Effects Of HDAC9 On Proliferation And Angiogenesis Of EA.hy926 Endothelial Cells

BackgroundStroke,one of the major diseases that endanger human health,which marked by characteristics of high incidence,high mortality,high recurrence and high morbidity rate,had exerted heavy burden to our society.The veins that nourish the brain been cut by vascular occlusion or rupture plaque was considered to be the main reason to cause clinical symptom of stroke.Ischemic stroke(IS)is the most common type of stroke,accounting for 87% of stroke cases.At present a number of studies have shown that genetic factor is one of the risk factors IS.Analysis of genome-wide association studies(GWAS)found that the single nucleotide polymorphisms(SNPs)of histone deacetylase 9(HDAC9)correlate to the large artery atherosclerosis stroke(LAA-S)in cases of stroke.LAA-S is one of the main types of IS,and the main pathogenesis of IS is atherosclerosis(AS).For now,researchers had showed inflammatory reaction together with endothelial dysfunction and vascular smooth muscle cell proliferation played an important role in the formation and development of AS.Among them,endothelial dysfunction and persistent inflammation are considered to be the initiating factor of AS,which can lead to the formation and progression of atherosclerotic plaques.Autophagy,which is highly conserved in mammalian cells and plays an important role in cell protection,can be induced by various stress responses such as cellular stress,reactive oxygen species(ROS),starvation,the accumulation of abnormal proteins,and damaged organelles and so on,to achieve material and energy recycling process and maintain cell integrity and dynamic balance through the parcel and degradation of intracellular excess,aging or damaged organelles,and intracellular pathogens and other biological macromolecules to.Studies have shown that autophagy can be involved in AS formation and progression by mediating endothelial cell injury and dysfunction.However,the role of autophagy on vascular endothelial cells has not been clear.HDAC9 is one member of the II class HDACs,which can work as deacetylase,so it can catalyze the acetylation of N-teminal lysine residues of histone and non-histone proteins.By the way,HDAC9 could moderate gene transcription,cell cycle,differentiation and the other physiological functions in cells.Studies have shown that HDAC9 is highly expressed in atherosclerotic plaques,and HDAC9 knockout mice have a lighter damage in aorta.However,whether HDAC9 could impact the function of endothelial cells or not and the role it played in the pathogenesis and development of AS are still unclear.Objective In this study,we are meant to observe the effect of HDAC9 brought to the function of EA.hy926 endothelial cells,and preliminarily explored whether HDAC9 can affect the functions of endothelial cells by mediated autophagy.Methods 1.HDAC9 in EA.hy926 endothelial cells was overexpressed by adeno-associated virus and MTT was used to detect the effect of the overexpression of HDAC9 on proliferation of EA.hy926 endothelial cells.2.Flow Cytometry(PE Annexin V/7-ADD double staining)and western blot were used to measure the rate of apoptosis and the expression level of Cleaved Caspase3 in EA.hy926 endothelial cells which were overexpression of HDAC9,to determine whether HDAC9 can inhibit the apoptosis of EA.hy926 endothelial cell.3.The effect of HDAC9 on the function of tube formation of EA.hy926 endothelial cells was observed by the tubular formation assay.4.The expression of LC3,p62,which are the key molecules in autophagy signaling pathway in EA.hy926 endothelial cells were detected by Western blot,to explore whether HDAC9 is involved in the regulation of EA.hy926 endothelial cell proliferation and cell apoptosis by mediating cell autophagy.Results 1.The proliferation activity of the two groups increased in first day to fifth day.Compared with the Ad-GFP-NC group,the proliferation activity of Ad-GFPHDAC9 group was higher,and the difference between two groups was significant both fourth day and fifth day(P < 0.05);2.Apoptosis detection: Flow cytometry revealed that,compared with Ad-GFP-NC group,the early,late apoptotic rates and the necrosis rate of Ad-GFP-HDAC9 group were significantly decreased(P <0.05),western blot showed that Cleaved Caspase3 protein level in Ad-GFP-HDAC9 group was significantly lower than that of Ad-GFP-NC group(P <0.05).3.Compared with Ad-GFP-NC group,the number of tubule-forming nodes,the number of branches and the total branch length of Ad-GFP-HDAC9 group were significantly increased(P <0.05).4.Compare with control group,HDAC9 significantly increased the expression of LC3 to inhibit the apoptosis of EA.hy926 endothelial cells(P<0.05).Conclusions 1.HDAC9 can enhance the proliferation activity of EA.hy926 endothelial cells.2.HDAC9 can inhibit the apoptosis of EA.hy926 endothelial cells.3.HDAC9 can promote EA.hy926 endothelial cells to format tubes.4.HDAC9 may be inhibit the apoptosis of EA.hy926 endothelial cells and promote angiogenesis in vitro by induced autophagy.