance of an external magnet above the infarct area. Cardiac function, myocardial fibrosis, and the apoptosis of cardiomyocytes were observed at 4 weeks after treatment. In addition, EPC retention and the angiogenesis of ischemic myocardium were evaluated. Labeling with magnetic nanoparticles exhibited minimal influence to the biological functions of EPCs. The transplantation of magnetized EPCs guided by an external magnet significantly improved the cardiac function, decreased infarction size, and reduced myocardial apoptosis in MI rats. Moreover, enhanced aggregations of magnetized EPCs in the infarcted border zone were observed in rats with external magnet-guided transplantation, accompanied by the significantly increased density of microvessels and upregulated the expression of proangiogenic factors, when compared with non-external-magnet-guided rats. The magnetic field-guided transplantation of magnetized EPCs was associated with the enhanced aggregation of EPCs in the infarcted border zone, thereby improving the therapeutic efficacy of MI.

MicroRNA-212 promotes the recovery function and vascular regeneration of endothelial progenitor cells in mice with ischemic stroke through inactivation of the notch signaling pathway via downregulating MMP9 expression.

Ischemic stroke is a refractory disease caused by cerebral ischemic injury, which results in brain dysfunction. This study intends to investigate the effects of microRNA-212 (miR-212) on the recovery function and vascular regeneration of endothelial progenitor cells (EPCs) by inactivation of the Notch signaling pathway by binding to matrix metallopeptidase 9 (MMP9) in mice with ischemic stroke. According to the results of database retrieval systems and data analysis, MMP9 was predicted as a gene related to ischemic stroke and miR-212 is a potential regulating mRNA of MMP9. All 72 healthy adult C57BL6 mice were selected for middle cerebral artery occlusion (MCAO) establishment. Cerebral infarction was observed under triphenyltetrazolium chloride staining. A series of inhibitors, activators, and siRNAs were introduced to the verified regulatory functions for miR-212 governing MMP9 in ischemic stroke. Cell proliferation was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and tube-forming ability by tubule formation test. Reverse transcription quantitative polymerase chain reaction and Western blot analysis were used to detect the expressions of miR-212, MMP9, Hes-1, and Notch-1. The corresponding results demonstrated that the area of cerebral infarction and the number of neuronal necrosis increased in the MCAO group in contrast to the sham group. Meanwhile, upregulation of miR-212 or downregulation of MMP9 decreases the expressions of MMP9, Hes-1 Notch-1, increases cell proliferation and tube-forming ability and improves the pathological conditions of EPCs. Our study suggests that miR-212 promotes recovery function and vascular regeneration of EPCs through negative regulation of the Notch signaling pathway via downregulating expression of MMP9, thus provides a clinical theoretical basis for ischemic stroke therapy.

Bone-marrow-derived endothelial progenitor cells contribute to vasculogenesis of pregnant mouse uterus†.

Angiogenesis is essential for cyclic endometrial growth, implantation, and pregnancy maintenance. Vasculogenesis, the formation of new blood vessels by bone marrow (BM)-derived endothelial progenitor cells (EPCs), has been shown to contribute to endometrial vasculature. However, it is unknown whether vasculogenesis occurs in neovascularization of the decidua during pregnancy. To investigate the contribution of BM-derived EPCs to vascularization of the pregnant uterus, we induced non-gonadotoxic submyeloablation by 5-fluorouracil administration to wild-type FVB/N female mice recipients followed by BM transplantation from transgenic mice expressing green fluorescent protein (GFP) under regulation of Tie2 endothelial-specific promoter. Following 1 month, Tie2-GFP BM-transplanted mice were bred and sacrificed at various gestational days (ED6.5, ED10.5, ED13.5, ED18.5, and postpartum). Bone-marrow-transplanted non-pregnant and saline-injected pregnant mice served as controls (n = 5-6/group). Implantation sites were analyzed by flow cytometry, immunohistochemistry, and immunofluorescence. While no GFP-positive EPCs were found in non-pregnant or early pregnant uteri of BM-transplanted mice, GFP-positive EPCs were first detected in pregnant uterus on ED10.5 (0.12%) and increased as the pregnancy progressed (1.14% on ED13.5), peaking on ED18.5 (1.42%) followed by decrease in the postpartum (0.9%). The percentage of endothelial cells that were BM-derived out of the total endothelial cell population in the implantation sites (GFP+CD31+/CD31+) were 9.3%, 15.8%, and 6.1% on ED13.5, ED18.5, and postpartum, respectively. Immunohistochemistry demonstrated that EPCs incorporated into decidual vasculature, and immunofluorescence showed that GFP-positive EPCs colocalized with CD31 in vascular endothelium of uterine implantation sites, confirming their endothelial lineage. Our findings indicate that BM-derived EPCs contribute to vasculogenesis of the pregnant mouse decidua.

