Effects And Mechanism Of HDAC4Phosphorylation In The Process Of Angiogenesis After Cerebral Ischemia

Background and Objective:The major three causes of death in human disease is cerebrovascular disease,cardiovascular disease and cancer. Leading to death or severe neurologicaldysfunction, stroke has become a great danger of the disease which brings heavyburden to families and society. Epidemiological data show that ischemic strokeaccounts for about eighty percent of all cerebral vascular disease. There is still nogood treatment for ischemic stroke, including early thrombolysis, neurotrophic andneural function rehabilitation therapy. But these treatments are poor prognosis andmany serious complications. Therefore, some scholars propose that promoting theformation of new blood vessels to restore ischemic regional blood flow as soon aspossible, not only beneficial to save the penumbra area of neurons, also provide agood micro-environment for neural stem cells survival, proliferation, and remodelingcapabilities in repairing process, making the treatment of ischemic cerebral apoplexycan maximum limit to improve prognosis. So how to restore blood flow as soon aspossible to promote the formation of new blood vessels for the treatment of ischemicstroke is significant.Angiogenesis is the complex process of new blood vessels formation from anexisting network of blood vessels. Regulated by a variety of growth factors together,angiogenesis is a common multi-step synergistic result by varieties of related genes.VEGF and HIF-1had been widely used in studies of ischemic disease. HIF-VEGFsignal pathway is an important mechanism for regulating angiogenesis in ischemichypoxia, had been confirmed that can activate HIF downstream gene expression ofVEGF when ischemia and hypoxia, and play a key effect in self-repair process.Epigenetics modification also plays a very important role in the process ofregulation angiogenesis. Histone modification is an important regulation of epigeneticmodifications, in which the histone acetyltransferase and histone deacetylase isessential. In the research of tissue development and tumor angiogenesis found that theactivation of key HDACs plays a key regulatory role for the occurrence and improvement of angiogenesis. HDAC4is one of the members of the family ofHDACs, and has multiple phosphorylation sites. Other study found that HDAC4arephosphorylated and exported from the nucleus to the cytoplasm in response to VEGFsignaling, which activating the expression of downstream genes associated withangiogenesis. However, is phosphorylation of HDAC4also involved in regulation ofangiogenesis when ischemic cerebral stroke happen?Accordingly, the thesis was to investigate the change expression of signalingpathways proteins in vivo ischemia or in vitro hypoxia, such as HIF-1α, VEGFa,HDAC4and P-HDAC4632. To explore the function and mechanism of HDAC4phosphorylation in the process of angiogenesis after cerebral ischemia. It is not onlycontributed to further understand the process of angiogenesis, but also provide solidtheoretical basis for the treatment of ischemic stroke.Methods:1. In vivo experiment1.1Middle cerebral artery occlusion (MCAO) models were prepared byobstructing MCA using a single strand nylon thread.Spraue-Dawle (SD) rats wererandomly divided into MCAO-24h group and MCAO-48h group.Rats were sacrifiedat24h and48h after reperfusion. The brain was removed for inspection.1.2Frozen sections of brain sample by Immunofluorescence staining were usedto detect the expression of CD31to understand the microvessel density of theischemic and contralateral cortex after24h and48h ischemia reperfusion.1.3Western blot were used to detect the protein expression of HIF-1α, VEGFa,HDAC4and P-HDAC4632in each brain ischemic and the contralateral cortex after24h and48h ischemia reperfusion.2. In vitro experiment2.1Construction of HMEC-1cells to hypoxia and drug intervention model.Cells were randomly divided into normal oxygen group (N), hypoxia group (H),phosphorylase inhibitor pretreatment and hypoxia group (H+G).2.2Western blot were used to detect the protein expression of HIF-1α, VEGFa,HDAC4and P-HDAC4632in different cell groups.2.3The qRT-PCR was used to detect the mRNA expression of RCAN2in different cell groups.2.4To observe tube capacity of HMEC-1cells by hypoxia or drug intervention.Results1. In vivo experiment1.1Spraue-Dawle (SD) rats showed different degrees of neuroloical deficits,such as contralateral hemiplegis, ptosis, walking around in place, tilt to opposite side,and raised tail sideways to the opposite side after cerebral ischemia. The brainshowed normal red tissue and pale ischemic region by2,3,5-triphenyl-2h-tetrazoliumchloride (TTC) staining.1.2Compared with the24h ischemia reperfusion groups, new microvasculardensity is significantly higher in the48h ischemia reperfusion group.1.3Western blot analysis indicated that after24h ischemia reperfusion,ischemia side half dark cortex HIF-1α and VEGFa protein expression significantlyhigher than that of the contralateral cortex, while P-HDAC4632protein is lower thanthe contralateral cortex; But with the reperfusion time extended to48h, the ischemicpenumbra lateral cortex P-HDAC4632proteins are also significantly higher than thecontralateral cortex; among various groups of totle HDAC4protein levels have littlestatistical significance.2. In vitro experiment2.1Use hypoxic conditions (1%O2concentration,5%CO2concentration) toHMEC-1(human microvascular endothelial cells) in vitro, in which a group ofhypoxia group (H group), another group of use G6976pretreatment cell30minutesbefore hypoxia (H+G group). Normoxic (21%O2concentration,5%CO2concentration) as the control groupCompared with control group, whether or not useof drugs G6976, Western blot detection of HIF-1α and VEGFa protein were highlyexpressed under hypoxic conditions. In each group of which little change HDAC4protein expression levels, but the expression of P-HDAC4632protein is not consistentwith the change of HIF-1α and VEGFa protein; H group of P-HDAC4632significantlyhigher expression than normoxic group, however, H+G group of P-HDAC4632proteinexpression decreased.2.2Compared with control group, RCAN2gene was highly expressed in H group; However, after using phosphorylase inhibitor G6976pretreatment, theexpression of RCAN2gene was significantly reduced in H+G group.2.3Compared with control group, H group of tube capacity of HMEC-1cellsmarkedly increased; while H+G group of cells is restricted.Conclusions:Detecting microvessel density and changes expression of the related proteins ofischemic and contralateral cerebral cortex at different time points by MCAO model,we observed that expression of HIF-1α and VEGFa proteins increase in ischemiccerebral cortex, and also observed that the higher P-HDAC4632protein levels, highermicrovessel density. Building HMEC-1cells to hypoxia model in order to furtherexplore effects and mechanism of HDAC4phosphorylation levels on angiogenesisafter cerebral ischemia, we confirmed that expression of HIF-1α, VEGFa andP-HDAC4632proteins were upregulated in hypoxia. However, phosphorylase inhibitorGO6976weakened expression of downstream of VEGF gene associated withangiogenesis and vascular capacity of endothelial cells in hypoxic by inhibiting thephosphorylation of HDAC4.In conclusion, phosphorylation of HDAC4proteinclosely associated with the expression of HIF-1α and VEGFa proteins after ischemichypoxia, which may play a key regular role in brain tissue after ischemia andhypoxia.