The Effect And Mechanism Of Endothelial Cell Injury Mediated By Serum Exosome MiR-202-3p In The Aortic Dissection

Objective:Aortic dissection(AD)is a fatal cardiovascular disease with few clinical predictors and unknown molecular mechanisms of pathogenesis.The main objectives of this study include(1)to screen for exosomal mi RNAs differentially expressed in the serum of AD patients and the signalling pathways involved in their target genes.(2)To assess the diagnostic efficacy of differentially expressed exosomal mi RNAs and proteins for aortic dissection.(3)To investigate the pathogenic effect of AD serum exosomes on aortic dissection.(4)To explore the targets and molecular mechanisms of target mi RNAs in AD serum exosomes that induce endothelial cell injury for the development of AD.Methods:Part I: After collecting serum from AD patients and healthy subjects for exosome isolation and purification and identification,exosomal mi RNAs were sequenced and analysed.Differentially expressed mi RNAs were analysed using DEGseq,Target Scan predicted the target genes of exosomal differentially expressed mi RNAs,and DAVID analysed the GO enrichment process and KEGG signalling pathway in which the target genes are involved.Part II: Exosomal mi RNAs with high differential expression ploidy and associated with AD pathogenesis were selected by screening the differentially expressed exosomal mi RNAs in part I for further expanded validation using RT-PCR.Proteins associated with AD pathogenesis(MMP-9/12,etc.)were subjected to ELISA.ROC curves were plotted after statistical analysis to assess the diagnostic value of mi RNAs and proteins.The correlation between the markers was also analysed by spearman.On this basis,mi RNA and protein were selected as potential biomarkers for combined diagnosis,and the combined diagnostic efficacy was assessed.Part III: Serum exosomes from patients with aortic dissection and healthy subjects were extracted and characterised by western blotting,concentration particle size analysis and transmission electron microscopy.Exosomes were labelled with PKH26 and fluorescence inverted microscopy was used to observe the effect of exosome uptake by endothelial cells.An ex vivo injury model of aortic coarctation was constructed usingβ-aminopropionitrile and Ang-II,and the effects of exosomes on endothelial cell apoptosis,proliferation,migration and angiogenesis were studied by western blotting,TUNEL,CCK-8,scratch assay and tube formation assay.Part IV: Exosomes from AD patients can promote endothelial cell injury.Pre-sequencing results revealed differences in the expression of exosomal mi RNAs in serum from patients with aortic dissection compared to healthy populations,from which we infer that the pathogenic effect of AD exosomes is caused through mi RNAs in exosomes.Based on the results in Part I and Part II,we selected mi R-202-3p,which has a high differential expression ploidy,good diagnostic efficacy and is associated with the pathogenesis of AD,as the focus of our study,and verified the release of mi RNA from exosomes by RT-PCR.mi R-202-3p was overexpressed and its effects on endothelial cell apoptosis,proliferation,migration and angiogenesis were investigated by flow-through apoptosis assay kit,CCK-8,scratch assay and tube formation assay.The mi R-202-3p target gene was predicted using the Target Scan database and validated with a dual luciferase gene reporter.The expression of mi R-202-3P and target genes was inhibited and the regulation of mi RNA-202-3p on target genes and signalling pathways was investigated by western blotting and RT-PCR.Results:Part I: When P<0.05 and |log2foldchange(FC)|>0.5,231 differentially expressed exosomal mi RNAs were screened,130 with up-regulated expression and 101 with down-regulated expression.Bioinformatics enrichment analysis revealed that the differentially expressed exosomal mi RNAs target genes were mainly involved in cell adhesion,protein acetylation,cell biosynthesis and other processes.Signalling pathways such as Ras signalling pathway,extracellular matrix adhesion and epithelial cell signalling were involved.Part II: RT-PCR further expanded the validation of the eight screened exosomal mi RNAs and found that the expression of mi R-499a-5p,mi R-543,mi R-206,mi R-4433b-3p,mi R-744-5p,mi