Superparamagnetic iron oxide (SPIO) nanoparticles labeled endothelial progenitor cells (EPCs) administration inhibited heterotopic ossification in rats.

Heterotopic ossification (HO) is a painful disease characterized by unwanted bone ectopic formation outside of the skeleton after injury. SPIO nanoparticles therapy has been widely used in diverse orthopedic diseases. However, the effect of SPIO nanoparticles on heterotopic ossification remains unknown. Here, we prepared the SPIO nanoparticles carrying mothers against decapentaplegic homolog 7 (SMAD7) and evaluated their mechanism function to HO in a rat model. The results revealed that SPIO nanoparticles containing SMAD7 treatment lead to a decrease in epithelial-mesenchymal transition (EMT) relevant protein expression in vitro. Moreover, SPIO nanoparticles labeled EPCs transplantation effectively prevented heterotopic ossification and inhibited endothelial-mesenchymal transition (EndMT) in HO rats. In addition, SPIO nanoparticles labeled EPCs transplantation suppressed osteogenic and adipogenic differentiation of embryonic fibroblasts (EFs) in HO rats. Our results demonstrated that administration of SPIO nanoparticles labeled EPCs could inhibit heterotopic ossification in rats, which might be a potential therapy method for a medical intervention to treat HO in clinic.

Sweeteners modulate bioactivity of endothelial progenitor cells but not induce detrimental effects both on inflammation and behavioural changes.

This study sought to determine the possible detrimental effects of several low- or non-caloric sweeteners on endothelial progenitor cells (EPCs), inflammation and behavioural changes in mice. C57BL/6 male mice received low and high dose of natural and artificial sweeteners for 4 weeks. EPCs, physical and biochemical variables, inflammation and behavioural changes were evaluated. A significant reduction of about 25% of EPCs was found when mice received a moderate amount of all sweeteners (p < .05). This reduction was more strongly significant when a double dose of glucose, aspartame, rebaudioside A and cyclamate (p < .005) in comparison to fructose and sucrose (p < .05) was administered. During inflammation carrageenan-induced, all sweeteners produced a significant increase of EPCs compared to the control group (p < .05). Consumption of glucose and sugar substitutes affect mouse EPC number according to the absence or presence of an inflammatory status, but does not induce detrimental effects on inflammation and behavioural changes.

Endothelial progenitor cells from aged subjects display decreased expression of sirtuin 1, angiogenic functions, and increased senescence.

Studies have demonstrated that aging is associated with a substantial decline in numbers and angiogenic activity of endothelial progenitor cells (EPCs). In view of senescence being an important regulator of age-related cell survival and function, in the current study, we correlated EPCs numbers and functions with their senescence status and mechanisms in young and elderly subjects. Healthy young subjects (n = 30, below 60 y) and old subjects (n = 30, equal to or above 60 y) participated in the study. Subjects had no significant disease or risk factors of disease and aging was the only risk factor in the aged subjects. Enumeration of CD34-vegfr2 dual positive EPCs was performed. The ex vivo culture of EPCs was done to study colony formation, migration, and senescence-associated beta-galactosidase activity. The expression of cell cycle and senescence regulatory proteins including, p53, p21, and sirtuin 1 (SIRT1), a deacetylase protein was studied in cultured EPCs by RT-PCR and immunofluorescence staining. In vivo proliferation, ex vivo colonies, migration, and secretory ability of EPCs was significantly higher in young subjects than that in elderly subjects. EPCs in old subjects showed enhanced senescence and decreased expression of SIRT1 in comparison to that observed in young subjects. An inhibition of SIRT1 in EPCs of young subjects led to significant increase in senescence and reduction of cell differentiation. The study suggests that EPCs have decreased proliferation and functions in aged subjects due to increased senescence which may be attributable to decreased expression of SIRT1.

Sphingosine-1-phosphate promotes the proliferation and attenuates apoptosis of Endothelial progenitor cells via S1PR1/S1PR3/PI3K/Akt pathway.

