Antioxidants increase number of progenitor endothelial cells through multiple gene expression pathways

To date, there is no report on the effect of antioxidants on endothelial progenitor cells (EPCs). This study shows that in vitro incubation of EPCs with vitamin C and E reverted the already well documented lowering effect of TNF-α on EPC number and increased p-p38 expression levels. In order to document major changes of gene expression levels and gain insight into signalling pathways, microarray analysis was performed and a significant variation of the expression of 5389 genes in EPCs following antioxidant treatment was detected. Also in vivo evidence is provided about the positive effect of antioxidant vitamins on EPCs, since vitamin C and E supplementation potentiated the physical training-induced increase of EPC number and VEGF levels. Together, these data indicate that antioxidant treatment ameliorates EPC number and causes major changes of gene expression within these cells in vitro. Furthermore, concomitant antioxidant supplementation and physical training in vivo raised the levels of circulating EPCs and serum VEGF more than physical training alone.     

Physical exercise attenuates age-associated reduction in endothelium-reparative capacity of endothelial progenitor cells by increasing CXCR4/JAK-2 signaling in healthy men

Endothelial progenitor cells (EPCs) play an important role in repairing endothelial injury. Aging is associated with EPC dysfunction. Physical exercise has a beneficial impact on EPC activity. However, whether physical exercise can enhance the endothelial repair capacity of EPCs in healthy men with aging is not clear. Here, we investigated the effects of physical exercise on reendothelialization capacity and CXC chemokine receptor four (CXCR4) signaling in human EPCs. Before and after 12-week exercise, EPCs were isolated from elderly and young men. In vitro function and in vivo reendothelialization capacity of EPCs in a mouse model of carotid artery injury were measured. The expression of CXCR4 and its downstream signaling target Janus kinase-2 (JAK-2) were determined. Before exercise, in vitro function and in vivo reendothelialization capacity of EPCs were significantly reduced in elderly men compared with young men. After exercise intervention, in vitro function and in vivo reendothelialization capacity of EPCs from elderly men were markedly enhanced. Physical exercise increased a higher CXCR4 protein expression and higher JAK-2 phosphorylation levels of EPCs. The augmentation in reendothelialization capacity of EPCs was closely correlated with the upregulation of CXCR4/JAK-2 signaling and improvement of endothelial function. This study demonstrates for the first time that physical exercise attenuates age-associated reduction in endothelium-reparative capacity of EPCs by increasing CXCR4/JAK-2 signaling. Our findings provide insight into the novel mechanisms of physical exercise as a lifestyle intervention strategy to promote vascular health in aging population.     

Enhancement of angiogenic and vasculogenic potential of endothelial progenitor cells by haptoglobin

Endothelial progenitor cells (EPCs) were transfected with the haptoglobin (Hp) gene to investigate the effect of Hp on cell function. Hp potentiated the gene expression of various pro-angiogenic factors in the EPCs. The Hp-modified EPCs also increased in vitro tube formation on Matrigel compared with control cells. In hindlimb ischaemia models, Hp-EPCs showed a greater ability for improving blood perfusion and recovery from ischaemic injury. These results indicate that Hp improves EPC function in neovasculogenesis, which suggests that ex vivo modification of EPCs with the Hp gene can be applied to the treatment of vascular damage.     

VEGF contributes to postnatal neovascularization by mobilizing bone marrow-derived endothelial progenitor cells.

Vascular endothelial growth factor (VEGF) has been shown to promote neovascularization in animal models and, more recently, in human subjects. This feature has been assumed to result exclusively from its direct effects on fully differentiated endothelial cells, i.e. angiogenesis. Given its regulatory role in both angiogenesis and vasculogenesis during fetal development, we investigated the hypothesis that VEGF may modulate endothelial progenitor cell (EPC) kinetics for postnatal neovascularization. Indeed, we observed an increase in circulating EPCs following VEGF administration in vivo. VEGF-induced mobilization of bone marrow-derived EPCs resulted in increased differentiated EPCs in vitro and augmented corneal neovascularization in vivo. These findings thus establish a novel role for VEGF in postnatal neovascularization which complements its known impact on angiogenesis.     

