Single-cell Sequencing Combined With Multi-omics Technology To Analyze The Regulatory Mechanism Of Vascular Smooth Muscle Cell Transformation To Osteogenic Phenotype In Atherosclerosi

Background:Cardiovascular disease(CVD),including coronary,cerebrovascular,and peripheral vascular diseases,is the leading cause of death and disability in China.The main pathophysiological basis of CVD is atherosclerosis(AS).According to statistics,about 2.4 million people die from atherosclerotic cardiovascular disease in China every year,which is a major public health problem that needs to be solved urgently.In the process of plaque progression,plaque calcification is an important risk factor leading to plaque rupture,secondary thrombosis and cardiovascular and cerebrovascular events.Recent studies have shown that intraplaque calcification may be an active and complex pathophysiological process similar to bone development and highly regulated.The osteogenic phenotype transformation of Vascular smooth muscle cells(VSMCs)is a key link in the process of vascular calcification.However,the specific molecular regulation mechanism has not been elucidated.In this study,single-cell sequencing combined with multi-omics analysis was used to explore the pathophysiological and molecular regulatory mechanisms of phenotypic transformation of VSMCs,and to identify key targets for early intervention,which has important scientific significance for the clinical diagnosis and treatment of vascular intimal calcification.Methods:Three cases of carotid intimal plaque tissues were collected for tissue isolation and Single-cell RNA sequencing(scRNA-seq).Standard flow analysis of scRNA-seq data was performed in R using Seruat to obtain cell clustering results.The specific highly expressed genes of each cell population were calculated,and the identity of each cell was annotated by combining classical marker genes and SingleR automatic annotation method.Pseudo-time-series analysis with Monocle2 was used to investigate cell differentiation trajectories.Singleseqgset,AUCell,GO,KEGG and other methods were used for functional pathway enrichment analysis of each cell group.VSMCs were extracted to perform cell subpopulation analysis to identify the gene markers and enriched pathways of each phenotype of VSMCs.To explore the origin of osteogenic phenotype cells and the differentiation direction of VSMCs by integrating scRNA-seq data of human carotid artery and coronary artery,combined with single-cell data of SMC lineage tracking AS model mice.scRNA-seq and single-cell sequencing assay for transposase-accessible chromatin data were combined for multi-omics analysis of scMEGA.To identify the core transcription factors and target genes for the differentiation of contractile SMC into osteogenic phenotype.The osteogenesis in carotid plaque was identified by histopathological staining.The VSMCs osteogenic model was established by using osteogenic differentiation medium.The changes of osteogenic markers and osteogenic ability of VSMCs were observed after knocking down IRF9 by small interfering RNA and using TGF-β pathway inhibitor/activator.Results:Immune cells were the main cell type in carotid plaque.VSMCs showed obvious phenotypic heterogeneity,including normal contractile phenotype,macrophage phenotype,foam cell phenotype,NK cell phenotype,mesenchymal phenotype,osteogenic transition state phenotype,and osteoblast/chondrocyte phenotype.Anchor point matching analysis of human-mouse data suggested that the osteogenic phenotype SMC in human AS tissues was differentiated from the contractile phenotype SMC.Pathway enrichment analysis showed that TGF-β signaling pathway was the key pathway for osteogenic differentiation of contractile SMC.Multi-omics analysis of scRNA combined with scATAC data identified IRF9 as a key transcription factor regulating the osteogenic phenotype differentiation of SMC,and TGFB1 was the transcriptional target gene of IRF9.Histological experiments have demonstrated the existence of osteogenesis in human carotid atherosclerotic calcified plaques.Knocking out IRF9 in vitro reduced the expression of osteogenic markers and osteogenic ability of VSMCs.Conclusions:VSMCs have different phenotypes,such as normal contractile phenotype,osteogenic phenotype,and fibroblastic phenotype,which are characterized by different gene expression profiles.Cells with an osteoblast-like phenotype differentiate from SMCS with a contractile phenotype and undergo a differentiation process from a contractile phenotype to an osteogenic transition state to an osteoblast-like phenotype.TGF-βsignaling pathway is an important signaling pathway for the differentiation of SMC into osteogenic phenotype.IRF9 is a key transcription factor for the differentiation of contractile SMC into osteogenic phenotype.