Sphingosine-1-phosphate (S1P) is a bioactive lysophospholipid that involves in numerous pathophysiological processes. Endothelial progenitor cells (EPCs) play a crucial role in endothelial repair and tumor angiogenesis. The aim of study was to determine the effects of S1P on proliferation and anti-apoptosis of EPCs and their signaling pathways. In this study, we showed that S1P, SEW2871 (a selective S1P receptor 1 (S1PR1) agonist), or CYM5541 (a selective S1P receptor 3 (S1PR3) allosteric agonist promotes the proliferation and attenuates apoptosis of bone marrow (BM)-derived EPCs. Futhermore, it was showed that S1P could promote EPCs proliferation, which could be significantly inhibited by pretreatment with CAY10444 (an S1PR3 antagonist), VPC23019 (a selective S1PR(1)/S1PR(3) antagonist), or LY294002 (a PI3K inhibitor). Moveover, we discovered that S1P could significantly attenuate H2 O2 -induced apoptosis and activation of caspase-3 in vitro, while W146 (an S1PR1 antagonist), VPC23019, or LY294002 could significantly increase the activation of caspase-3 and subsequent augmented apoptosis. Our results indicated that the protective effect of S1P is mediated by activating the PI3K/Akt pathway. In addition, S1P promotion of EPCs proliferation was observed to be mainly mediated through S1PR3 and attenuation of EPCs apoptosis induced by H2 O2 was mainly mediated through S1PR1; both of these effects are mediated by activating the PI3K/Akt pathway, which provides potentially useful therapeutic targets for coronary artery disease, diabetes mellitus, and cancer treatment.

Chronic stress is associated with reduced circulating hematopoietic progenitor cell number: A maternal caregiving model.

BACKGROUND: Chronic psychological stress is a risk factor for cardiovascular disease and mortality. Circulating hematopoietic progenitor cells (CPCs) maintain vascular homeostasis, correlate with preclinical atherosclerosis, and prospectively predict cardiovascular events. We hypothesize that (1) chronic caregiving stress is related to reduced CPC number, and (2) this may be explained in part by negative interactions within the family. METHODS: We investigated levels of stress and CPCs in 68 healthy mothers - 31 of these had children with an autism spectrum disorder (M-ASD) and 37 had neurotypical children (M-NT). Participants provided fasting blood samples, and CD45+CD34+KDR+ and CD45+CD133+KDR+ CPCs were assayed by flow cytometry. We averaged the blom-transformed scores of both CPCs to create one index. Participants completed the perceived stress scale (PSS), the inventory for depressive symptoms (IDS), and reported on daily interactions with their children and partners, averaged over 7 nights. RESULTS: M-ASD exhibited lower CPCs than M-NT (Cohen's d=0.83; p⩽0.01), controlling for age, BMI, and physical activity. Across the whole sample, positive interactions were related to higher CPCs, and negative interactions to lower CPCs (allp's<0.05). The adverse effects of group on CPCs were significantly mediated through negative interactions with the child (indirect ß=-0.24, p⩽0.01). In the full model, greater age (ß=-0.19, p=0.04), BMI (ß=-0.18, p=0.04), and negative interactions with the child (ß=-0.33, p<0.01) were independently associated with lower CPCs. M-ASD had a less healthy lipid profile (total cholesterol/HDL), which in turn, was associated with lower CPCs. CONCLUSIONS: Chronic stress adversely impacts CPC number, an early-stage biomarker that predicts subclinical atherosclerosis and future CVD events, independent of traditional cardiovascular risk factors and inflammatory factors. Among maternal caregivers, child-related interpersonal stress appears to be a key psychological predictor of stress-related CVD risk.

Effects of transplanted circulating endothelial progenitor cells and platelet microparticles in atherosclerosis development.

