Abstract Body: A defining feature of atherosclerosis is the uptake of oxidized lipids by cells, leading them to differentiate into foam cells. Foam cells were once believed to originate primarily from macrophages. However, recent research suggests that over 70% are derived from vascular smooth muscle cells (VSMCs). While the ability of VSMCs to undergo phenotypic modulation into foam cells is established, the transcriptomic distinctions and regulatory mechanisms regulating this transition remain poorly characterized.

We hypothesized that given their phenotypic and functional differences; VSMC-derived foam cells would have a distinct gene expression and regulatory signature compared to contractile VSMC.
To investigate this, we conducted bulk RNA sequencing of atherosclerotic plaques from conditional VSMC-lineage tracing LDLR^-/- mouse models maintained on a high-fat diet. Using LipidTox staining VSMC and VSMC-derived foam cells were identified, and isolated using flow cytometry at key time points representing late and advanced stages of disease progression. Computational analyses revealed distinct differentially expressed genes, key molecular regulatory functions, and enriched biological pathways, including pathways such as cell cycle signalling, integrin interactions and interleukin signalling, that differentiate VSMC-derived foam cells from their contractile counterparts.

Our findings also reveal that the expression of the Basic Helix-Loop-Helix Family Member E40, Bhlhe40 (also known as Dec1), a pro-inflammatory transcription factor, is activated in VSMC-derived foam cells during advanced stages of atherosclerosis and enriched in modulated and proliferative VSMC-derived cells measured at scRNA-seq of atherosclerotic lesions. Upstream regulator analysis suggests that Bhlhe40 may play a pivotal role in driving the differentiation of VSMCs into foam cells. Functional experiments revealed that Bhlhe40 knockdown in vitro inhibits VSMC phenotypic modulation and lipid uptake in response to cotreatment withTNFα and MBD-Cholesterol for 48 hours which promotes foam cell formation.

These findings reveal a modulatory role for Bhlhe40 in the VSMC response to atherosclerotic stress, positioning it as a potential therapeutic target for regulating VSMC-derived foam cell formation in atherosclerosis.