Procyanidin B2 improves endothelial progenitor cell function and promotes wound healing in diabetic mice via activating Nrf2

One of the major reasons for the delayed wound healing in diabetes is the dysfunction of endothelial progenitor cells (EPCs) induced by hyperglycaemia. Improvement of EPC function may be a potential strategy for accelerating wound healing in diabetes. Procyanidin B2 (PCB2) is one of the major components of procyanidins, which exhibits a variety of potent pharmacological activities. However, the effects of PCB2 on EPC function and diabetic wound repair remain elusive. We evaluated the protective effects of PCB2 in EPCs with high glucose (HG) treatment and in a diabetic wound healing model. EPCs derived from human umbilical cord blood were treated with HG. The results showed that PCB2 significantly preserved the angiogenic function, survival and migration abilities of EPCs with HG treatment, and attenuated HG-induced oxidative stress of EPCs by scavenging excessive reactive oxygen species (ROS). A mechanistic study found the protective role of PCB2 is dependent on activating nuclear factor erythroid 2-related factor 2 (Nrf2). PCB2 increased the expression of Nrf2 and its downstream antioxidant genes to attenuate the oxidative stress induced by HG in EPCs, which were abolished by knockdown of Nrf2 expression. An in vivo study showed that intraperitoneal administration of PCB2 promoted wound healing and angiogenesis in diabetic mice, which was accompanied by a significant reduction in ROS level and an increase in circulating EPC number. Taken together, our results indicate that PCB2 treatment accelerates wound healing and increases angiogenesis in diabetic mice, which may be mediated by improving the mobilization and function of EPCs.     

Downregulation of MicroRNA-130a Contributes to Endothelial Progenitor Cell Dysfunction in Diabetic Patients via Its Target Runx3

Dysfunction of endothelial progenitor cells (EPCs) contributes to diabetic vascular disease. MicroRNAs (miRs) have emerged as key regulators of diverse cellular processes including angiogenesis. We recently reported that miR-126, miR-130a, miR-21, miR-27a, and miR-27b were downregulated in EPCs from type II diabetes mellitus (DM) patients, and downregulation of miR-126 impairs EPC function. The present study further explored whether dysregulated miR-130a were also related to EPC dysfunction. EPCs were cultured from peripheral blood mononuclear cells of diabetic patients and healthy controls. Assays on EPC function (proliferation, migration, differentiation, apoptosis, and colony and tubule formation) were performed. Bioinformatics analyses were used to identify the potential targets of miR-130a in EPCs. Gene expression of miR-103a and Runx3 was measured by real-time PCR, and protein expression of Runx3, extracellular signal-regulated kinase (ERK), vascular endothelial growth factor (VEGF) and Akt was measured by Western blotting. Runx3 promoter activity was measured by luciferase reporter assay. A miR-130a inhibitor or mimic and lentiviral vectors expressing miR-130a, or Runx3, or a short hairpin RNA targeting Runx3 were transfected into EPCs to manipulate miR-130a and Runx3 levels. MiR-130a was decreased in EPCs from DM patients. Anti-miR-130a inhibited whereas miR-130a overexpression promoted EPC function. miR-130a negatively regulated Runx3 (mRNA, protein and promoter activity) in EPCs. Knockdown of Runx3 expression enhanced EPC function. MiR-130a also upregulated protein expression of ERK/VEGF and Akt in EPCs. In conclusion, miR-130a plays an important role in maintaining normal EPC function, and decreased miR-130a in EPCs from DM contributes to impaired EPC function, likely via its target Runx3 and through ERK/VEGF and Akt pathways.     

Anti-inflammatory properties of alpha- and gamma-tocopherol

Natural vitamin E consists of four different tocopherol and four different tocotrienol homologues (alpha,beta, gamma, delta) that all have antioxidant activity. However, recent data indicate that the different vitamin E homologues also have biological activity unrelated to their antioxidant activity. In this review, we discuss the anti-inflammatory properties of the two major forms of vitamin E, alpha-tocopherol (alphaT) and gamma-tocopherol (gammaT), and discuss the potential molecular mechanisms involved in these effects. While both tocopherols exhibit anti-inflammatory activity in vitro and in vivo, supplementation with mixed (gammaT-enriched) tocopherols seems to be more potent than supplementation with alphaT alone. This may explain the mostly negative outcomes of the recent large-scale interventional chronic disease prevention trials with alphaT only and thus warrants further investigation.     