BACKGROUND INFORMATION: Atherosclerosis is an inflammatory disease, in which risk factors such as hyperlipidemia and hypertension affect the arterial endothelium, resulting in dysfunction, cell damage or both. The number of circulating endothelial progenitor cells and microparticles provides invaluable outcome prediction for atherosclerosis disease. However, evidence for the therapeutic potential of endothelial progenitor cells and microparticles in atherosclerosis development is limited. Our study was designed to investigate the possible protective role of a cell therapy-based approach, using endothelial progenitor cells and the dual behaviour of circulating platelet microparticles, on atherosclerosis development in hypertensive-hypercholesterolemic hamster model. Consequently, control hamsters received four intravenous inoculations of: (1) 1×10(5) endothelial progenitor cells of healthy origins in one dose per month, during four months of diet-induced atherosclerosis, and after hypertensive-hypercholesterolemic diet for further four months; (2) in a second set of experiments, 1×10(5) endothelial progenitor cells of healthy origins or/and 1×10(5) platelet microparticles of atherosclerotic origins were inoculated every other month during hypertensive-hypercholesterolemic diet. RESULTS: Endothelial progenitor cell treatment had the following effects: (1) re-established plasmatic parameters: cholesterol and triglyceride concentrations, blood pressure, heart rate, cytokine and chemokine profiles, platelet microparticle pro-thrombotic activity and endothelial progenitor cell paracrine activity reflected by cytokine/chemokine detection; (2) reduced lipid, macrophage and microparticle accumulation in liver; (3) reduced atherosclerosis development, revealed by decreased lipid, macrophage and microparticle content of arterial wall; (4) induced the recruitment and incorporation of endothelial progenitor cells into liver and arterial wall; (5) improved arterial dysfunction by increasing contraction and relaxation; (6) reduced the protein expression of specific pro-inflammatory molecules in liver and arterial wall. Platelet microparticle transplantation aggravated the above-mentioned biomarkers and atherosclerosis process, which were partially reverted with co-inoculation of platelet microparticles and endothelial progenitor cells. CONCLUSIONS: With this study, we demonstrate in a hypertensive-hypercholesterolemic hamster model, that the endothelial progenitor cell-based therapy suppresses the development of atherosclerosis and reduces hepatic lipid and macrophage accumulation with the consequent alleviation of dyslipidaemia and hypertension. SIGNIFICANCE: Our results support the notion that increasing the number of circulating endothelial progenitor cells by different ways could be a promising therapeutic tool for atherosclerosis.

Quercitrin treatment protects endothelial progenitor cells from oxidative damage via inducing autophagy through extracellular signal-regulated kinase.

Atherosclerosis is a disease resulting from impaired endothelial function, often caused by oxidant injury or inflammation. Endothelial progenitor cells (EPCs) play a critical role in repairing damaged endothelium and protecting against atherosclerosis. Quercitrin, a plant-derived flavonoid compound, displays antioxidant and anti-inflammatory activities. In this study, we showed that quercitrin treatment reduced the apoptosis of EPCs caused by oxidized low-density lipoprotein (ox-LDL) in a dose-dependent manner. Quercitrin improved tube formation, migration and adhesion of ox-LDL-treated EPCs. To determine the effect of quercitrin in vivo, EPCs treated with or without ox-LDL and quercitrin were locally injected into the ischemic hind limb muscle of nude mice. Those injected with EPCs treated with ox-LDL and quercitrin showed significantly increased local accumulation of EPCs, blood flow recovery and capillary density compared with the control and ox-LDL only groups. Furthermore, we showed that quercitrin enhanced autophagy and upregulated mitogen-activated protein kinase and ERK phosphorylation in a dose-dependent manner in vitro. Autophagy inhibitors, chloroquine and 3-methyladenine, abrogated quercitrin-enhanced autophagy caused by ox-LDL as evidenced by decreased numbers of branch points, migratory cells and adherent cells, and increased numbers of apoptotic cells. The ERK inhibitor PD98059 abrogated quercitrin-enhanced autophagy, as identified by decreased autophagosome formation and downregulated ERK phosphorylation. The inhibition of ERK did not affect the expression of Rac1, but enhanced phosphorylation of Akt. Quercitrin treatment also increased the expression of E-cadherin, and PD98059 abrogated the upregulation of E-cadherin induced by quercitrin. Our findings suggested that autophagy is a protective mechanism in EPCs exposed to oxidative damage. Quercitrin can promote autophagy through the activation of ERK and the ERK signaling pathway is therefore thought to play a pivotal role in mediating the protective effects on EPCs.

Metformin improves the angiogenic functions of endothelial progenitor cells via activating AMPK/eNOS pathway in diabetic mice.

BACKGROUND: Endothelial dysfunction has been suggested as a possible causal link between hyperglycemia and microvascular complications in diabetes mellitus. The effect of metformin on endothelial progenitor cells (EPCs) is still unclear. This study was designed to test the hypothesis that metformin could accelerate wound healing by improving the impaired EPC functions in streptozotocin-induced diabetic mice. METHODS: Streptozotocin (STZ, 60 mg/kg/d × 5 d, i.p.) was injected to induce type 1 diabetes in male C57BL/6 mice. Mice were treated with metformin (250 mg/kg/d, i.g.) for consecutive 14 days. Wound closure was evaluated by wound area and number of CD31 stained capillaries. Functions of bone marrow-endothelial progenitor cells (BM-EPCs) were assessed by tube formation and migration assays, and expression of AMP-activated protein kinase (AMPK) and endothelial nitric oxide synthase (eNOS) was determined by western blot analysis. RESULTS: Metformin accelerated wound closure and stimulated angiogenesis in diabetic mice. The number of circulating EPCs was increased significantly in metformin treated diabetic mice. Abilities of tube formation and migration of BM-EPCs were impaired in diabetic mice, which were improved by metformin. Expression of both phosphorylated-AMPK and phosphorylated-eNOS was significantly increased, and nitric oxide (NO) production was enhanced by metformin in BM-EPCs of diabetic mice. In vitro, metformin improved impaired BM-EPC functions, and increased phosphorylated-eNOS expression and NO production in cultured BM-EPCs caused by high glucose, which was prevented by the AMPK inhibitor compound C. CONCLUSIONS: Our results suggest that metformin could improve BM-EPC functions in STZ-induced diabetic mice, which was possibly dependent on the AMPK/eNOS pathway.

