Sexually Dimorphic Transdifferentiation of Endothelial Cells in Atherosclerosis

Aims: Sex differences in atherosclerosis are well recognized clinically, yet the underlying mechanisms remain incompletely understood. Given the pivotal role of endothelial cell (EC) dysfunction in atherogenesis, this study aimed to delineate and compare EC fate trajectories and uncover transcriptional networks driving atherosclerosis progression in males and females. Methods and results: EC-specific lineage-tracing mice (Apoe−/−/Cdh5-CreERT2/Rosa26-mTmG) of both sexes were generated. Following tamoxifen-induced Cre recombination, mice were fed a high-fat diet (HFD) to induce hypercholesterolemia and atherosclerosis. Single-nucleus RNA sequencing (snRNA-seq) of plaque-bearing aortae identified and validated nine distinct EC subpopulations. Differential abundance analysis using miloR revealed that female aortae were enriched for capillary and lymphatic ECs expressing Sox17, Myh11, Mki67, or Ly6a, whereas male aortae were enriched for ECs expressing Col8a1 and Fmo2. Fate inference by GeneTrajectory and single-cell lineage tracing demonstrated that approximately a quarter of GFP⁺ ECs transdifferentiated into adipocyte-like cells, smooth muscle cell (SMC)-like cells, and fibroblast-like cells. Female ECs exhibited a greater propensity for EC-to-SMC-like conversion, whereas male ECs showed a stronger inclination toward EC-to-adipocyte-like transition. Regulatory network analysis using pySCENIC identified Klf2 and Creb5 as key transcription factors governing EC fate decisions in atherosclerosis. Conclusions: Endothelial cells exhibit sex-specific transdifferentiation trajectories during atherosclerosis progression, contributing to distinct vascular phenotypes in male and female plaques and revealing potential targets for precision therapeutics.