Female-Biased VSMC GRNs Predict MYH9 as Regulator of Fibrous Plaque Phenotype

BACKGROUND: Atherosclerosis, an inflammatory driver of coronary artery disease, manifests as unstable atheromatous plaques and stable fibrous plaques. Although atheromatous plaques have been extensively studied, fibrous plaques, particularly in women aged <50 years, where erosion contributes significantly to coronary thrombosis, remain less understood. The molecular mechanisms underlying sex differences in plaque biology, including vascular smooth muscle cell contributions, are incompletely defined. METHODS: Sex-specific gene regulatory networks (GRNs) were constructed from RNA-sequencing data of cultured human vascular smooth muscle cells isolated from 119 male and 32 female heart transplant donors. Network preservation analyses identified female-biased GRNs, which were evaluated in single-cell RNA-sequencing data sets from human carotid atherosclerotic plaques. Bayesian network modeling and proteomic analyses were used to identify and validate regulatory drivers. RESULTS: Two female-biased vascular smooth muscle cell networks, GRN floralwhite and GRN yellowgreen , were enriched for inflammatory and actin remodeling pathways, respectively. Single-cell RNA-sequencing confirmed sex-specific network activity in plaque vascular smooth muscle cells. Subcellular phenotyping identified a sex-specific gene expression program within GRN yellowgreen enriched for contractile and vascular development pathways. Bayesian network modeling identified MYH9 (myosin heavy chain 9) as a key driver gene. Elevated MYH9 abundance was associated with increased smooth muscle cell content and reduced lipid content in female carotid plaques compared with males, consistent with fibrous plaque features. Proteomic analyses confirmed MYH9 upregulation in female fibrous plaques and association with stable plaque characteristics. CONCLUSIONS: These findings identify MYH9 as a regulator of female-biased fibrous plaque biology and highlight the importance of sex-specific network regulation in atherosclerosis.