Effects And Mechanisms Of Hypoxia On Activity Of Endothelial Progenitor Cells From Peripheral Blood

Accumulating evidence suggests that damaged organs repair is driven not only by migration and proliferation of adjacent cells,but also with the contribution of ectopic stem cells.Endothelial progenitor cells(EPCs) are a cell population that have the capacity to proliferate,and differentiate into mature endothelial cells,but have neither acquired characteristic mature endothelial markers nor formed a lumen.EPCs appear to be characterized by the expression of CD34,the vascular endothelial growth factor receptor (VEGFR)-2 and CD133.Recent reports documented that endothelial progenitor cells(EPCs) significantly contributed to neovascularization after tissue ischemia and reendothelialization after vascular injury.EPCs derive from the bone marrow(mainly) and can be mobilized to the peripheral circulation(mobilization) upon A series of stimulus including tissue ischemia through the release of growth factors.EPCs are recruited and stay at the site of vascular repair or neovascularization(homing),where they differentiate into endothelial cells(differentiation) and release of cytokines that promote the proliferation and migration of local endothelial cells.EPCs transplantation is suitable for a wide range of implications in treatment of cardiovascular disease,which has been shown to improve cardiac function,reduced ventricular remodeling in patients with myocardial infarction and increase pain-free walking time in patients with leg ischemia.Hypoxia is associated with angina pectoris,myocardial infarction,heart failure,and peripheral artery disease.Hypoxia and tissue ischemia are caused by either arterial obstruction or functional and anatomical capillary rarefaction resulting from hypertension. Diminished oxygen concentration induces programmed responses,such as endothelial proliferation and angiogenesis that ultimately relieve tissue hypoxia.Several signaling molecules have been reported to be involved in the response to hypoxia.Hypoxia-inducible factor-1(HIF-1) has been regarded as the key mediator of cellular responses to reduced oxygen availability,previous studies have demonstrated hypoxic microenvironments(such as ischemic tissue and injured vessel) are a conditional EPCs niche,in which many proteins are directly regulated by hypoxia-inducible factor-1(HIF-1),such as vascular endothelial growth factor(VEGF) and the chemokine stromal cell-derived factor-1(SDF-1)which facilitate EPCs mobilization,recruitment and retention in site of hypoxia.However, hypoxia on EPCs migration and survival are not well known.On the basis of these considerations,we hypothesized that hypoxia affected EPCs functional activity,thus influenced endothelial repair process and neovascularization, which impacts of a series of cardiovascular disease development.To test this hypothesis, we measured the functional activity of EPCs exposed to hypoxia at first.Then,we studied the mechanisms by which hypoxia affected EPCs functional activity.Part 1:Effects of Stromal cell-derived factor-la on function activity of endothelial progenitor cells from peripheral bloodThe aim of this study is to investigate the effects of hypoxia on functional activity of EPCs from peripheral blood.Total mononuclear cells(MNCs) were isolated from peripheral blood by Ficoll density gradient centrifugation,and then the cells were plated on fibronectin-coated culture dishes.After 7 days cultured,attached cells were cultured in normoxic or hypoxic conditions(2%O₂) for 24 hours.EPCs were characterized as adherent cells double positive for DiLDL-uptake and lectin binding by direct fluorescent staining under a laser scanning confocal microscope.EPCs were further documented by demonstrating the expression of CD34,VEGFR-2 and AC133 with flow cytometry.Cell viability,migration and apoptosis induced by serum deprivation were assayed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay,modified Boyden chamber assay,Terminal deoxynucleotidyl transferase-mediate dUTP nick-end labeling (TUNEL) and annexin V-PI dual-color flow cytometry respectively.We observed that EPC significant increase in migration to SDF-1a(100ng/mL) under hypoxic conditions(58.57±10.49 vs 38.57±7.48).The induction of apoptosis upon serum withdrawal was reduced when cells were cultured under hypoxic conditions.In contrast,if the cells were in normoxia and serum starved,there was significant increase in cell apoptosis(27.87%± 2.03%vs 32.43%±3.11%).The changes in cell viability was similar to cell apoptosis(0.74±0.13 vs 0.62±0.09).These results indicated that hypoxia can enhance migratory ability and improve EPCs viability and suppress serum withdrawal-induced EPCs apoptosis.Part 2:Mechanisms of hypoxia on function activity of endothelial progenitor cells from peripheral bloodRecent studies have demonstrated several signaling molecules are involved in the response to hypoxia such as PI3K/Akt,MAPK,HIF-1,AP-1.These signaling molecules regulate a variety of cellular functions such as cell migration,proliferation,survival and angiogenesis.In present study,we aimed to determine the phosphoinositide 3-kinase (PI3K)/Akt,mitogen-activated protein kinases(MAPKs) and Hypoxia-inducible factor-1 a(HIF-1a) signaling in the effect of hypoxia on EPC migration and apoptosis induced by serum deprivation.EPCs were isolated from peripheral blood and characterized.We showed that Hypoxia promoted up-regulation of CXCR4 expression in endothelial progenitor cells by flow cytometric analysis and reverse transcription-PCR(RT-PCR).The enhanced migratory ability and the up-regulation of CXCR4 expression were nearly completely abolished by PI3K inhibitor(LY294002) and AMD3100(the CXCR4 antagonist).Whereas,inhibitors of MAPK(ERK1/2) had no significant effect on these effect.PI3K inhibitor(LY294002) also could inhibit hypoxia suppressed the apoptosis induced by Serum Withdrawal in EPCs.Quiescent EPCs exposed to hypoxia resulted in time-dependent Akt,ERK1/2,Glycogen synthase kinase-3β(GSK-3β) phosphorylations and HIF-1a expression.These findings suggested that PI3K/Akt activation,but not MAPK(ERK1/2) activation,is required for the up-regulation of CXCR4 expression under hypoxic conditions.Inhibition of apoptosis by hypoxia also require PI3K/Akt activity.