Exploring the various aspects of the pathological role of vascular endothelial growth factor (VEGF) in diabetic retinopathy.

Diabetic retinopathy, a sight-threatening microvascular complication of diabetes mellitus, is initiated by retinal endothelial dysfunction and succeeded by various pathological events, eventually resulting in vision-loss. These events are regulated by numerous mediators, including vascular endothelial growth factor (VEGF), which induces the progression of various events characterizing diabetic retinopathy, such as neovascularization and macular edema. VEGF is physiologically required for regulating proliferation and assembling of endothelial cells, during vasculogenesis, as well as for their maintenance and survival throughout the lifetime of blood vessels. However, various pathological conditions are induced in the body during diabetes (such as ischemia, oxidative stress and overactivation of protein kinase C), which upregulate the expression of VEGF, thereby deviating it from its physiological role and leading to various pathological demonstrations such as angiogenesis, increased permeability of endothelium, decreased inhibition of pro-apoptotic proteins and activation of various other inflammatory mediators. Such events disrupt vascular homeostasis and play key roles in the pathophysiology of diabetic retinopathy. Hence, acknowledging various VEGF-mediated pathways helps in understanding the deeper aspects related to progression of this disorder. Targeting and inhibiting VEGF-mediated disease progression might provide an effective alternative therapy and hence prove beneficial in the treatment of diabetic retinopathy.

Endothelial progenitor cells and pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disease characterised by lung endothelial cell dysfunction and vascular remodelling. A number of studies now suggest that endothelial progenitor cells (EPCs) may induce neovascularisation and could be a promising approach for cell based therapy for PAH. On the contrary EPCs may contribute to pulmonary vascular remodelling, particularly in end-stage pulmonary disease. This review article will provide a brief summary of the relationship between PAH and EPCs, the application of the EPCs to PAH and highlight the potential clinical application of the EPCs cell therapy to PAH.

Exosomal hsa_circ_0093884 derived from endothelial progenitor cells promotes therapeutic neovascularization via miR-145/SIRT1 pathway.

Therapeutic neovascularization is a strategy to promote blood vessel growth and improve blood flow, which is critical to tissue repair and regeneration in ischemic diseases. Here, we investigated the role of endothelial progenitor cell - derived exosomes (EPC-Exos) in therapeutic neovascularization and clarified the mechanism of hsa_circ_0093884 in EPC-Exos mediated neovascularization. Injection of EPC-Exos improved mouse ischemic hindlimb perfusion, promoted angiogenesis in Matrigel plugs and mouse skin wound healing. In vitro coculture with EPC-Exos improved HUVEC proliferation, angiogenic and migration ability, while alleviated hypoxia-induced apoptosis. hsa_circ_0093884 was identified from eleven types of circRNA derived from SIRT1 and proved to be enriched in EPC-Exos. Overexpression of hsa_circ_0093884 in EPC-Exos further enhanced the angiogenic capacity, while knockdown of hsa_circ_0093884 abolished the benefits. Mechanistically, EPC-Exos mediated shuttling of hsa_circ_0093884 induced cytoplasmic sponge of miR-145, thereby releasing repression of SIRT1. In vitro co-transfection indicated silence of miR-145 further strengthened the angiogenic effect of hsa_circ_0093884, while overexpression of miR-145 inhibited hsa_circ_0093884 mediated angiogenesis and abolished the beneficial effect of EPC-Exos. Furthermore, in vivo experiments using endothelial specific SIRT1 conditional knockout mice indicated hsa_circ_0093884 overexpressing EPC-Exos failed to promote therapeutic neovascularization in SIRT1cKO mice. Collectively, our results demonstrated that EPC-Exos promoted therapeutic neovascularization through hsa_circ_0093884/miR-145/SIRT1 axis.