| Increasing evidence suggests endothelial progenitor cells (EPCs) improve neovascularization and endothelium regeneration via the production of paracrine factors. Vascular endothelial growth factor (VEGF) and interleukin-8(IL-8) are major cytokines for EPC-based angiogenesis and re-endothelialization.Hyperhomocysteinemia has been recognized as a major risk factor of cardiovascular diseases. Although the mechanism by which Hey damages the vessels wall and induces atherosclerosis is complicated. Hey may injure EPC function and therefore inhibit the therapeutic effects of EPCs. Hey reduced EPC numbers and impairs their functional activity including proliferative, migratory, adhesive and in vitro vasculogenesis capacity. Hey accelerated the onset of EPC senescence, which involved inhibition of telomerase activity and Akt phosphorylation in EPCs. However, whether Hey cloud affect the paracrine function of EPCs needs further investigation. Therefore, we aimed to explore the effect of Hey on EPC paracrine function and the effect of paracrine factors from EPCs on in vivo post-injury reendothelialization in rats. Moreover, the mechanism of Hey involved in Hcy-induced EPC paracrine impairment is also under investigation. Pioglitazone (PIO), the peroxisome proliferator-activated receptors (PPARs) agonist, could improve the migratory response and the adhesive capacity of EPCs in patients with diabetes mellitus and coronary artery disease. PIO was proved to prevent apoptosis of EPCs and increase in vivo neoangiogenesis in mice. In addition, PIO ameliorated Ang Ⅱ-induced senescence of EPC. However, none of previous studies have investigated the effect of PIO on EPC cytokine production and related mechanisms.Based on these considerations, we aimed to investigate the expression and the secretion of cytokines in EPCs under high Hcy, and to study the effects of PIO on Hcy-injured EPCs and possible mechanism that involved in this progress. Finally, we observed the effect of knockdown of p67and Nox2on EPC function.Part1The effect of high Hcy on paracrine function of endothelial progenitor cellsObjective:To investigate the effect of high Hcy on paracrine function of endothelial progenitor cells.Methods:EPCs, isolated from peripheral blood of healthy volunteer, were cultured on fibronectin-coated dishes. EPCs were characterized as adherent cells double positive for DiLDL-uptake and lectin binding under a laser scanning confocal microscope. They were further documented by demonstrating the expression of VE-cadherin, KDR, CD34and AC133by flow cytometry. EPCs-derived conditioned medium (EPC-CM) was obtained from culture EPCs subjected to trophic deprivation24h. The expression of cytokine mRNAs was measured by realtime PCR. VEGF and IL-8in cell culture supernatants were detected by enzyme-linked immunosorbent assay (ELISA). Cells were treated with Hcy (to make a series of final concentrations:10,50,100,200and500μmol/L) for24h. EPCs were also stimulated by200μmol/L Hey for respective time points (3,6,12,24and36h). After balloon injury, male Sprague-Dawley rats were divided into four groups to assay the re-endothelialization effect of EPC-CM:(1) normal rats as control;(2) balloon-injured vessels treated with EBM-2;(3) balloon-injured vessels treated with EPC-CM (4) balloon-injured vessels treated with EPC-CM collected from Hcy-stimulated EPCs. After treatment with EPC-CM for1week, the re-endothelialized area in balloon-injured arteries was observed.Result:compared to normal EGM-2, EBM-2, which is without cytokines or FBS (fetal bovine serum), stimulated EPCs to secrete a great quantity of VEGF and IL-8, but other cytokines such as IGF, HGF, SDF-1, PDGF-BB and Tβ4remained unchanged. Concentrations of VEGF and IL-8in EPC-CM were260.54±32.09pg/ml/1×106EPCs,15724.54±1817.24pg/ml/1×106EPCs, respectively. High Hey inhibited the secretion of VEGF and IL-8from EPCs.200μmol/L Hey inhibited VEGF and IL-8secretion in a time-dependent manner. The re-endothelialized area in balloon-injured arteries was increased significantly by EPC-CM. However, EPC-CM from Hcy-treated EPCs could not promote the reendothelialization process.Conclusion:VEGF and IL-8were the major cytokines secreted by EPCs. These paracrine cytokines could promote the reendothelialization of balloon-injured arteries. Hey induced a significant inhibition of VEGF and IL-8from EPCs, and inhibited the indirect