The Mechanistic Study Of IL-6 Regulating Vascular Smooth Muscle Cell Phenotypic Transformation And Promoting Atherosclerosis Through TWIST1

BackgroundAtherosclerosis(AS)is a chronic inflammatory disease characterized by the formation of core necrotic plaques and narrowing of lumen based on lipid metabolism disorders.The pathogenesis is related to environmental factors,genetic factors and physical and chemical damage.The transformation of vascular smooth muscle cells(VSMC)from contractility to synthesis is an early event and key pathological change of AS.At the same time,macrophage infiltration promotes local inflammatory reaction and lipid deposition,further exacerbates the instability of plaque,and induces complications of coronary heart disease and stroke.The exact pathogenesis of AS is unclear so far,which is a hotspot and difficulty in the study for the basic and clinical atherosclerosis research.The cellular composition of AS plaques is complex,with multiple cell types undergoing pathological transformation and interacting with each other to influence the onset and progression of AS.Therefore,an in-depth exploration of the composition of the AS plaque microenvironment and the interactions between cells can help us better understand the pathogenesis of AS and provide more valuable action targets for drug development.Single-cell RNA sequencing(scRNA-seq)can analyze the characteristics of transcriptome at the single cell level and is suitable for investigating the heterogeneity of complex tissue microenvironments and cell subpopulations.This technique has been widely used in the pathogenesis of cardiovascular and cerebrovascular diseases,and can help identify pathological cell subpopulations,elucidate cell differentiation trajectories and pathogenesis,etc.Thus,it is a powerful tool help us resolve the pathogenesis of AS.ObjectiveWe utilized scRNA-seq to construct a single-cell atlas of human AS plaque to identify the characteristics of microenvironment,deeply explore the pathological transformation of VSMCs and macrophages,and elucidate the interactions between cells in the progression of AS.Our study aims to provide new theoretical supports for the pathogenesis of AS and new targets for clinical drugs.Method(1)In this study,plaque tissue samples were obtained from 6 core areas and 3 adjacent areas from carotid atherosclerosis(CA)patients.The plaque samples were digested with a complex enzyme,prepared single cell suspensions,and were performed for scRNA-seq using the 10X genomics platform.(2)ScRNA-seq data were possessed using Seurat package to identify the cellular composition in the plaques based on cell type marker genes.The proportion of different cell types were further compared between plaque core and adjacent regions.The expression of cardiovascular disease(CVD)risk genes in different cell types was also analyzed to identify key cell types associated with AS pathogenesis.(3)We further identified heterogeneity among VSMC and macrophage subsets.The transition trajectories of VSMC subsets were analyzed using Monocle2 package.Differentially expressed genes were calculated to explore the gene expression characteristics of different subsets.Gene set variation analysis(GSVA)was used to calculate the level of pathway activity in different cell subsets.We used Nitchnet software to construct a communication network between macrophages and VSMC and to identify the macrophage-derived ligands that affect VSMC phenotypic transformation.(4)Clinical plaque tissues were collected to validate key cell subsets and target molecules by immunohistochemistry and immunofluorescence.Smooth muscle cell lines(T/G HA-VSMC)were stimulated with different cytokines,and the expression levels of molecules related to VSMC phenotypic transformation were estimated using q-PCR and Western Blot.CCK8 and transwell migration assay were used to detect the proliferation and migration ability of VSMC.(5)Lentivirus was used to construct TWIST1 knockdown T/G HA-VSMC lines.After IL-6 stimulation,the expression level of transcription factor TWIST1 was detected by q-PCR and WB,and the binding of TWIST1 to the COL1A1 promoter region was validated using dual-luciferase reporter assay.Results(1)In this study,we successfully constructed the single cell landscape of human CA,obtained transcriptome data of 54,271 high-quality cells,and identified 16 cell types.VSMC included three subsets:contractile-SMC,SMC-derived intermediated cells(SEM)and fibrochondrocytes(FC),and myeloid cells included monocytes,macrophages and dendritic cells.The proportion of contractile-SMC in the plaque core area was decreased and the proportion of macrophages was increased compared to the adjacent area.In addition,VSMC and myeloid subsets expressed multiple CVD risk genes.The results suggested that VSMC and macrophages in CA plaques may be the key cell types involved in the pathogenesis of AS.(2)The developmental trajectories analysis showed that contractile-SMC first transformed into SEM and then differentiated into FC subsets in AS plaques.The three VSMC subsets showed different gene expression characteristics.Inflammatory response,ossification,and extracellular matrix pathway activity were increased in SEM and FC cell subsets compared to contractile-SMC.Functionally heterogeneous subsets were also found in the myeloid cells.The proportion of monocytes was decreased in the core region of the plaque,and CX3CR1~+macrophages and SPP1~+macrophages was increased compared to adjacent area.CX3CR1~+macrophages highly expressed various inflammatory factors,antigen presenting genes and phagocytosis genes,and SPP1~+macrophages highly expressed foam cell marker genes,lipid metabolism genes and matrix metalloproteinase,respectively,suggesting that CX3CR1~+macrophages had strong pro-inflammatory and phagocytic effects,while SPP1~+macrophages might form"foam"macrophages and degraded extracellular matrix,promoting plaque instability.(3)Intercellular communication analysis showed that macrophage-derived IL-6 could potentially regulate the VSMC phenotypic transformation process.An independent data set further confirmed the correlation between IL-6 and IL-6 signaling pathways and the progression of CA plaques.In vitro experiments showed that IL-6 could significantly down-regulate the contractile genes,up-regulate the expressions of fibrosis and ossification genes,and promote the proliferation and migration of VSMC,suggesting that IL-6 promoted phenotypic transformation of VSMC.(4)Bioinformation analysis revealed that TWIST1 was a specific transcription factor for FC,potentially up-regulating extracellular matrix and cytokine-related pathways and down-regulating VSMCs contractile pathways.In vitro experiments showed that IL-6 could up-regulate TWIST1 transcription and protein expression levels.TWIST1 knock down on VSMC could reverse the effect of IL-6 on phenotypic transformation of VSMC.Dual-luciferase reporting assay confirmed that TWIST1 could bind to the COL1A1 promoter,thereby regulating COL1A1 expression and promoting the fibrochondrocyte-like phenotype of VSMC.ConclusionMacrophage-derived IL-6 promotes transcriptional regulation of the downstream target gene COL1A1 through TWIST1,which promotes VSMCs fibrochondrocyte-like phenotype and is involved in the pathogenesis of AS.The present study may provide a new theoretical basis for the study of the pathogenesis of AS and the search for effective targets.