Identification Of Smooth Muscle Cell Apoptosis Activity And IL-1β Expression Level Changes In The Progression Of Mouse Thoracic Aortic Dissection Based On Single-Cell RNA Sequencing

Objective:Thoracic aortic dissection （TAD） is an extremely dangerous vascular disease,with inconspicuous clinical symptoms in the early stages and rapid progression,high surgical risk,high mortality rate,and heavy financial burden on patients and their families.Therefore,it is crucial to identify effective early diagnostic factors and drug targets in the diagnosis and treatment of TAD.In recent years with the maturity of high-throughput sequencing technology and the trend of data sharing,the molecular biology mechanisms of TAD have become a hot research topic.In this study,we analyzed the single-cell transcriptome sequencing data of mouse TAD models from the NGDC database,and used various bioinformatics methods for data mining to understand the complex cell ecosystem in TAD tissue and organs, with the aim of providing important basis for disease mechanisms,drug development,and personalized medicine.Method:The study analyzed the "Single-cell RNA-seq of thoracic aortic aneurysm and dissection in mouse" dataset shared by the National Center for Cardiovascular Diseases of the Chinese Academy of Medical Sciences on the NGDC database using bioinformatics analysis.The data induced thoracic aortic dissection by daily oral administration of BAPN （0.5 g/kg/d） to male C57BL/10 mice at 3 weeks of age,and the aortic tissue was collected for single-cell RNA sequencing on the 7th,14th,and 21st day of the experiment,which dynamically simulated the occurrence of TAD.The data.were downloaded and subjected to quality control using R language （4.2.1）.A total of 80,514 cells were obtained from six samples,and these cells were subjected to dimensionality reduction clustering （PCA t-SNE,UMAP） processing,cell annotation,and division into eight cell types.The proportion of each cell type was calculated in each group of cells and visualized,and the results were analyzed.Genes with an absolute valuue of inter-group differential expression fold change> 0.5 were selected as differentially expressed genes.GO and KEGG enrichment analysis were performed on these differentially expressed genes to examine changes in gene function and pathways during TAD induction.The AUCell package was used to analyze smooth muscle cell apoptosis using the mouse apoptosis gene set obtained from the GSEA database.Finally,the expression levels of IL-1β,MMP-9,MMP-2,TIMP-2,and CD147 in thoracic aortic tissue at each stage were visualized.Results:1.Cell annotation of single-cell sequencing data revealed that the mouse aortic tissue is mainly composed of vascular smooth muscle cells,endothelial cells,fibroblasts:macrophages,neutrophils,monocytes,T cells, and B cells.When a dissection occurs a large number of vascular smooth muscle cells are lost,and there is asignificant infiltration of inflamm atory cells such as macrophages and neutrophils.2.A total of 256 differentially expressed genes were identified in the smooth muscle cells of the BAPN₂1d group compared to the control group.The top 5 upregulated genes were Malat1,Dmd, Lyz2,Gm42418,and Ubb,while the top 5 downregulated genes were Fos,Ogn,Sost, Wdr89,and mt-Nd41.GO and KEGG enrichment analysis was performed on these 256 differentially expressed genes.3.The apoptotic activity of vascular smooth muscle cells was reduced in the BAPN₂1d group.4.With the in crease of TAD induction time,the expression of IL-1β and MMP9 in the aortic tissue was initially low,but became significantly increased at 21d.However,the expression of CD147,MMP2,and TIMP2 gradually decreased.Conclusion:1.Under normal conditions,the aortic tissue is mainly composed of vascular smooth muscle cells.However,during the occurrence of TAD,smooth muscle cells decrease while inflammatory cells,incliding macrophages,neutrophils,monocytes,and lymphocytes,heavily infiltrate the tissue.This indicates that inflammation plays an important role in the pathogenesis of TAD.Therefore,anti-inflammatory treatment should be highly emphasized in the perioperative treatment of TAD patients.2.In this study,through analysis of differential gene expression in 21d smooth muscle cells a total of 256 differentially expressed genes were screened with the main upregulated genes being Malatl Dmd Lyz2,Gm42418,and Ubb,and the main downregulated genes being Fos,Ogn,Sost,Wdr89,and mt-Nd41.These DEGs can provide some guidance and direction for subsequent research.3.Through GO and KEGG enrichment analysis,we found that differentially expressed genes mainly enriched in pathways such as the structure and organization of actin,regulation of cell-matrix adhesion,extracellular matrix,and transcriptional regulator inhibitors,the importance of which has been confirmed in previous studies.In addition,we also found that these DEGs were enriched in the ribosome,parathyroid hormoine,and oxytocin,signaling pathways:the relationship of which with TAD is unclear.Therefore,further in-depth study of these pathways may lead to more research directions.4.In this study,we observed that during the establishment of a model of thoracic aortic dissection,the apoptosis activity of smooth muscle cells gradually decreased.However,previous studies have shown that the number of smooth muscle cells in aortic dissection tissue specimens decreased.Therefore,we considered two possibilities:first,SMC apoptosis plays a role in TAD,and cell apoptosis occurs,thereby relatively reducing the response to the activated gene set;second,SMC apoptosis is not related to the occurrence of TAD,and the absence of SMCs in aortic tissue is achieved through other means.5.IL-1β is highly expressed in TAD aortic tissue and is accompanied by an increase in MMP-9,showing a positive correlation with disease progression,while CD147,MMP2,and TIMP2 show a negative correlation with disease progression.IL-1β may be used to assist in the diagnosis and monitoring of thoracic aortic dissection,while IL-1β receptor antagonists may serve as perioperative treatment drugs for TAD.