A Single-Cell Atlas Of Brain Arteriovenous Malformations And Preliminary Study On TIMP-1 In Regulating Vascular Homeostasis

Background:Brain arteriovenous malformation(bAVM)is a kind of malformed vascular mass that is prone to rupture and is the main cause of hemorrhagic stroke in children and young people.The dynamic changes of cell components and their interactions in the vascular microcirculation are the causes of the formation,progression and rupture of bAVM.Vascular related endothelial cells,smooth muscle cells,pericytes and perivascular fibroblasts are the main cell components of the vascular microenvironment,and also the main sites driving development of bAVM.With the advent of the era of"omics",the disease-related features of bAVM such as somatic mutations,gene expression differences and cell phenotype transformation have been revealed.However,the abundances of cell components and potential functional characteristics of bAVM vascular related cells have not been explored in detail.Single-cell RNA sequencing(scRNA-seq)can analyze the characteristics of transcriptome at single-cell resolution,and is suitable for exploring the heterogeneity of cells in complex vascular microenvironment.The application of single cell technology in the field of bAVM can carry out detailed characterization of the main cell components of bAVM,help identify new cell subclusters,clarify the cell differentiation track,and is a powerful means to analyze the pathogenesis of bAVM.Objective:To construct the bAVM single cell transcriptome atlas,investigate the heterogeneity of bAVM endothelial cells,vascular smooth muscle cells(VSMC),pericytes(PC)and perivascular fibroblasts(PVFB),clarify the potential pathogenic subcluster and characterize their potential function.Furthermore,to screen the potential pathogenic molecule in bAVM and verify its function through subsequent experiments.We hope that this study could provide new theoretical support for bAVM pathogenesis research and clinical drug development.Methods:In this study,4 temporal lobe tissue from patients undergoing temporal-lobe resection for epilepsy and 6 bAVMs single cell data were included.The two groups of data were jointly analyzed,and the cell components were identified by data cleaning and dimension reduction clustering.In order to study the unique transcriptional characteristics of bAVM,the differences of the two groups in endothelial cells,VSMC,PC and PVFB were compared from the gene expression,functional activation,transcriptional activity and differentiation trajectory.Furthermore,the potential pathogenic molecule was screened by bioinformatics and verified in bAVM tissue.Finally,its functions were preliminarily explored by cell experiments.Results:1.Through the quality control of single cell data,87229 effective single cell data were obtained,and the single cell atlas of bAVM was constructed.Through dimensionality reduction and clustering of the data,a total of 12 main cell components were identified.The proportion analysis showed that there were significant differences in the composition of blood vessel cells and peripheral tissue cells in bAVM.2.The endothelial cells were annotated into 6 subclusters with the characteristics of arteries,veins,capillaries/venules.The abundance of Art＿like and Venous＿2 subclusters increased significantly in bAVM.Functional enrichment suggested that the activation of many bAVM disease-related pathways increased with the progression of bAVM rupture,and the highest activation degree was found in Atr＿like subcluster,suggesting that Atr＿like is a potential pathogenic endothelial cell subcluster of bAVM.It was found that most of the Art＿like and Venous＿2 in bAVM were at the differentiation trajectory starting point,and the expression of HES1 and NES with stemness and differentiation potential was the highest at the differentiation trajectory starting point,and gradually decreased at the end.Immunofluorescence showed that the number of coexpression of NES and endothelial cell marker gene CD31 in bAVM was significantly higher than that in the control group.3.A total of 6 subclusters were identified in VSMC.The binary classification characterization of contraction and synthesis phenotype of VSMC in our data is not accurate.There were obvious gene expression and functional heterogeneity in the six subclusters.Pseudotime analysis showed that there were two obvious differentiation trajectories in VSMC system.Although both of them were characterized by the gradual down-regulation of contractile genes,the differentiation characteristics of the two tracks were different.Inflammation,SMC proliferation and endothelial cell migration pathways were gradually enhanced in the differentiation trajectory including SMC1-SMC4.In the other trajectory containing SCM5-6,it was found that the expression of PDGFRB and SMTN decreased gradually,while the expression of IGF1R increased gradually,and the distribution of functional score was consistent with the expression distribution of the above functional genes.4.After detailed annotation of PC,it was found that the proportion of PC3 cells increased significantly in bAVM.The overall gene expression level and functional activity were lower,and the proportion of G1 phase in cell cycle was higher in PC3.Further functional annotation and transcriptional activity analysis suggested that apoptosis and inflammatory cell chemotactic phenotype activation,suggesting that PC3 may be a specific subcluster of pericyte apoptosis or quantitative loss in bAVM.Based on the analysis of the new cell group PVFB identified in the recent cerebrovascular single cell study,it was found that it could be divided into three subgroups,of which PVFB3 subcluster were significantly enriched in bAVM.Functional characterization suggests that it is related to inflammatory response,vascular-related cell differentiation and tissue remodeling.5.The differential gene analysis of vascular cells showed that the number of differential genes in endothelial cells was the largest,and TIMP-1 was significantly highly expressed in these cells,especially in endothelial cells.Further analysis found that TIMP-1in endothelial cells was significantly correlated with End MT.Subsequently,cell experiments showed that TIMP-1 and End MT markers were significantly up-regulated under 6dyn/cm~2 shear stress.Overexpression of TIMP-1 under static conditions was not enough to induce End MT.Knocking down TIMP-1 under 6dyn/cm~2 shear stress could significantly inhibit the expression of End MT markers.Conclusion:Through our data,we identified the transcriptional characteristics of subclusters related to the pathogenic phenotype of vascular cells in bAVM.These data provide high-resolution insights into the complexity and heterogeneity of bAVM,and suggest that specific subclusters in various vascular cells may have different contributions to the pathogenesis of bAVM.It is further found that TIMP-1 may cause vascular homeostasis disorder in bAVM by regulating mesenchymal transformation of endothelial cells,thus promoting the progress of bAVM.