Single-Cell Spatial Transcriptomics Reveals Sex-Specific Differences Driving Carotid Atherosclerotic Plaque Instability

BACKGROUND: Ischemic stroke remains a leading contributor to cardiovascular disease, and unstable atherosclerotic plaques in the carotid arteries are an important contributor to its pathogenesis. Notably, sex differences exist in plaque composition and morphology; whereas males are more likely to develop unstable plaques, females present with worse outcomes poststroke. As such, this study aims to elucidate sex-specific cellular and molecular mechanisms driving carotid plaque instability. METHODS: We performed single-cell RNA sequencing on carotid endarterectomy specimens from male and female patients with stable or unstable plaques, profiling over 64 000 cells. To spatially contextualize these findings, we integrated single-cell RNA sequencing data with CosMx high-plex spatial transcriptomics using the human discovery 6k RNA panel to map gene expression and cell type localization within key histological regions defining instability. RESULTS: Single-cell RNA sequencing revealed that vascular smooth muscle cells (VSMCs), T lymphocytes, and macrophages comprised ≈88% of plaque-resident cells, with distinct sex-specific differences in relation to instability. In males, unstable plaques showed depletion of contractile VSMCs and an abundance of inflammatory synthetic and macrophage-like VSMCs, alongside increased inflammatory macrophages and activated naive T lymphocytes. In contrast, female unstable plaques retained relatively more contractile VSMCs but exhibited greater expansion of cytotoxic T lymphocytes and foam-cell macrophages. Sex-specific cell-cell interaction analysis demonstrated increased VSMC-to-stromal interactions and elevated macrophage-macrophage signaling in male and female unstable plaques, respectively. Through spatial transcriptomics, inflammatory synthetic VSMCs identified by single-cell RNA sequencing dominated the lipid core in both male and female unstable plaques. However, only in males did these cells also replace the fibrotic cap, forming immune-rich niches among themselves and with macrophage-like VSMCs. CONCLUSIONS: Overall, these findings uncover distinct cellular and spatial mechanisms driving plaque destabilization in males and females, highlighting the need for sex-specific approaches to the diagnosis and treatment of carotid atherosclerosis.