In vivo enhancement of angiogenesis by adenoviral transfer of HIF-1alpha-modified endothelial progenitor cells (Ad-HIF-1alpha-modified EPC for angiogenesis)

Hypoxia inducible factor (HIF)-1alpha over-expression may have beneficial effects in cell therapy of hypoxia-induced pathophysiological processes, such as ischemic disease. Our previous study showed the feasibility of ex vivo modification of endothelial progenitor cells (EPCs) by HIF-1alpha transfection. In this study, we sought to determine if such ex vivo modified EPCs facilitated functional therapeutic neovascularization. Ad-HIF-1alpha was transduced in human EPC in vitro. HIF-1alpha-transduced EPCs were administered to nude mice with hindlimb ischemia. BrdU-labeling of these EPCs showed that they enhanced neovascularization in vivo. Limb and toe necrosis was significantly reduced in HIF-1alpha-EPC group compared to GFP-EPC group and medium control group at 14 days after transplantation (both P<0.05). A statistically significant difference was still observed in the HIF-1alpha group until 1 and 2 months of follow-up. Neovascularization was improved by both histological and physiological assessments. Exogenous EPC homing was observed. HIF-1alpha over-expression enhanced its mRNA and protein expression in the ischemia zone. The expression of genes downstream of HIF-1alpha was examined to explore the possible mechanism of EPC homing. In conclusion, HIF-1alpha-EPC gene transfer augments impaired neovascularization in experimentally induced mouse hindlimb ischemia in vivo.     

Damage to liposomal lipids: protection by antioxidants and cholesterol-mediated dehydration

Liposomes composed of egg phosphatidylcholine (EPC) (13.4%, of the acyl chains being polyunsaturated fatty acids (PUFA)) and EPC/cholesterol (10:1 mol/mol) were studied for factors that affect liposomal lipid oxidative damage and hydrolysis upon long-term (16 months) storage. Factors studied include: (1) levels of lipid/water interface hydration, related to the presence of cholesterol in the lipid bilayer; (2) the membrane-associated antioxidant vitamin E; (3) the water-soluble antioxidant Tempol; and (4) exposure to light. Liposomal dispersions were stored at room temperature, either exposed to or protected from daylight, for a period of 16 months. Chemical and physical changes were monitored at several time points to assess oxidative and hydrolytic degradation of liposomal lipids. The conclusions of the study are: (1) PUFA are the most sensitive component of the liposome bilayer to oxidative degradation damage during long-term storage; (2) EPC liposomes are more sensitive to degradation during storage than EPC cholesterol liposomes, the presence of cholesterol in the lipid bilayer having a protective effect, probably due to its effect in decreasing the lipid-bilayer hydration; (3) oxidative degradation is the major process during long-term storage, having an earlier onset than the hydrolytic degradation: and (4) Tempol provided significantly better protection than vitamin E to EPC liposomal PUFA against oxidative damage during long-term storage. The relevance of cholesterol's presence, as a 'drying agent', in membranes containing PUFA to resistance of biological membranes to oxidative damage is discussed.     

Angiogenesis by transplantation of HIF-1 alpha modified EPCs into ischemic limbs

Hypoxia inducible factor-1 alpha (HIF-1 alpha) is a key determinant of oxygen-dependent gene regulation in angiogenesis. HIF-1 alpha overexpression may be beneficial in cell therapy of hypoxia-induced pathophysiological processes, such as ischemic heart disease. To address this issue, human peripheral blood mononuclear cells (PBMNCs) were induced to differentiate into endothelial progenitor cells (EPCs), and then were transfected with either an HIF-1 alpha-expressing or a control vector and cultured under normoxia or hypoxia. Hypoxia-induced HIF-1 alpha mRNA and protein expression was increased after HIF-1 alpha transfection. This was accompanied by VEGF mRNA induction and increased VEGF secretion. Hypoxia-stimulated VEGF mRNA induction was significantly abrogated by HIF-1 alpha-specific siRNA. Functional studies showed that HIF-1 alpha overexpression further promoted hypoxia-induced EPC differentiation, proliferation and migration. The expressions of endothelial cell markers CD31, VEGFR2 (Flk-1) and eNOS as well as VEGF and NO secretions were also increased. Furthermore, in an in vivo model of hindlimb ischemia, HIF-1 alpha-transfected EPCs homed to the site of ischemia. A higher revascularization potential was also demonstrated by increased capillary density at the injury site. Our results revealed that endothelial progenitor cells ex vivo modification by hypoxia inducible factor-1 alpha gene transfection is feasible and may offer significant advantages in terms of EPC expansion and treatment efficacy.     

