Effects Of High-fat Diet On Aortic Cell Composition And Function In Mice And Its Mechanism

The inflammatory response induced by a high-fat diet influences the development of cardiovascular disease.The aorta,as the main artery of the body,plays an important role in maintaining blood pressure,blood flow,oxygen exchange,and other physiological functions,highlighting the significance of the entire cardiovascular system.On the one hand,the aorta contains many cell types that undergo phenotypic transformation,gene expression,and functional differences during inflammatory responses that constantly influence the development of cardiovascular disease.On the other hand,the aorta is divided into 4 segments from the upper thorax to the lower abdomen:ascending,arch,thoracic,and abdominal.The internal structure and microenvironment of the arterial vessels are different,and there are differences in the response to inflammation,leading to a bias in the development of aortic disease.However,the differences in cellular composition and function of different segments of the aorta during high-fat diet-induced aortic inflammation have not been fully evaluated.Therefore,this study investigates the segmental variability,cellular heterogeneity,and key target proteins involved by investigating the cellular composition and function of the aorta affected by a high-fat diet,as well as the mechanisms involved.The main findings are as follows.1.Revealing the effect of high-fat diet on aortic cell composition in mice.By single-cell transcriptome sequencing of the four segments of the aorta in mice on normal and high-fat diets,respectively,10 major cell types were identified,including endothelial cells,smooth muscle cells,fibroblasts,monocytes/macrophages/dendritic cells,T cells,B cells,neutrophils,neuronal cells,erythrocytes,and pericytes.After a high-fat diet,the percentage of fibroblasts in the four segments of the mouse aorta increased.An increase in the percentage of monocytes/macrophages/dendritic cells was observed only in the ascending and thoracic aortic segments,and a decrease in the percentage of smooth muscle cells was observed only in the ascending and thoracic aortic segments.The proportions of’synthetic’EC(Endothelial cell)＿1subpopulation of antigen-binding,’pro-inflammatory’Fibro(Fibroblast)＿1 and Fibro＿3subpopulations in the thoracic aortic segment showed different degrees of increase,while the ascending aortic segment showed’anti-inflammatory’SMC(Smooth muscle cell)＿2 and SMC＿5 subpopulations,the proportions of which were decreased,and the proportions of Mono Macro DC(monocyte\macrophage\Dendritic cells)＿4 subpopulation and T cells in the’pro-inflammatory’type were increased.This suggests that the effect of high-fat diet on the cell composition of the ascending and thoracic segments of the aorta is more pronounced,mainly in terms of changes in the proportion of cell subpopulations responding to the inflammatory response.2.Revealing the effect of high-fat diet on aortic cell function in mice.By analyzing the functional enrichment of cell subpopulations and changes in gene expression,combined with vascular muscle tone,RT-PCR and tissue immunofluorescence assays,it was found that high-fat diet altered the function of the EC＿2 subpopulation in the thoracic aortic segment,weakening the diastolic function of blood vessels;altered the function of the smooth muscle cell subpopulations in the ascending aortic segment,triggering abnormal aortic systolic function;altered the function of the fibroblast subpopulations in the thoracic and abdominal aortic segments,which were significantly enriched for biological functions such as leukocyte chemotaxis and monocyte migration,and also altered the biological functions of monocyte/macrophage/dendritic cell subpopulation in the ascending aortic segment,which enriched inflammatory responses.In addition,intercellular communication analysis identified the Mono Macro DC＿1 subpopulation as the main subpopulation of cells secreting pro-inflammatory factors,and the CXCL signaling pathway plays an important role in regulating vascular inflammation.This suggests that the differential effect of high-fat diet on the function of different segments of the aorta is reflected in the heterogeneity of cellular function and gene expression.3.Identification of Klf2(Krüppel-like factor 2)as a key regulator of aortic inflammation in Vcam1~+(Vascular cell adhesion molecule 1)EC subpopulation.The study demonstrated the presence of an’activated’endothelial cell subpopulation(Vcam1~+EC subpopulation)with anti-inflammatory effects through single-cell transcriptome sequencing and tissue immunofluorescence assays.The subpopulation was identified to be involved in biological functions such as lymphocyte migration,cell adhesion,blood flow shear,and atherosclerosis during high-fat diet-induced aortic inflammation,with a significant alteration in the expression of genes involved in these functions.Furthermore,GAWS analysis and tissue immunofluorescence assays in the coronary population revealed a significant downregulation of Klf2 in the Vcam1~+EC subpopulation,indicating its key role in regulating vascular inflammation.4.Activation of the KLF2-S1PR1(Sphingosine-1-phosphate Receptor 1)signaling axis ameliorates the inflammatory response of vascular endothelial cells.Protein-protein interaction network analysis of differentially expressed genes in Vcam1~+EC subpopulation identified Klf2downstream target protein S1pr1 was identified.It was verified by WB and RT-PCR assays that Klf2 and S1pr1 m RNA and protein levels were reduced only in Vcam1~+endothelial cells under a high-fat diet,with no significant changes observed in Vcam1-endothelial cells.By constructing a Klf2 overexpression plasmid that was transfected into HUVEC and conducting RT-PCR,cell adhesion,and cell migration assays,it was found that LPS induced an inflammatory response in endothelial cells.However,overexpression of Klf2 decreased the expression levels of inflammatory factors,reduced the number of endothelial cells that adhered to monocytes,and enhanced the migration ability of endothelial cells during the inflammatory response.In addition,in vivo tests in mice showed that oral administration of atorvastatin significantly improved serum lipid levels,reduced the expression of inflammatory factors in endothelial cells,and decreased the extent of macrophage invasion of the vessel wall in mice on a high-fat diet.This suggests that both gene and drug therapies targeting Klf2 can improve the inflammatory response of endothelial cells.