Single-Cell Analysis Reveals a Critical Role for Macrophage Epsins in Regulating the Origin of Foam Cells in Atherosclerosis

Background:Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of lipid-laden foam cells and plaques within the arterial wall. Dysfunctional vascular smooth muscle cells (VSMCs), fibroblasts, endothelial cells (ECs), and macrophages contribute to disease progression. Here, we report that macrophage-specific expression of epsins, highly conserved endocytic adaptor proteins involved in clathrin-mediated endocytosis, accelerates atherosclerosis in Western diet (WD)-fed mice.Methods:Apoe-deficient (WT/Apoe−/−) mice and littermates with a myeloid-specific deletion of epsin 1/2 on an Apoe−/− background (LysM-DKO/Apoe−/−) were generated and fed a WD for 16 weeks. Single-cell RNA sequencing (scRNA-seq) was conducted to investigate the cellular and molecular mechanisms regulated by macrophage epsins during atherosclerosis. Findings from scRNA-seq were validated through metabolic profiling, qRT-PCR, immunostaining, and co-culture experiments to assess associated phenotypic changes.Results:LysM-DKO/Apoe−/− mice exhibited significantly reduced atherosclerotic foam cell formation compared to WT/Apoe−/− controls. scRNA-seq analysis identified 19 major cell types, including six VSMC and five macrophage subpopulations. Modulated VSMC1 and VSMC2 subtypes were associated with inflammation, migration, and VSMC-to-macrophage transition. These populations, along with foamy-Trem2 and inflammatory macrophages, were markedly reduced in LysM-DKO/Apoe−/− mice. Transition of modulated VSMC2 subtype into macrophages was significantly inhibited, as confirmed by both computational analysis and experimental validation. Additionally, macrophage epsin deletion reversed endothelial dysfunction, suppressed cholesterol- and glucose-mediated signaling, and reduced expression of pro-inflammatory ligands IL-1β and TNF-α.Conclusion:Macrophage epsin deletion limits foam cell formation and preserves VSMC and endothelial cell phenotypes and functions. These findings reveal a potential therapeutic strategy targeting macrophage epsins to combat atherosclerosis.