Effect of One-Year Vitamin C- and E-Supplementation on Cerebrospinal Fluid Oxidation Parameters and Clinical Course in Alzheimer’s Disease

Antioxidant vitamins are being widely discussed as a therapeutic option in Alzheimer??s disease (AD). We recently found that supplementation with vitamin C and E over 1?month leads to an increase of their levels in cerebrospinal fluid (CSF) and a reduction of CSF lipid peroxidation. In the present study, we followed-up the biochemical and clinical effect of vitamin C and E supplementation in an open clinical trial over 1?year. Twelve AD patients stably taking a cholinesterase inhibitor were supplemented with vitamin C (1,000?mg/day) and E (400 I.U./day), while 11 patients taking cholinergic medication only served as a control group. Cognition was assessed at baseline, after 6 months and 12 months using the Mini-Mental State Examination; a more detailed testing of cognitive function was performed at baseline and after 12 months. From eight of the vitamin-supplemented patients, CSF was taken at baseline, after 1?month and after 1?year to measure the antioxidant effect of vitamin supplementation on CSF lipids using a recently established in vitro oxidation assay. CSF antioxidant vitamins were significantly increased after 1?month and 1?year of supplementation, while in vitro oxidation of CSF lipids was significantly reduced only after 1?year of the supplementation. The clinical course of AD did not significantly differ between the vitamin and the control group. We conclude that supplementation with vitamins E and C did not have a significant effect on the course of AD over 1?year despite of a limited antioxidant effect that could be observed in CSF.     

Brown fat thermogenesis and exercise: two examples of physiological oxidative stress?

Both brown fat tissue (BAT) and skeletal muscle experience large increases of oxygen consumption and oxygen radical generation during activation. This, together with the relatively low activities of antioxidant enzymes in these two tissues and the high lipid content and free fatty acid liberation of BAT, can produce a physiological oxidative stress. Increases of in vivo or in vitro (BAT) lipid peroxidation have been described in these tissues after activation. They react to this oxidative stress in an adaptive way after chronic stimulation. Cold acclimation increases antioxidant enzymes, ascorbate, and especially reduced glutathione (GSH) in BAT. There is controversy about the variations of antioxidants in skeletal muscle after acute exercise. Nevertheless, exercise training seems to increase muscle antioxidant enzymes and GSH. Many reports show that vitamin E levels decrease in the muscle and increase in plasma during exercise. Studies of vitamin E deficiency and supplementation strongly suggest that this vitamin is of protective value during exercise.     

Alcohol Consumption Negates Estrogen-mediated Myocardial Repair in Ovariectomized Mice by Inhibiting Endothelial Progenitor Cell Mobilization and Function

We have shown previously that estrogen (estradiol, E2) supplementation enhances voluntary alcohol consumption in ovariectomized female rodents and that increased alcohol consumption impairs ischemic hind limb vascular repair. However, the effect of E2-induced alcohol consumption on post-infarct myocardial repair and on the phenotypic/functional properties of endothelial progenitor cells (EPCs) is not known. Additionally, the molecular signaling of alcohol-estrogen interactions remains to be elucidated. This study examined the effect of E2-induced increases in ethanol consumption on post-infarct myocardial function/repair. Ovariectomized female mice, implanted with 17β-E2 or placebo pellets were given access to alcohol for 6 weeks and subjected to acute myocardial infarction. Left ventricular functions were consistently depressed in mice consuming ethanol compared with those receiving only E2. Alcohol-consuming mice also displayed significantly increased infarct size and reduced capillary density. Ethanol consumption also reduced E2-induced mobilization and homing of EPCs to injured myocardium compared with the E2-alone group. In vitro, exposure of EPCs to ethanol suppressed E2-induced proliferation, survival, and migration and markedly altered E2-induced estrogen receptor-dependent cell survival signaling and gene expression. Furthermore, ethanol-mediated suppression of EPC biology was endothelial nitric oxide synthase-dependent because endothelial nitric oxide synthase-null mice displayed an exaggerated response to post-acute myocardial infarction left ventricular functions. These data suggest that E2 modulation of alcohol consumption, and the ensuing EPC dysfunction, may negatively compete with the beneficial effects of estrogen on post-infarct myocardial repair.     

