Single-cell Analysis Of High Salt-induced Hypertensive Mouse Aortae Reveals Cellular Heterogeneity And Function Changes

As the largest and most critical arterial vessel in the human body,the aorta can be classified into four segments based on its anatomical path: the ascending aorta,aortic arch,descending aorta,and abdominal aorta.Each aortic segment has distinct regional susceptibility to various aortic diseases.Unfortunately,surgical intervention is currently the only effective treatment for aortic diseases,which are the most aggressive type of cardiovascular disease,and no clinically available drugs have been found to be effective.Epidemiological studies have shown that high blood pressure,caused by excessive salt intake,is prevalent in most countries and is closely associated with cardiovascular disease,particularly aortic pathology.The Chinese consume 2.5 times the recommended amount of dietary salt,one of the highest salt intakes worldwide.However,the effects of hypertension induced by a high-salt diet on different segments of the aorta and alterations in the composition and function of vascular cells remain inadequately described.In this study,we employed single-cell transcriptome sequencing technology to map normal aortic cells and changes in each vascular cell subpopulation under a high-salt diet.Our findings are as follows:(1)A high-salt diet administered to mice for 10 weeks resulted in a gradual increase in blood pressure.Wire myography assay revealed that the endothelium-dependent diastolic function of aortic segments was impaired in the hypertensive group,with the aortic arch and thoracic aortic segments showing the most severe impairment.Furthermore,the vasoconstriction of aortic segments in hypertensive mice was enhanced,particularly in the aortic arch,thoracic aorta,and abdominal aorta segments.In addition,the contractile function of aortic segments in control mice also varied,with the aortic arch segment exhibiting the strongest contractile ability.Evans blue staining showed that hypertension induced by high salt diet increased the permeability of the aortic arch segment.Therefore,compared to other segments of the aorta,hypertension induced by high salt diet caused the most significant damage to the aortic arch segment.(2)Single-cell transcriptome sequencing of normal mouse aortic arch tissue identified 19 cell clusters,including fibroblasts,smooth muscle cells,endothelial cells(ECs),and a substantial population of immune cells.Endothelial cells in the aortic arch were found to comprise three distinct subpopulations with strengths in extracellular matrix production and integrin signaling,lipid processing and angiogenesis,and lymphatic function,respectively.Three smooth muscle subpopulations were identified,with subpopulation 1 being contractile smooth muscle,subpopulation 2 being synthetic smooth muscle,and subpopulation 3 being a previously unreported subpopulation.Immunostaining experiments confirmed the presence of these three smooth muscle subpopulations.Of the three fibroblast subpopulations,subpopulation 1 appeared to be associated with external stimuli,while subpopulation 2 had a multifunctional role,and subpopulation 3 differed from the other two in the production of extracellular matrix proteins.(3)Integrative analysis of single-cell transcriptome sequencing data from the aortic arch of normal and high-salt diet mice revealed an increase in T cells,smooth muscle cells,and endothelial cells in the vascular tissue of the high-salt diet group.Subsequent analysis of these three cell types showed that EC 1 and EC 3 subpopulations had higher proliferation rates in the hypertensive state,while EC 1 and EC 2 had higher expression of ROS-related enzymes,collagen,and contractile genes.The number of all three smooth muscle subpopulations expanded during hypertension development,partly due to increased proliferative efficiency and partly due to endothelial-mesenchymal transition of some endothelial cells,a key mechanism underlying aortic remodeling.In addition,the phenotype of smooth muscle subpopulation 1 shifted from contractile to synthetic in the hypertensive state.Among T-cell subsets,Th17 and Th2 subpopulations exhibited significantly increased expression of chemokine genes in hypertensive tissues,which could recruit distal T cells and cause vascular inflammation.Analysis of intercellular communication revealed more frequent cellular communication between aortic cells in the hypertensive group of mice.(4)Integration of single-cell transcriptome analysis with Genome wide association study(GWAS)data has identified potential cellular subpopulation targets for the treatment of hypertension in the aorta,including endothelial cells,smooth muscle cells,and fibroblasts.A total of 93 GWAS genes associated with hypertension characteristics were identified.Analysis of the blood pressure profile revealed that the highest number of GWAS genes were highly expressed in endothelial cells,followed by smooth muscle cells,while fibroblasts had the least number of dominantly expressed candidate genes.By overlaying the differentially expressed genes of the three cell types in the control and hypertensive groups with the GWAS genes associated with blood pressure traits,21 genes were identified that may be involved in the generation and development of hypertension.Among them,consistent changes across subpopulations were observed in Cfdp1,Zeb1,Nadk,and Cpeb4 genes,which were upregulated in each mural cell subpopulation of the hypertensive aortic arch.These genes may be key targets for the treatment of hypertension.