SMAD4 Gene Polymorphism Rs12455792 And The Relationship Between Macrophage And Thoracic Aortic Aneury Sm Development

Objective:This study was to further explore the potential mechanism of SMAD4 gene rs12455792 in the development of aortic aneurysms and the relationship between SMAD4 gene expression and macrophage recruitment and development of aortic aneurysms.Methods:In vitro experiments:1.Human aortic smooth muscle cells(HASMC)were transfected with siRNA-NC and siRNA-SMAD4 respectively.(1)The changes of canonical and non-canonical TGF-? signals were detected by q-PCR and Western-blot.(2)The expression of chemokines such as CCL2,CCL5,CXCL3,CXCL10,CXCL12,CXCL13,CX3CL1 and IL-lb were detected by the q-PCR.(3)Transwell assay detected the migration of macrophages.2.Western-blot assay was used to detect the changes of classic and non-classical TGF-? signaling molecules in aortic aneurysms of different genotypes CC,CT,TT.3.The different genotypes of aortic aneurysm tissue immunohistochemistry were detected in different genotypes of macrophages location,number and infiltration changes.4.Immunofluorescence detection macrophages in different genotypes of the location,the number and the correlation between SMAD4 expression and macrophage recruitment in different genotypes and the relationship between chemokine expression.In vivo experiments:In vivo experiments were selected six male wild type mice,six male SMAD4 KD mice.The two groups of mice were intraperitoneally injected with angiotensin ?,to establishment the aortic aneurysm model,compared the incidence of aortic aneurysm in both groups of mice and macrophage cell recruitment,as well as chemokines and matrix metalloproteinases Express the situation.Results:The qPCR results showed that the expression of CXCL10,CXCL9,CXCL3 and CCL2 in siRNA-SMAD4 group was significantly higher than that of siRNA-NC group(P<0.05).The results of qPCR showed that the expressions of CTGF in the TGF-?signaling pathway of siRNA-SMAD4 group were significantly higher than those in siRNA-NC group(P<0.01).The results of Western-blot experiment showed that the expression of p-SMAD2 and p-SMAD3 in the classical TGF-? signaling pathway in the siRNA-SMAD4 group was significantly higher than that in the siRNA-NC group,the expression of p-ERK and p-JNK in the non classical TGF-? signaling pathway was significantly higher than that of the siRNA-NC group,P<0.01,the difference was statistically significant.The results of classical and non-classical TGF-? signaling in Western-blot,CC,CT and TT genotypes showed that rs12455792C>T mutation affected classical SMAD2 pathway and non-canonical pathway-mediated JNK phosphorylation.Tranwell assay showed that macrophage recruitment in siRNA-SMAD4 group was significantly higher than that in siRNA-NC group(P<0.05).The results of immunofluorescence staining showed that the infiltration of macrophages in CT and TT aorta tissues was significantly higher than that of CC type,and the M1 type macrophage polarization markers(TNF-?)in the TT type aorta was significantly higher than that of the CC type.The results of in vivo experiments showed that the aortic arch of SMAD4 KD mice was thick and covered with plaques and the incidence of aortic aneurysm was significantly higher than that of wild type mice(P<0.01).Conclusions:1.When the expression of co-Smad SMAD4 in classical TGF pathway is reduced,rs12455792C>T mutation influences classical TGF pathway mediated SMAD2,SMAD3 and non-classical TGF pathway,and mediates the phosphorylation of molecular JNK and ERK.It is suggested that the abnormal activation of TGF-? signaling pathway contributes to the formation of neointima and the development of aortic aneurysm.2.The C>T mutation of rs12455792 site of SMAD4 gene promoted the expression of chemokine CXCL10,CXCL9,CXCL3 and CCL2,thereby promoting the adhesion and aggregation of macrophages in the vascular wall.3.The C>T mutation of the rs12455792 site of the SMAD4 gene promotes the expression of M1 macrophages in the vascular wall,and plays a major role in the inflammatory response.4.The SMAD4 KD mice are more likely to form thoracic aortic aneurysms compared with the control group,and the aortic layer is severely damaged,tearing,forming dissection,and having a large number of inflammatory cell infiltration.