Chronic administration of NMU into the paraventricular nucleus stimulates the HPA axis but does not influence food intake or body weight

C-reactive protein (CRP), a predictor of future cardiovascular diseases, has been reported to damage the vascular wall by inducing endothelial dysfunction and inflammation. This proatherogenic CRP was speculated to have a role in attenuating angiogenic functions of human endothelial progenitor cells (EPCs), possibly impairing vascular regeneration and increasing cardiovascular vulnerability to ischemic injury. Herein, we investigated the direct effect of CRP on angiogenic activity and gene expression in human EPCs. Incubation of EPCs with human recombinant CRP significantly inhibited EPC migration in response to vascular endothelial growth factor, possibly by decreasing the expression of endothelial nitric oxide synthase and subsequent nitric oxide production. In addition, CRP-treated EPCs showed the reduced adhesiveness onto an endothelial cell monolayer. When assayed for the gene expression of arteriogenic chemo-cytokines, CRP substantially decreased their expression levels in EPC, in part due to the upregulation of suppressors of cytokine signaling proteins. These results suggest that CRP directly attenuates the angiogenic and possibly arteriogenic functions of EPCs. This CRP-induced EPC dysfunction may impair the vascular regenerative capacity of EPCs, thereby leading to increased risk of cardiovascular diseases.     

Impaired in vivo vasculogenic potential of endothelial progenitor cells in comparison to human umbilical vein endothelial cells in a spheroid-based implantation model

Objectives:  Neovascularization represents a major challenge in tissue engineering applications since implantation of voluminous grafts without sufficient vascularity results in hypoxic cell death of implanted cells. An attractive therapeutic approach to overcome this is based on co-implantation of endothelial cells to create vascular networks. We have investigated the potential of human endothelial progenitor cells (EPC) to form functional blood vessels in vivo in direct comparison to vascular-derived endothelial cells, represented by human umbilical vein endothelial cells (HUVEC).
Materials and methods:  EPCs were isolated from human peripheral blood, expanded in vitro and analysed in vitro for phenotypical and functional parameters. In vivo vasculogenic potential of EPCs and HUVECs was evaluated in a xenograft model where spheroidal endothelial aggregates were implanted subcutaneously into immunodeficient mice.
Results:  EPCs were indistinguishable from HUVECs in terms of expression of classical endothelial markers CD31, von Willebrand factor, VE-cadherin and vascular endothelial growth factor-R2, and in their ability to endocytose acetylated low-density lipoprotein. Moreover, EPCs and HUVECs displayed almost identical angiogenic potential in vitro , as assessed by in vitro Matrigel sprouting assay. However in vivo , a striking and unexpected difference between EPCs and HUVECs was detected. Whereas implanted HUVEC spheroids gave rise to formation of a stable network of perfused microvessels, implanted EPC spheroids showed significantly impaired ability to form vascular structures under identical experimental conditions.
Conclusion:  Our results indicate that vascular-derived endothelial cells, such as HUVECs are superior to EPCs in terms of promoting in vivo vascularization of engineered tissues.     

Dysregulated miR-361-5p/VEGF Axis in the Plasma and Endothelial Progenitor Cells of Patients with Coronary Artery Disease

Dysfunction and reduction of circulating endothelial progenitor cell (EPC) is correlated with the onset of cardiovascular disorders including coronary artery disease (CAD). VEGF is a known mitogen for EPC to migrate out of bone marrow to possess angiogenic activities, and the plasma levels of VEGF are inversely correlated to the progression of CAD. Circulating microRNAs (miRNAs) in patient body fluids have recently been considered to hold the potential of being novel disease biomarkers and drug targets. However, how miRNAs and VEGF cooperate to regulate CAD progression is still unclear. Through the small RNA sequencing (smRNA-seq), we deciphered the miRNome patterns of EPCs with different angiogenic activities, hypothesizing that miRNAs targeting VEGF must be more abundant in EPCs with lower angiogenic activities. Candidates of anti-VEGF miRNAs, including miR-361-5p and miR-484, were enriched in not only diseased EPCs but also the plasma of CAD patients. However, we found out only miR-361-5p, but not miR-484, was able to suppress VEGF expression and EPC activities. Reporter assays confirmed the direct binding and repression of miR-361-5p to the 3′-UTR of VEGF mRNA. Knock down of miR-361-5p not only restored VEGF levels and angiogenic activities of diseased EPCs in vitro, but further promoted blood flow recovery in ischemic limbs of mice. Collectively, we discovered a miR-361-5p/VEGF-dependent regulation that could help to develop new therapeutic modalities not only for ischemia-related diseases but also for tumor angiogenesis.