Pathological Response Heterogeneity Of Endothelial Cell Subpopulations And Angiogenesis After Cerebral Ischemia-Reperfusion In Mice

BackgroundStroke has a high mortality and disability rate,and is now the leading cause of death in China.After revascularization treatment,oxidative stress and various inflammatory damages are induced,and endothelial cells(ECs)that constitute the inner wall of blood vessels are an important pathological basis of the post-stroke injury process.However,the characteristics of the pathological responses of different vascular ECs and the formation of angiogenesis after cerebral ischemia are still unclear.Based on the development of single-cell sequencing technology,the gene expression of each cell can be studied at the single-cell level,revealing the heterogeneity between cells and providing a new research method for the in-depth study of disease development mechanisms.In this study,we further analyzed the heterogeneity of the pathological responses of different endothelial cell subpopulations in different periods after stroke and investigated the tissue distribution and functional characteristics of new endothelial cells based on the results of endothelial cell-specific transgenic mice transcriptomic data at day 2,day 5 and day 14 after transient middle cerebral artery occlusion.ObjectiveTo investigate the pathological response heterogeneity of EC subpopulations after cerebral ischemia-reperfusion in mice,the histological distribution of newly generated cerebral vessels and effectively perfused vessels,characteristics of angiogenesis,and recovery of effective perfusion function.MethodsTo map the single-cell transcriptome atlas of murine ECs in different periods after transient middle cerebral artery occlusion(MCAO),combined the group’s previous studies on transcriptome data at different periods after MCAO in Cdh5-CreERT2/+;Rosa26-mTmG transgenic mice.To investigate the heterogeneity of pathological responses across EC subpopulations using GSVA and cell trajectory analysis.Immunofluorescence staining of brain tissue sections was performed to verify the characteristics of angiogenesis and the function of sprouting endothelial cells after stroke by selecting endothelial cell subpopulation-specific markers and dyes for the assessment of vascular perfusion function.ResultsBy analyzing the ECs transcriptome sequencing results,a total of 11 brain EC subpopulations were obtained,including the venous,capillary,and arterial ECs as well as the newly emerged tip cells,stalk cells,and pre-artery ECs.It was found that there was heterogeneity in the pathological responses of each EC subpopulation after cerebral ischemic injury.Venous ECs were enriched in inflammatory response-related pathways and significantly upregulated in BBB dysfunction.Arterial ECs were mainly associated with ECs morphogenesis.Capillary ECs were involved in anti-inflammatory response and BBB structural reconstruction.Tip cells,venous and arterial ECs were significantly up-regulated in response to VEGF.The increased tip cells,whose TGF-βsignaling pathway and basement membrane formation pathway were significantly upregulated after ischemia induction,had the highest differentiation potential.Stalk cells had overlapping genetic profiles and functional characteristics with venous endothelial cells and biological processes involved in EC development and angiogenesis regulation in pre-arterial ECs.In addition,Cdh5-labeled ECs were significantly increased at the hippocampus and cortex after ischemia,but hippocampal regions lacking tip cell generation did not have significant perfusion function in their neovascularization,whereas tip cells at the cortex with stalk cells derived from venous ECs promoted the formation of arteries in the cortex and were associated with restoration of effective perfusion function of vessels at the cortex.ConclusionEach EC subpopulation had a unique pathological response in different periods after MCAO.Simultaneously,angiogenesis of the hippocampus and cortex on the ischemic side was particularly pronounced in the subacute phase.However,newly generated tip cells at the cortex only promote the formation of arteries and were associated with the restoration of effective vascular perfusion function at the cortex.