Background:Atherosclerosis (AS) is a chronic inflammatory disease involving various cell types, cytokines, and adhesion molecules. Herein, we aimed to uncover its key molecular mechanisms by single-cell RNA-seq (scRNA-seq) analysis.Methods:ScRNA-seq data of cells from atherosclerotic human coronary arteries were analyzed using the Seurat package. Cell types were clustered, and differentially expressed genes (DEGs) were screened. GSVA (Gene Set Variation Analysis) scores of hub pathways were compared among different cell clusters. DEGs in endothelial cells between apolipoprotein-E (ApoE)−/−mice and specific TGFbR1/2 KO ApoE−/−mice fed with high-fat diet were overlapped with those from human AS coronary arteries. In fluid shear stress and AS, hub genes were determined based on the protein–protein interaction (PPI) network, which were verified in ApoE−/−mice. Finally, hub genes were validated in three pairs of AS coronary arteries and normal tissues by histopathological examination.Results:ScRNA-seq identified nine cell clusters in human coronary arteries, namely, fibroblasts, endothelial cells, macrophages, B cells, adipocytes, HSCs, NK cells, CD8+T cells, and monocytes. Among them, endothelial cells had the lowest fluid shear stress and AS and TGF-beta signaling pathway scores. Compared to ApoE−/−mice fed with normal diet, fluid shear stress and AS and TGF-beta scores were both significantly lower in endothelial cells from TGFbR1/2 KO ApoE−/−mice fed with normal or high-fat diet. Furthermore, the two hub pathways had a positive correlation. Three hub genes (ICAM1, KLF2, and VCAM1) were identified, and their expression was distinctly downregulated in endothelial cells from TGFbR1/2 KO ApoE−/−mice fed with normal or high-fat diet than in those from ApoE−/−mice fed with a normal diet, which were confirmed in human AS coronary artery.Conclusion:Our findings clarified the pivotal impacts of pathways (fluid shear stress and AS and TGF-beta) and genes (ICAM1, KLF2, and VCAM1) in endothelial cells on AS progression.