Risk Gene Screening And Pathogenic Mechanism Of Aortic Dissection

Background:Aortic dissection is a life-threatening disease and one of the leading causes of sudden cardiac death.Although some susceptibility genes related to aortic dissection have been identified,the genetic causes of the disease in many patients are still unknown.While single nucleotide polymorphisms are the main tool in current genetic studies,copy number variations(CNVs)also play an important role in the genetic research of many diseases.However,there is little research exploring the contribution of CNVs to aortic dissection.Therefore,through whole-genome sequencing,this study aims to explore the role of CNVs in sporadic thoracic aortic dissection,in order to further understand the genetic mechanism of the disease.Methods:To explore new genetic mechanisms of sporadic aortic dissection,we first recruited 100 patients with sporadic type A aortic dissection and 132 controls for wholegenome sequencing in the discovery cohort to identify novel CNVs.To validate whether these novel CNVs were associated with sporadic type A aortic dissection,we included three independent cohorts comprising 171 patients with sporadic type A aortic dissection,157 patients with sporadic type B aortic dissection,and 886 healthy controls,and assessed the pathogenicity of these CNVs using multiplex ligation-dependent probe amplification.We also used immunoblotting to detect gene expression in aortic tissue from patients with aortic dissection.To further investigate whether the genes involved in the selected CNVs were involved in the development of aortic dissection,we used CRISPR/Cas9 technology to generate gene knockout mice and induced different types of aortic dissection in these mice using BAPN or PCSK9+AngⅡ.We collected the mice’s aortas and assessed the degree of aortic dissection using hematoxylin-eosin staining and Elastin-Van Gieson’s staining.TUNEL staining was used to measure the level of apoptosis in vascular smooth muscle cells(VSMCs)in the aortic wall,while immunofluorescence was utilized to examine the expression levels of molecules related to VSMCs apoptosis and phenotypical switch in the aortic tissue.To further investigate the involvement of screened CNV-associated genes in the occurrence and progression of aortic dissection,we isolated VSMCs from target gene knockout or control mice and treated them with BAPN(1 mM)or AngⅡ(1 μmol/L)+ox-LDL(150 ng/ml),and assessed the degree of cell apoptosis using flow cytometry.The expression levels of proteins related to apoptosis and VSMCs phenotypical switch were also detected using immunoblotting.RNA sequencing was performed to explore changes in the gene expression profile of VSMCs after gene knockout,and VSMC proteins were extracted from knockout and control mice for mass spectrometry analysis to identify the target proteins of the genes.Results:In the discovery cohort,we identified four CNVs and related genes(DSCAM,APP,LINC00907,PROCR)that may be potential pathogenic factors for aortic dissection.These four candidate genes showed significant differences in three independent validation cohorts.Notably,compared to the control group,the expression of APP in the aortic tissue of aortic dissection patients was significantly decreased.Furthermore,we constructed an App gene knockout mouse model and induced aortic dissection using BAPN or PCSK9+AngII.The results showed that the incidence and mortality of aortic dissection were significantly increased in the App knockout mice,and elastic fiber fractures were more severe.Compared to the control group,the loss of App promoted the expression of VSMC apoptosis markers(cleaved caspase 3 and cleaved PARP)and secretion phenotype markers(MMP2,MMP9,type I collagen,and type III collagen),while reducing the expression levels of contraction phenotype markers(SM22α/transgelin,α-SMA,calponin-1,and smoothelin).In addition,the number of apoptotic cells increased significantly.When primary VSMCs from the mice were treated with BAPN and AngⅡ+ox-LDL,the primary VSMCs in the App knockout group showed a higher apoptotic ratio than the control group,and the expression levels of apoptotic-related proteins and secretion phenotype-related proteins were significantly increased,while the expression levels of contraction phenotype-related proteins were significantly reduced.RNA sequencing analysis of changes in the gene expression profile of VSMCs after App knockout showed consistent results with the observed VSMCs apoptosis and phenotypical switch.Further mass spectrometry analysis and immunoblotting showed that the expression level of Anxal was significantly downregulated in smooth muscle cells with App knockout,suggesting that App may regulate VSMCs apoptosis and phenotypical switch by regulating the expression of Anxa1.Conclusions:Through this study,we have reported for the first time the association between APP-related CNVs and aortic dissection and found that the loss of APP promotes the occurrence and development of aortic dissection by affecting the apoptosis and phenotypical switch of VSMCs in both human and mouse aortas.This finding suggests that APP may be a genetic risk factor and potential therapeutic target for aortic dissection,providing new directions and strategies for further exploration of the genetic mechanisms and the development of novel treatments for this disease.