The Regulation Mechanism Of The Abnormal Regulation Mechanism In The Acute Type A Aortic Dissection Tissue

Objective: Through GO function analysis,KEGG signal pathway analysis,PPI network establishment and other biological information methods,the potential key genes and pathways in the development of ATAAD were screened.The expression of VE-cadherin in patients with Acute type A aortic dissection(ATAAD)was studied by aortic tissue samples,blood samples and in vitro cell experiments.To explore the mechanism of VE-cadherin regulation by VEGF165 and its role in the occurrence and development of ATAAD.Methods:(1)The GE0 dataset GSE153434(a total of 20 samples for transcriptome sequencing,including 10 ATAAD samples and 10 patients undergoing coronary artery bypass grafting)was screened by edge R model(Padj<0.05,log Fc absolute value >2)Differentially expressed genes(DEGs)were obtained,and PPI network map was made through STRING database.Visualization analysis was carried out on Cytoscape,Mcode algorithm was used to calculate the high density connection points,and the genes with the highest connectivity were selected.The corresponding KEGG and GO pathways and their P values were obtained from David database.The significantly enriched pathways were selected and the bubble map was used as the graphical representation of protein gene KEGG and GO enrichment analysis.The coexpression of core genes was analyzed by Gene MANIA database.(2)Clinical patients in our department were collected from January 2021 to January 2023,Among them,24 aortic wall tissue specimens and blood samples from patients undergoing ATAAD surgery were used as ATAAD group,and 12 aortic wall tissue specimens and blood samples from patients undergoing coronary artery bypass surgery were used as control group.The relative expression level of VE-cadherin、VEGFA protein was detected by western blot.HE staining was used to observe the morphology of aortic wall tissue,and immunohistochemistry was used to detect the VE-cadherin expression level and localization.(3)Human umbilical vein endothelial cells(HUVECs)were cultured and treated with VEGF165.CCK-8 was used to detect the cell viability and determine the optimal concentration.HUVECs was divided into control group and VEGF165 treated group for cell culture.The permeability of endothelial cells in each group was detected by FITC-Dextran infiltration method.Western blot was used to detect the relative expression levels of VE-cadherin,p-VE-cadherin,VEGF-R2,p-VEGF-R2,src,p-src,MMP9 and MMP-2,and immunofluorescence was used to detect the expression level of VE-cadherin and its location.Results:(1)The GEO dataset GSE153434(a total of 20 samples underwent transcriptome sequencing,including 10 ATAAD samples and 10 coronary artery bypass grafts)was screened by edge R model(Padj<0.05,log Fc absolute value >2)797differentially expressed genes were obtained,and the PPI network map was made through the STRING database.Visualization analysis was carried out on Cytoscape,Mcode algorithm was used to calculate high-density connection points,and 28 genes including ACE,ADIPOQ,CCL2,CCL20,CCL7,CD68,CDH5 and VEGFA with the highest connectivity were selected(Table 1).The average edge connectivity of cluster formed by these genes was 17.07.The P values of KEGG and GO pathways and each pathway were obtained through David database,and the corrected P values [false discovery rate(FDR)] based on Fisher’s exact test of hypergeometric distribution were obtained.The threshold value of 0.05 was selected as the significantly enriched pathway,and the bubble map was used as the graphical representation of protein gene KEGG and GO enrichment analysis.Coexpression analysis of 28 core genes was conducted through Gene MANIA database.Among the genes related to endothelial cell function,CDH5 had a strong correlation,indicating that it plays an important role in endothelial cell related studies.(2)Western blot analysis showed that VE-cadherin and VEGFA were expressed in both the dissection group and the control group,and the expression of VE-cadherin in the ATAAD group was significantly lower than that in the control group(P<0.05),VEGFA expression in ATAAD group was significantly higher than that in control group(P<0.05);In blood samples,the expression of VE-cadherin in the interlayer group was significantly higher than that in the control group(P<0.05 and P <0.01);In histopathologic sections,HE staining results showed that the arrangement and morphology of the inner membrane and media membrane of the sandwich group were obviously disordered,while the inner membrane and media membrane of the control group were tightly structured with regular morphology.Immunohistochemical results showed that the expression of VE-cadherin in the sandwich group was significantly lower than that in the control group(P<0.05 and P<0.01),and exists in the intima layer.(3)Cell experiments showed that the optimal concentration of VEGF165 on HUVECs detected by CCK-8 was 6ng/ml,and the optimal time was 48 h.FITC-Dextran infiltration method showed that the permeability of endothelial cells increased significantly after adding VEGF165(P<0.05).Western blot analysis showed that the expressions of VE-cadherin,VEGF-R2 and Src in VEGF165 treatment group were lower than those in control group(P<0.05 and P<0.01),the expressions of p-VE-cadherin,p-VEGF-R2,p-Src,MMP9 and MMP-2 in VEGF165 treatment group were higher than those in control group(P<0.05 and P<0.01),immunofluorescence detection showed that VE-cadherin expression in VEGF165 treatment group was significantly lower than that in control group(P<0.05).Conclusion: VEGE165 may be involved in the occurrence and development of aortic dissection by mediating VE-cadherin phosphorylation and affecting endothelial cell permeability.