R-4488 and mi R-202-3p were significantly upregulated in serum exosomes from patients with aortic dissection(P<0.05),and the expression trends of these mi RNAs were consistent with the sequencing results.In addition,changes in the expression levels of the proteins MMP-9,MMP-12,TGF-β and D-dimer,which are associated with the pathogenesis of AD,were also found.The diagnostic efficacy of differentially expressed exosomal mi RNAs and proteins was further assessed.The highest diagnostic sensitivity was found for mi R-744-5p(93.3%,AUC=0.831)and the highest diagnostic specificity was found for mi R-4433b-3p(86.7%,AUC=0.861).And the sensitivity and specificity of mi R-499a-5p,mi R-4433b-3p and mi R-206 were found to be above 80%.On the basis of correlation analysis,mi RNAs and proteins were used as potential biomarkers for combined diagnosis,and the combination with the highest sensitivity in combined diagnosis was mi R-202-3p+TGF-β(96.7%,AUC=0.844)and the combination with the highest specificity was mi R-499a-5p+mi R-4433b-3p(85%,AUC=0.840).Functional enrichment of target genes for mi RNAs with high diagnostic efficacy revealed possible involvement in the regulation of signaling pathways such as amyloid-β metabolism,T-cell migration,Fox O,synaptic vesicle recycling and PI3K-Akt.Part III: Analysis of serum exosome expression in the two groups revealed that serum exosome expression was increased in AD patients and that exosomes could be taken up by endothelial cells,internalised and thus function in intercellular communication.When serum exosomes from AD patients(AD-Exo)and healthy subjects(Con-Exo)were transfected into endothelial cells,it was found that AD-Exo significantly promoted endothelial cell apoptosis and inhibited endothelial cell proliferation,migration and angiogenesis(P<0.05),while inhibition of exosomes reduced the extent of endothelial cell damage by AD-Exo(P<0.05).Part IV: Expression of exosomal mi R-202-3p was elevated in the serum of mice with aortic dissection model and AD patients,and exosomes acted as carriers to encapsulate and release mi R-202-3p.overexpression of mi R-202-3p in endothelial cells resulted in increased apoptosis and decreased proliferation,migration and angiogenesis of endothelial cells,consistent with the injury results of AD-Exo(P<0.05).PI3 KCA was identified as a downstream target gene of mi R-202-3p and negatively regulated by mi R-202-3p by Target Scan prediction and dual luciferase gene reporter assays.Endothelial cell apoptosis,proliferation,migration and angiogenesis were partially restored under exogenous intervention with mi R-202-3p in combination with PI3 K inhibitors.PI3K/AKT/Fox O1 signalling pathway activation and axial changes in gene expression levels were observed in the aorta of AD patients and mouse AD models.After overexpression of mi R-202-3p,western blotting reaction and RT-PCR detected axial changes in the expression levels of PI3 KCA,p-AKT and p-Fox O1,with decreased expression levels of PI3 KCA and p-AKT and increased expression levels of p-Fox O1.Conclusion:(1)The 231 differentially expressed exosomal mi RNAs were screened by high-throughput sequencing in the serum of AD patients,and their target genes were mainly involved in protein acetylation,cell biosynthesis and other signalling pathways such as Ras signalling pathway,extracellular matrix adhesion and epithelial cell signalling.(2)mi R-499a-5p and mi R-202-3p were expressed at elevated levels in serum exosomes of patients with aortic dissection and had high diagnostic efficacy for aortic dissection.The combined diagnostic efficacy of mi R-499a-5p+mi R-4433b-3p was superior to that of a single index.(3)Patients with aortic dissection have elevated levels of exosomal proteins in their serum,which can be taken up and internalised by endothelial cells.This in turn affects the processes of endothelial cell apoptosis,angiogenesis,migration and proliferation,suggesting that AD-Exo mediates endothelial cell injury involved in the pathogenesis of aortic dissection.(4)The differentially expressed exosome mi R-202-3p in the serum of AD patients exerts its endothelial cell damaging effects through the target gene PI3 K to negatively regulate the PI3K/AKT/Fox O1 signalling pathway and promote the development of aortic dissection.