Role Of Sema7A In The Changes Of Cell Functional Heterogeneity In Carotid Artery Induced By Disturbed Blood Flow

BackgroundAtherosclerosis is a multifactorial chronic inflammatory disease of the vascular wall.Atherosclerosis remains one of the leading causes for the high morbidity and mortality of cardiovascular disease in the world,despite extensive research in the prevention and treatment of atherosclerosis.Disturbed blood flow(d-flow)is characterized by low and oscillatory shear stress,which plays a vital role in the development of atherosclerosis.In regions of d-flow,the blood vessels and the cells of the arterial wall undergo significant changes,including endothelial dysfunction,inflammation,angiogenesis,EndMT,apoptosis,VSMCs(vascular smooth muscle cells)migration,and extracellular matrix(ECM)remodeling.These changes increase the risk of atherosclerosis in the vascular wall.Using DNA microarray and bulk RNA-seq,previous studies identified some mechanosensitive genes and certain cellular pathways in the major arterial cell types of the vasculature.However,the molecular profile,and the heterogeneity of individual cells under d-flow are poorly understood.In recent years,using single-cell RNA sequencing technology,researchers have discovered functional heterogeneity in both immune cells and vascular wall cells in atherosclerotic plaques.However,whether disturbed blood flow can alter the functional heterogeneity of vascular wall cell subpopulations is unclear.Our recent work found that Sema7A promotes atherosclerosis in response to d-flow,but whether Sema7A affects the function of d-flow-related cell subpopulations remains unclear.In order to further study the mechanism of disturbed flow and Sema7A involved in atherosclerosis,we established the single-cell atlas of all cells in the carotid artery under d-flow,and found that disturbed flow can induce the generation of novel vascular wall cell subpopualtions,and Sema7A mainly affects the functional heterogeneity of d-flow-induced smooth muscle cells.AimsTo elucidate the role of Sema7A in the changes of cell functional heterogeneity in carotid artery induced by disturbed blood flow.Methods(1)To generate d-flow in vivo,partial carotid artery ligation(PCL)was performed;(2)To obtain a single-cell suspension,we used a two-step digestion with collagenase/DNase combined with trypsin to digest the mouse carotid artery;(3)We use the 10×Genomics platform to load the single cell suspension,then build and sequence the library;(4)We use the Seurat package to perform an integrated analysis of the scRNA-seq raw data;(5)Gene set variation analysis(GSVA)was used to analyze the biological processes of each cell subpopulation;(6)Cell cycle analysis was used to calculate the cell cycle in which a subpopulation of cells was located;(7)Single-cell trajectories analysis was performed to predict the transition among cell subpopualtions;(8)Western blot,immunofluorescence and en face immunostaining techniques were performed for identification and localization of d-flow-related cell subpopulations;(9)We use the Seurat package to calculate the number of cells in each sample and analyze the relative abundance;(10)Construction of Sema7A-overexpressing human smooth muscle cell line to explore the effect of Sema7A on the transformation of smooth muscle cells;(11)Stimulation of human-derived smooth muscle cells by purified Sema7A recombinant protein to determine the phenotypic transformation of smooth muscle cells;(12)We used the bulk RNA-seq to explore Sema7A-mediated mechanisms.Results(1)The integrated analysis identified that mainly six cell types,including VSMCs,fibroblasts,ECs,and d-flow-induced immune cell populations(M(p/DCs,granulocytes,and lymphocytes)existed in the vascular wall of left carotid artery.(2)We found that Dkk2hi ECs and Cd36hi ECs were two novel subpopualtions of endothelial cells induced by d-flow.Among then,Dkk2hi ECs was a mechanosensitive EC subpopulation,and single-cell trajectories analysis showed that Klk8hi ECs might directly transition to Dkk2hi ECs under d-flow.Meanwhile,Dkk2hi ECs and Cd36hi ECs were also found in the lesser curvature of the aortic arch where d-flow usually occurs or in high-fat diet induced atherosclerotic plaques.(3)Three macrophage subpopualtions including Trem2hi Mφ,Res-like Mφ and Birc5hi Mφ existed in the vessel wall under d-flow.Of note,the novel Birc5hi Mcp was identified as a potential contributor to the accumulation of macrophages in atherosclerosis.(4)The CD4+T cells,CXCR6+T cells,NK cells and granulocytes existed in the carotid artery when the d-flow occurs and appeared to be endowed with specific functions such as T cell differentiation and apoptosis,responses to oxygen levels,insulin,and hormone,cell killing,and leukocyte aggregation.(5)D-flow-induced Spp1hi VSMCs subpopulation had a particular set of functions such as osteoblast differentiation,collagen biosynthetic process,blood vessel remodeling,and aging compared to two other origianl VSMC subpopulations.(6)Relative-abundance analysis showed that the proportion of smooth muscle cells was decreased while the fibroblasts and macrophages were increased after PCL surgery.However,Sema7A deletion decreased the proportion of fibroblasts and increased the proportion of smooth muscle cells.(7)Sema7A deficiency prevented Dkk2hi ECs from executing multiple biological functions such as integrin-mediated cell adhesion,cell migration and chondrocyte differentiation.In Cd36hi ECs,Sema7A had specific biological functions such as angiogenesis,hypoxia response,mechanical response,and cell differentiation.(8)Sema7A deletion reduced the expression of collagen-related molecules Co13a1 and Col1a1,and promoted the expression of smooth muscle cell-related markers Acta2 and Tagln in Trem2hi Mφ.Furthermore,Sema7A deficiency suppressed the expression of Birc5hi Mφ proliferation markers Top2a,Ube2c and Mki67.(9)Sema7A deficiency significantly reduced the transition of d-flow-induced Spp1hi VSMCs to fibrochondrocyte-like cells.(10)Overexpression of Sema7A in smooth muscle cells and addition of recombinant Sema7A protein reduced the expression of smooth muscle cell markers α-SMA and Cnnl,and increased the expression of fibrochondrocyte-like cell markers Col1a1 and Col3a1.Conclusions1.By combining the PCL surgery that generates d-flow in vivo with the powerful single-cell RNA sequencing(scRNA-seq)technology,we uncovered 10 distinct d-flow-associated cell clusters:Dkk2hi ECs,Cd36hi ECs,Spp1hi VSMCs,Trem2hi Mφ,DCs,Birc5hi Mφ,CD4+T cells,CXCR6+T cells,NK cells and granulocytes.2.Dkk2hi ECs is a mechanosensitive EC subpopulation,while Birc5hi Mφ exhibits a high proliferation ability.Furthermore,Dkk2hi ECs and Cd36hi ECs present in the lesser curvature(LC)rather than the greater curvature(GC)of the aortic arch and in the atherosclerotic plaques,implying their potential role in atherosclerosis.Of note,d-flow-induced Spp1hi VSMCs had a particular set of functions such as osteoblast differentiation,and collagen biosynthesis.3.Sema7A deletion reduced the proportion of d-flow-induced fibroblasts by attenuating the transition of d-flow-induced Spp1hi VSMCs to fibrochondrocyte-like cells.In vitro experiments confirmed that Sema7A promotes the transition of smooth muscle cells to fibrochondrocyte-like cells.