therapy of EPCs on balloon-injured vessels. Part2Pioglitazone ameliorates Hcy-induced dysfunctions of EPCs via inhibiting the activation of PKC and NADPH oxidase Objective:To investigate the effect and mechanism of pioglitazone on Hcy-treated EPCsMethods:EPCs, isolated from peripheral blood of healthy volunteer, were cultured on fibronectin-coated dishes for7d. EPCs-derived conditioned medium(EPC-CM) was obtained from culture EPCs subjected to trophic deprivation24h. Cells were treated with Hcy (200μM) for24h. PIO (10μM) was added30min before Hcy treatment. The expression and secretion of VEGF and IL-8were measured by realtime-PCR and ELISA. The expression of PKC-α/βⅡ,p-PKC-α/βⅡ,PKC-α,p-PKC-α,PKC-βⅡ and p-PKC-βⅡ were detected by Western blot. EPCs were pre-incubated with PKC inhibitor GF (5μM) for30min before the addition of Hcy (200μM) for24h. Intracellular ROS levels were measured by flow cytometry with the fluorescent probe, H2DCF-DA. The lucigenin-derived enhanced chemiluminescence assay was used to determine NADPH oxidase activity. EPCs were pre-incubated with PIO (10μM), NADPH oxidase inhibitor DPI (5μM) and GF (5μM) for30min before the addition of Hcy (200μM) for24h. We observed the effect of PIO, DPI and GF on ROS and NADPH oxidase. The effects of PKC inhibitor GF and NADPH oxidase inhibitor DPI on both protein and mRNA production of VEGF and IL-8by EPCs after co-cultured with Hcy were analyzed by ELISA and real-time RT-PCR. The effects of PIO, DPI and GF on EPC migration and adhesion were assayed in8.0-μm pore size transwells and fibronectin-coated culture dishes, respectively.Result:PIO normalized VEGF and IL-8secretion and expression which were decreased by Hcy. Hcy treatment increased phosphorylation of PKC in a time-dependent manner. We found Hcy stimulated PKCβⅡ phosphorylation but did not affect phosphorylation of PKCα. Moreover, PIO inhibited PKC activation induced by Hcy. PIO and GF pretreatment significantly reduced Hcy-induced ROS and NADPH oxidase activation, which was consistent with the results of DPI. Pretreatment with DPI and GF restored not only Hcy-mediated VEGF and IL-8reduction but also injured adhesion and migration of EPCs.Conclusion:Addition of PIO to Hcy treated EPCs attenuated dysfunctions. PIO attenuated EPC paracrine dysfunction induced by Hcy by inhibition of NADPH oxidase and PKC. Part3The effect of pioglitazone on NADPH oxidase of Hcy-treated EPCsObjective:To investigate the effect of pioglitazone on expression of NADPH oxidase subunits.Methods:EPCs were cultured on fibronectin-coated dishes for7d. Cells were treated with Hcy (10,50,100and200μM) for24h. EPCs were also pre-incubated with PIO (10μM), NADPH oxidase inhibitor DPI (5μM) and GF (5μM) for30min before the addition of Hcy (200μM) for24h. The expressions of p67and Nox2subunits were detected by Western blot. Transfection of EPCs with p67-siRNA and Nox2-siRNA resulted in abolishment of the expression of p67and Nox2. We observed the effect of down-regulation of NADPH subunits on EPC paracrine function. The productions of VEGF and IL-8by EPCs after transfection of siRNAs were analyzed by ELISA and real-time RT-PCR. The effects of down-regulation of p67and Nox2on EPC adhesion and migration were assayed in8.0-μm pore size transwells and fibronectin-coated culture dishes.Result:Western blotting data showed Hcy dose dependently increased expression of p67and Nox2. PIO may inhibit p67and Nox2by inhibition of PKC. Our data further demonstrated siRNAs against NADPH oxidase components, p67and Nox2, suppressed Hcy-mediated oxidative stress. P67-siRNA exerted a more significant effect than Nox2-siRNA on reversing paracrine dysfunction caused by Hcy. EPCs transfected with Nox2and p67siRNA showed apparently higher cell migration and adhesiveness than cells transfected with control siRNA under stimulation of Hey. Besides, PIO attenuated Hcy-induced EPC dysfunction, especially reduced expression and secretion of VEGF and IL-8by EPCs in a PPARy-independent manner.Conclusion:The mechanism of PIO-mediated down-regulation of p67and Nox2subunits involved inhibition of PKC. p67-siRNA reversed EPC paracrine dysfunction caused by Hey. EPCs transfected with Nox2or p67siRNA restored EPC migration and adhesiveness. Protective effect of PIO on cytokine secretion by EPCs could be unrelated to the activation of PPARy.
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