Comprehensive Multimodal Profiling of Atherosclerosis Reveals Bhlhe40 as a Potential Regulator of Vascular Smooth Muscle Cell Phenotypic Modulation

Ibikunle, C. O.; Xue, C.; Kim, E.; Yan, H.; Coronel, J.; Zhu, L. Y.; Cui, J.; Chung, A.; Bauer, R. C.; Sachs, N.; Maegdefessel, L.; Li, M.; Tall, A. R.; Bashore, A. C.; Reilly, M.

doi:10.1101/2025.05.20.655228

bioRxiv · 10.1101/2025.05.20.655228

Comprehensive Multimodal Profiling of Atherosclerosis Reveals Bhlhe40 as a Potential Regulator of Vascular Smooth Muscle Cell Phenotypic Modulation

Ibikunle, C. O.·Xue, C.·Kim, E.·Yan, H.·Coronel, J.·Zhu, L. Y.·Cui, J.·Chung, A.·Bauer, R. C.·Sachs, N.·Maegdefessel, L.·Li, M.·Tall, A. R.·Bashore, A. C.·Reilly, M.

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Abstract

BackgroundVascular 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. MethodsTo comprehensively define the phenotypic diversity of VSMCs during atherosclerosis progression, we performed in-depth profiling using cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) and bulk RNA sequencing in a VSMC lineage-tracing atherosclerotic mouse model. Insights from these datasets guided the design of targeted in vitro experiments to investigate candidate regulatory mechanisms. ResultsSingle-cell multi-omics 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 substantial population of VSMC-derived foam cells, comprising approximately 70% of all foam cells in the lesions. These cells exhibited activation of gene programs associated with lipid metabolism, proliferation, and tumor-like features. The transcription factor Bhlhe40 emerged as a key regulator of this phenotypic transition, with elevated expression in VSMC-derived foam cells during disease progression. Functional knockdown of Bhlhe40 suppressed VSMC phenotypic switching and foam cell characteristics, underscoring its potential role as a driver of VSMC modulation. ConclusionsThese findings advance our understanding of VSMC phenotypic modulation in atherosclerosis and highlight Bhlhe40 as a key regulator of this process. Elucidating the mechanisms governing VSMC plasticity may offer new therapeutic opportunities to reduce cardiovascular risk by targeting disease-driving cellular transitions.

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Ibikunle, C. O., Xue, C., Kim, E., Yan, H., Coronel, J., Zhu, L. Y., Cui, J., Chung, A., Bauer, R. C., Sachs, N., Maegdefessel, L., Li, M., Tall, A. R., Bashore, A. C., Reilly, M.. 2025-05-23. Comprehensive Multimodal Profiling of Atherosclerosis Reveals Bhlhe40 as a Potential Regulator of Vascular Smooth Muscle Cell Phenotypic Modulation. https://doi.org/10.1101/2025.05.20.655228

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