Abstract Body (Do not enter title and authors here): Background: Cardiovascular disease remains the leading cause of death worldwide. Unstable atherosclerotic plaques that reside in the carotid arteries are major etiological factors known to increase the incidence of ischemic strokes. Moreover, sex differences exist in plaque morphology and composition; males are more prone to develop unstable plaques and subsequent rupture, while females tend to develop more stable plaques but exhibit worse outcomes post-stroke. Despite these differences however, there is currently a lack of sex-specific guidelines for carotid atherosclerotic disease management.
Objective: To investigate the association between endogenous sex hormones and the sex-specific cellular and genetic signatures linked to plaque instability.
Methods: Carotid plaque specimens and pre-operative fasted blood samples were collected and analyzed (n=460; mean age of 71.0±9.0; 68.9% male) from recruited males and post-menopausal females who underwent carotid endarterectomy at McGill University-affiliated hospitals. Distribution and relative expression of sex hormones in plaques were evaluated using liquid chromatography mass spectrometry (LC-MS/MS)-based methods by quantifying levels of 17ß-estradiol (E₂) and testosterone (T). Plaque single-cell suspensions (n=10) underwent single-cell RNA sequencing (scRNA-seq) to characterize differences in plaque cell expression phenotypes by identifying distinct cell subpopulation clusters.
Results: Males with unstable plaques had significantly higher circulating T levels and T to E₂ ratio compared to males with stable plaques, with no significant differences in E₂ levels within all groups. Furthermore, scRNA-seq analysis of plaque samples revealed 15 subpopulations of cells; 6 non-immune cell clusters (smooth muscle cells [SMCs], myofibroblasts, and endothelial cells) and 9 immune cell clusters (macrophages, T-lymphocytes, B-lymphocytes, natural killer cells, and dendritic cells). In both sexes, stable plaques exhibited the highest abundance of myofibroblast-like SMCs and pro-fibrotic genes compared to unstable plaques. Similarly, female stable plaques exhibited significantly reduced number of myofibroblast-like SMCs compared to male stable atherosclerotic plaques.
Conclusions: Our findings demonstrate that taken together, increased circulating testosterone levels and reduced myofibroblast-like SMCs expressing pro-fibrotic genes in the plaque may be associated with increased carotid atherosclerotic plaque instability.