Atherosclerosis Profiling Reveals BHLHE40 as a Candidate Modulator of VSMC.

Abstract

Vascular smooth muscle cells (VSMCs) play a central role in atherosclerosis by undergoing phenotypic modulation from a quiescent, contractile state to a range of synthetic phenotypes, including fibroblast-like, macrophage-like, and lipid-laden foam cell-like states. However, a comprehensive multimodal characterization and understanding of the transcriptional programs driving these transitions remain incomplete. To comprehensively define the phenotypic diversity of VSMCs during atherosclerosis progression, we performed in-depth profiling using cellular indexing of transcriptomes and epitopes by sequencing and bulk RNA sequencing in a VSMC-lineage-tracing atherosclerotic mouse model. Insights from these data sets guided the design of targeted in vitro experiments to investigate candidate regulatory mechanisms. Single-cell multiomics revealed extensive cellular heterogeneity within atherosclerotic plaques, including a rare population of VSMC-derived macrophage-like cells, whose presence was confirmed by histological analysis. These studies also identified a large population of VSMC-derived foam cells that exhibited activation of gene programs associated with lipid metabolism, proliferation, and tumor-like features. The transcription factor BHLHE40 (basic helix-loop-helix family member e40) emerged as a candidate regulator of this phenotypic transition, with elevated expression and activity in VSMC-derived foam cells during disease progression and expression in modulated VSMC in human carotid atherosclerosis. Functional knockdown of <i>Bhlhe40</i> reprogrammed immune, cell cycle, and lipid homeostasis genes in cultured VSMC and suppressed VSMC phenotypic switching and foam cell characteristics, consistent with a potential regulatory role in VSMC modulation. These findings advance our understanding of VSMC phenotypic modulation in atherosclerosis and implicate BHLHE40 as a candidate transcriptional regulator of this process. Elucidating mechanisms governing VSMC plasticity may offer new therapeutic opportunities to reduce cardiovascular risk by targeting disease-driving cellular transitions.