The Effect Of Cx43 In Pulmanory Arterial Hypertension And Its Underlying Mechanism

Objective:To investigate the role of Cx43 in pulmonary arterial hypertension(PAH)and its related mechanisms.Method:1.PAH model was established by chronic hypoxia and intraperitoneal injection of monocrotaline(MCT,60mg/kg)in rats.The general condition of rats in each group was observed.Right ventricular hypertrophy index(RVHI)was calculated.HE staining was used to observe the pathological changes of pulmonary vasculature.The expression level of Cx43 was detected by western blot.2.The effect of Cx43 knocked down by lentivirus vector on the remodeling of pulmonary arteries in PAH rats was observed.Male rats were divided into four groups:Cont,MCT,Cx43-RNAi-LV+MCT and NC-GFP-LV+MCT group.The lentivirus vector(1×108TU,once a week,A total of 2 times)was injected through the tail vein before treatment with MCT.The structure of rat pulmonary artery was observed by HE staining.The expression of Cx43 in pulmonary artery was detected by immunohistochemistry and the expression level of Cx43 was detected by Western blot.3.To detect the effect of echinomycin(Ech),a HIF-1α inhibitor,on the transcriptional expression of Cx43 and cell proliferation of PASMCs under hypoxic conditions.PASMCs were divided into three groups:Cont,Hypoxia(CoCl250μmol/L,24h),Ech+Hypoxia(Ech100nmol/L).The mRNA level of Cx43 in PASMCs was detected by RT-PCR,and the expression level of Cx43 in PASMCs was detected by Western blot.The MTT assay was used to observe the proliferation of PASMCs at 24,48,and 72 hours.Results:1.Compared with the control group,the rats in the hypoxic group and MCT group gradually became darker,lost of appetite,and slowed weight gain.The RVHI of these two groups were significantly higher than that of control(p<0.001).Compared with control,the pulmonary small artery wall in hypoxia and MCT group was significantly thickened,the lumen was obviously narrowed,the pulmonary vascular density was decreased and the vascular remodeling occurred.The expression of Cx43 protein in hypoxia and MCTgroup was significantly increased compared with the control group(p<0.05).2.HE staining showed that compared with control,the neointima in MCT group and NC-GFP-LV+MCT group diffused into the lumen,the blood vessel wall was thickened,and the lumen was significantly narrowed.Neointimal hyperplasia was significantly reduced in the Cx43-RNAi-LV+MCT group compared with MCT group.Immunohistochemistry results showed that compared with the control group,the positive expression density of Cx43 protein in the pulmonary arterioles of MCT group and NC-GFP-LV+MCTgroup was significantly increased.But the positive expression density of Cx43 protein in the Cx43-RNAi-LV+MCT group was significantly lower than that of MCT group.Western blot analysis showed that the expression of Cx43 protein in MCT group was significantly increased compared with control(P<0.05),and the Cx43 protein expression was significantly decreased in Cx43-RNAi-LV+MCT treatment compared with MCT group(p<0.05).3.RT-PCR experiments found that hypoxic treatment can significantly increase the mRNA level of Cx43 in PASMCs cells.Interestingly,hypoxia-induced Cx43 mRNA elevation was significantly inhibited when cells were pretreated with echinomycin(Ech),a HIF-1α inhibitor(p<0.05).Western blot analysis showed that hypoxia could up-regulate the expression of Cx43 in PASMCs cells,echinomycin could also effectively inhibit the up-regulation of Cx43 induced by hypoxia(p<0.05).MTT assays showed that the proliferative capacity of PASMCs was significantly increased under hypoxic conditions,and echinomycin could also significantly inhibit hypoxia-induced proliferation of PASMCs(p<0.05).Conclusion:1.PAH model were successfully set by chronic hypoxia and MCT treatment in rats.Pulmonary vascular remodeling develops significantly and the expression level of Cx43 increases obviously in PAH model rats.2.Cx43-RNAi-LV significantly inhibits the pulmonary vascular remodeling and higher expression of Cx43 protein in PAH model,which indicates Cx43 is involved in the pulmonary vascular remodeling process of PAH.3.HIF-1α may involve the regulation of Cx43 gene expression and PASMCs proliferation under hypoxic condition.The results demonstrates that HIF-1α may be associated with the involvement of Cx43 in PAH pulmonary vascular remodeling.