Study On The Role Of Gax Inphenotypic Transformation Of Vascular Smooth Muscle Cells

Part 1 Study on expression of Gax in thoracic aortic aneurysm tissues and its role in the regulation of thoracic aortic smooth muscle cells phenotypic modulationObjective To investigate the expression of Gax in thoracic aortic aneurysm tissues and the effect of Gax on phenotypic modulation of thoracic aortic smooth muscle cellsMethods Aortic tissues were collected from patients who suffered from thoracic aortic aneurysm(16 cases)and underwent surgery in our hospital from September 2016 to September 2017.Normal Aortic tissue specimens(16 cases)were obtained from coronary artery disease patients who underwent coronary artery bypass grafting during the same period in our hospital.Real-time PCR and Western blot methods were used to detect the expression levels of Gax mRNA and protein in vascular tunica media from thoracic aortic aneurysm tissues respectively.VSMCs were isolated from normal aortic specimens or thoracic aortic aneurysm samples and primary cultured by the method of enzyme digestion and adherent.The immunofluorescent cytochemistry staining was used to detect the expressions of a-actin and Gax in VSMCs.VSMCs were transfected with Ad-Gax or Ad-Gax siRNA.The Western blot method was used to detect the effect of Gax over-expression on Calpoin and SM-MHC 11 proteins expressions in VSMCs with or without PDGF-BB stimuli.CCK8 method was used to detect the role of Gax over-expression or low-expression in VSMCs proliferation with or without PDGF-BB stimuli.Transwell method was used to detect the function of Gax over-expression or low-expression on VSMCs migration with or without PDGF-BB stimuliResults Real-time PCR and Western blot showed that the mRNA and protein levels of Gax were significantly decreased in aortic samples from thoracic aortic aneurysm,as compared with normal aortic tissues.The immunofluorescent cytochemistry staining showed that compared with VSMCs from normal aortic specimens,the expressions of Gax and a-actin proteins were significantly down-regulated in VSMCs from thoracic aortic aneurysm.VSMCs transfected with Ad-Gax showed significantly increased protein levels of Calpoin and SM-MHC 11 with or without PDGF-BB stimuli.CCK8 assay showed that over-expression of Gax could inhibit the proliferation of VSMCs with or without PDGF-BB stimuli whereas Gax silencing promoted cells proliferation Transwell method showed that over-expression of Gax could inhibit the migration of VSMCs with or without PDGF-BB stimuli whereas Gax silencing promoted cells migrationConclusions Gax expression was significantly down-regulated in tunica media tissues and VSMCs from thoracic aortic aneurysm samples.And the increased expression of Gax could induced phenotype modulcation of thoracic aortic smooth muscle cells from the synthetic type to contractile typePart 2 Study on the role of Gax-regulated Rap1A gene expression in VSMCs phenotypic modulationObjective To investigate inhibiting transcription factor Gax regulated phenotypic modulation of thoracic aortic smooth muscle cells via down-regulating Rap1A gene expression and reveal the pathogenensis of thoracic aortic aneurysmMethods The differentially expressed genes in VSMCs exposed to overexpression of transcription factor Gax were screened by cDNA array analysis and were confirmed by the methods of Real-time PCR and Western blot.Western blot was used to detect the expression levels of Rap1A protein in vascular tunica media from thoracic aortic aneurysm tissues.The immunofluorescent cytochemistry staining was used to detect the expression of Rap1A in primary cultured VSMCs from thoracic aortic aneurysm tissues.Recombinant lentiviral vector Lenti-Rap1A was constructed and identified VSMCs were transfected by Lenti-Rap1A and the expression of Rap1A was detected by Western blot.The Western blot method also was performed to detect the effect of Rap1A over-expression on the expressions of contractile phenotype markers such as Calpoin and SM-MHC 11 proteins in VSMCs with or without PDGF-BB stimuli CCK8 method was used to detect the role of Rap1A over-expression in VSMCs proliferation with or without PDGF-BB stimuli.Transwell method was used to detect the function of Rap1A over-expression on VSMCs migration with or without PDGF-BB stimuli.Then,VSMCs were transfected by Ad-Gax combined with Lenti-Rap1A.And Western blot was used to detect the effect of Rap1A over-expression on Calpoin and SM-MHC 11 proteins expression in VSMCs with Gax over-expression.CCK8 method was used to detect the role of Rap1A over-expression in proliferation of VSMCs with Gax over-expression.Transwell method was used to detect the function of Rap1A over-expression on migration of VSMCs with Gax over-expressionResults cDNA array analysis showed that Rap la was the most significant down-regulated gene in VSMCs with inhibiting transcription factor Gax over-expression.The fold change was 2.94 and there was significantly statistic difference(P<0.01).It was confirmed that inhibiting transcription factor Gax over-expression could significantly down-regulate Rap1A expression in VSMCs with or without PDGF-BB stimuli.Western blot showed that the protein level of Rap1A was significantly increased in aortic samples from thoracic aortic aneurysm,as compared with normal aortic tissues.The immunofluorescent cytochemistry staining showed that compared with VSMCs from normal aortic specimens,the expressions of Rap1A was significantly up-regulated in VSMCs from thoracic aortic aneurysmRecombinant lentiviral vector Lenti-Rap 1A was successfully constructed.The virus titer was 3.5×108 Tu/ml,Lenti-Rap1A was confirmed by PCR,enzyme digestion reaction and DNA sequencing.Rap1A over-expression was observed in VSMCs transfected by Lenti-Rap1A.The protein levels of VSMCs contractile phenotype markers such as Calpoin and SM-MHC 11 were significantly down-regulated in VSMCs with or without PDGF-BB stimuli after Lenti-Rap1A transfection.CCK8 assay showed that over-expression of Rap1A could promote the proliferation of VSMCs with or without PDGF-BB stimuli.Transwell method showed that over-expression of Rap1A could promote the migration of VSMCs with or without PDGF-BB stimuli.As compared with those in Ad-Gax transfection group and Ad-Gax+Lenti-EGFP transfection group,VSMCs contractile phenotype markers such as Calpoin and SM-MHC 11 in Ad-Gax+Lenti-Rap1A transfection group were significantly decreased whereas the abilities of cells proliferation and migration were significantly enhancedConclusions Rap1A protein was up-regulated in thoracic aortic aneurysm tissues And inhibiting transcription factor Gax-mediated phenotypic modulation of thoracic aortic smooth muscle cells was accomplished via down-regulating Rap1A gene expression.