Sexually Dimorphic Transdifferentiation of Endothelial Cells in Atherosclerosis

Abstract Objective To elucidate the sex-specific trajectories of endothelial cell (EC) transdifferentiation and their contribution to distinct atherosclerotic plaque phenotypes in males and females. Approach and Results We utilized Cdh5-CreER T 2 /Apoe −/− /Rosa26-mTmG lineage-tracing mice to perform single-nucleus RNA sequencing (snRNA-seq) of aortic tissues from male, female, and ovariectomized female mice following 12 weeks of high-fat diet. We mapped high-resolution EC fates, identifying distinct transdifferentiation routes into smooth muscle cell (SMC)-like, adipocyte-like, macrophage-like, and fibroblast-like cells. Females exhibited higher overall EC plasticity with a preference for SMC-like and metabolically active adipocyte-like trajectories, contributing to plaque stability. Conversely, male ECs favored inflammatory macrophage-like and stress-responsive fates. Trajectory analysis revealed a novel direct EC-to-adipocyte-like transition that bypasses the canonical endothelial-to-mesenchymal transition (EndoMT). Gene regulatory network analysis, validated by in vitro knockdown assays, identified KLF2 as a homeostatic brake on transdifferentiation, while CREB5 and ESRRG emerged as critical molecular switches governing the bifurcation between adipogenic and myogenic fates. Ovariectomy in females shifted the transcriptomic landscape toward a male-like, lipid-anabolic phenotype. Conclusions EC plasticity is a sex-dimorphic process driving differential plaque composition. We identify a novel, direct EC-to-adipocyte trajectory and pinpoint KLF2 and CREB5 as key regulators, offering new mechanistic insights into why premenopausal women develop more stable atherosclerotic lesions than men.