The Protective Effect Of Trail On Hypoxia-reoxygenation Injury Of Myocardial Microvascular Endothelial Cell And Its Possible Mechanism

IntroductionAcute Myocardial Infarction?AMI?is a worldwide acute ischemic heart disease.Its morbidity factor is mainly caused by thrombolytic fragments and plaques,which are formed by atherosclerosis.Then the coronary blood circulation was blocked and caused local myocardial ischemia.Myocardial cells were dead at the same time.Clinically,the treatments of AMI were mainly use of thrombolytic drugs or direct intracoronary surgery.However,after treatment of AMI,usually will inevitably occur myocardial ischemia and reperfusion?I/R?damage,and then cause a greater number of myocardial cell death.Studies have shown that,the myocardial microvascular endothelial cell?CMEC?was the first to be affected in I/R and the function was disordered.Endothelial cells have a strong secretion function,which can secrete a variety of bioactive substances to regulate the body's steady-state balance.The microcirculatory system plays an important role in maintaining and repairing cardiac function.Tumor necrosis factor-related apoptosis-inducing ligand?TRAIL?is a kind of TNF family type II trans-membrane protein,which plays an important role in regulating apoptosis,inflammatory and immune response.In recent years,people's attention to TRAIL is not only just its highly effective to tumor necrosis,but also attention to the complex physiological function in the cell.A large number of studies have shown that TRAIL plays an important role in vascular pathophysiological regulation and has potential cardiovascular protection effect.Studies have confirmed that one of mechanisms of cardiovascular protection was by activating phosphatidylinositol-2 kinase/protein kinase B/endothelial nitric oxide synthase?PI3K/Akt/eNOS?pathway of TRAIL.The eNOS can regulate the release of NO in endothelial cells,and NO has a strong role in the relaxation of blood vessels,which also can promote the proliferation of vascular smooth muscle,plays a protective effect of vascular endothelial cells.In the early stage of AMI,serum TRAIL concentration was significantly reduced,and there was a negative correlation between the level of TRAIL and the severity of myocardial infarction.In vitro and in vivo studies have shown that TRAIL has a potential protective effect on cardiovascular disease.In the process of myocardial infarction,the microvascular endothelial dysfunction is one of the major factors that leading to the myocardial infarction and reperfusion injury.There is not yet clear that whether the decrease of serum TRAIL concentration is a protective effect on vascular endothelial cells.Therefore,this study was aimed to investigate the effect of TRAIL on hypoxia/reoxygenation-induced CMEC function,and provide a new target for the treatment of cardiovascular disease.ObjectiveThe aims of this study were:?1?to establish isolation and culture of adult rats CMEC and identificate the specific surface antigen of CMEC,?2?to study the effect of TRAIL on CMEC under normoxic and hypoxia-reoxygenation conditions,?3?to explore the possible mechanisms of TRAIL on CMEC.Methods?1?The myocardium microvascular endothelial cells of Wistar male adult rats were isolated by enzymatic digestion.To identified whether the cell surface had factor VIII factor-related antigen and CD31 expression.Cell proliferation curve and Matrigel matrix gel were used to detect the proliferation and angiogenesis ability of CEMC and the express of DR5 on CMEC were detected by immunofluorescence and Western Blot.?2?Under normoxic condition,the TRAIL concentrations were set at 0,25,50,100ng/ml.LDH and MTS were used to detect the proliferation of CMEC.Transwell and scratch tests were used to detect the migration ability of CMEC.?3?Hypoxia workstation was used to establish hypoxia/reoxygenation model of CMEC.LDH and MTS were used to detect the proliferation of CMEC.?4?Investigated the effect of TRAIL on the migration and angiogenesis of CMEC induced by hypoxia/reoxygenation.The experiments were divided into normal group?normal?,hypoxia/reoxygenation group?H/R?,TRAIL treatment group?25,50,100ng/ml?.The changes of CMEC migration ability were detected by transwell chamber and scratches.RT-PCR was used to detect the expression of ICAM-1 and VCAM-1 mRNA in CMEC.Meanwhile,the change of CEMC angiogenesis was detected by Matrigel matrix gel,and RT-PCR was used to detect the expression of VEGF and Ang-1 mRNA in CMEC.?5?RT-PCR was used to detect the effect of TRAIL on the expression of CMEC inflammatory factors IL-1?,IL-6 and eNOS mRNA which was induced by hypoxia/reoxygenation.Results?1?The microvascular endothelial cells obtained by enzymatic digestion were cultured about 90%confluence and the cells were spindle-shaped or polygonal,and morphological cells were typical of"paving stones".Immunofluorescence identification of factor VIII-related antigen and CD31 were positive.The result of the cell proliferation curve was shown that.The cells were logarithmic growth and rapid proliferation at the firat 4 days,after that the cells into a stable growth state.Then on the 7^th day the number of cells began to reduce.Cell proliferation was consistent with the growth cycle.The matrigel matrix gel results showed that CMEC angiogenesis was well,and the lumen structure integrity.The results of immunofluorescence and WB showed that DR5 protein was expressed on the surface of CMEC.?2?Under normoxic condition,CMEC cells were treated with different concentrations of TRAIL for 24h,and the results showed that,at 50ng/ml concentration of TRAIL,the number of CMEC migrated is most and the rate of the scar healing is greatest.The results showed that TRAIL had the greatest effect on the migration ability of TRAIL at 50ng/ml.?3?CMEC hypoxia reoxygenation model was established.MTS and LDH showed that CMEC had the strongest effect on CMEC at hypoxia 4h and reoxygenation 2h.With the increase of reoxygenation time,the cell viability increased gradually.According to the results of Transwell chamber and scratches.Under the condition of H/R,the migration rate of CMEC increased gradually with the increase of TRAIL concentration,reached the maximum at50ng/ml,decreased at 100ng/ml;and similar results of ICAM-1 and VCAM-1 were detected by RT-PCR.?4?In the detection of CMEC angiogenesis capacity,TRAIL can promote H/R-induced CMEC angiogenesis.Increase the expression of angiopoietin Ang-1 in CMEC cells,and then control the maturation of blood vessels;The detection of inflammatory factors shows that TRAIL can inhibit the synthesis of H/R-induced CMEC inflammatory cytokines IL-1?and IL-6,reduce the inflammatory response of cells,promote the synthesis of eNOS and enhance the cell viability.ConclusionsThe rat microvascular endothelial cells were isolated by enzymatic digestion and identified successfully.TRAIL can promote the migration and proliferation of CMEC under normoxic and H/R conditions,and the mechanisms may be through the improvement of eNOS synthesis and inhibition of inflammatory factors IL-1?,IL-6 expression to play the role of endothelial cell protection.