SOX4 is a novel phenotypic regulator of endothelial cells during atherosclerotic diseases revealed by single-cell analysis

Hyperlipidemia-associated cytokines and disturbed flow upregulate SOX4 expression in ECs, where SOX4 promotes EndoMT and atherogenesis. Metformin pharmacologically suppresses SOX4 level in ECs. EC, endothelial cell; IL-1β, interleukin-1β; TGF-β1, transforming growth factor β1. • Hyperlipidemia enriched mesenchymal markers in EC cluster of atherosclerotic mice. • EC cluster can be partitioned into ‘ endothelial-like ’ and ‘ mesenchymal-like ’ subpopulations. • SOX4 was upregulated in mouse and human atherosclerotic tissues. • SOX4 upregulation was observed in human aneurysmal tissues. • EC-specific SOX4 overexpression promoted EndoMT, endothelial dysfunction and atherogenesis. • Hyperlipidemia-related cytokines and disturbed flow upregulated endothelial SOX4 level. Atherosclerotic complications represent the leading cause of cardiovascular mortality globally. Dysfunction of endothelial cells (ECs) often initiates the pathological events in atherosclerosis. In this study, we sought to investigate the transcriptional profile of atherosclerotic aortae, identify novel regulator in dysfunctional ECs and hence provide mechanistic insights into atherosclerotic progression. We applied single-cell RNA sequencing (scRNA-seq) on aortic cells from Western diet-fed apolipoprotein E-deficient ( ApoE −/− ) mice to explore the transcriptional landscape and heterogeneity of dysfunctional ECs. In vivo validation of SOX4 upregulation in ECs were performed in atherosclerotic tissues, including mouse aortic tissues, human coronary arteries, and human renal arteries. Single-cell analysis on human aortic aneurysmal tissue was also performed. Downstream vascular abnormalities induced by EC-specific SOX4 overexpression, and upstream modulators of SOX4 were revealed by biochemical assays, immunostaining, and wire myography. Effects of shear stress on endothelial SOX4 expression was investigated by in vitro hemodynamic study. Among the compendium of aortic cells, mesenchymal markers in ECs were significantly enriched. Two EC subsets were subsequently distinguished, as the ‘ endothelial-like ’ and ‘ mesenchymal-like ’ subsets. Conventional assays consistently identified SOX4 as a novel atherosclerotic marker in mouse and different human arteries, additional to a cancer marker. EC-specific SOX4 overexpression promoted atherogenesis and endothelial-to-mesenchymal transition (EndoMT). Importantly, hyperlipidemia-associated cytokines and oscillatory blood flow upregulated, whereas the anti-diabetic drug metformin pharmacologically suppressed SOX4 level in ECs. Our study unravels SOX4 as a novel phenotypic regulator during endothelial dysfunction, which exacerbates atherogenesis. Our study also pinpoints hyperlipidemia-associated cytokines and oscillatory blood flow as endogenous SOX4 inducers, providing more therapeutic insights against atherosclerotic diseases.