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, 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–fed mice. METHODS: WT/Apoe −/− (Apoe-deficient) mice and littermates with a LysM-DKO/Apoe −/− (myeloid-specific deletion of epsin 1/2 on an Apoe −/− background) were generated and fed a Western diet for 16 weeks. Single-cell RNA sequencing was conducted to investigate the cellular and molecular mechanisms regulated by macrophage epsins during atherosclerosis. Findings from single-cell RNA sequencing were validated through metabolic profiling, qRT-PCR, immunostaining, and coculture experiments to assess associated phenotypic changes. RESULTS: LysM-DKO/Apoe −/− mice exhibited significantly reduced atherosclerotic foam cell formation compared with WT/Apoe −/− controls. Single-cell RNA sequencing analysis identified 19 major cell types, including 6 VSMC and 5 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. In addition, macrophage epsin deletion reversed endothelial dysfunction, suppressed cholesterol- and glucose-mediated signaling, and reduced expression of proinflammatory ligands IL (interleukin)-1β and TNF-α (tumor necrosis factor α). CONCLUSIONS: 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.