MD-PhD Student McGill University Montréal, Quebec, Canada
Background: Ischemic stroke remains a leading cause of cardiovascular mortality worldwide, largely driven by unstable atherosclerotic plaques that reside in the internal carotid artery. While clinical guidelines recommend carotid endarterectomy based on stenosis severity, unstable plaques can cause strokes even with low to moderate stenosis, suggesting that factors beyond plaque stenosis may contribute to its instability. Among these, vascular smooth muscle cells (VSMCs) play a crucial role in atherosclerosis, demonstrating remarkable plasticity as they undergo phenotypic switching. Upon local environmental stimuli, VSMCs transition from a healthy contractile state to several others, which include adipogenic, inflammatory (synthetic), fibroblast-like, and foam cell-like phenotypes, but their role in influencing carotid plaque instability remains unclear. Therefore, this study explored the transcriptomic profiles of VSMCs and their corresponding phenotypes between stable and unstable carotid atherosclerotic plaques.
METHODS AND RESULTS: Stable (n=6) and unstable (n=6) carotid plaque specimens underwent single-cell RNA sequencing (scRNA-seq) to identify various cell subpopulations. Differential abundance testing, CellChat, and gene set enrichment analysis (GSEA) were performed to uncover distinct transcriptomic signatures, cellular interactions, and VSMC phenotypes linked to plaque instability. ScRNA-seq analysis of 71,205 cells revealed 14 distinct cell populations, with VSMCs representing the most abundant cell type in carotid plaques (44.28%). Differential abundance testing demonstrated a significant decrease in VSMCs in unstable plaques compared to stable plaques (49.51% vs. 19.36%; p = 2.65 x 10⁻88). Subclustering analysis revealed 6 distinct VSMC phenotypes, with contractile, fibroblast-like, and myofibroblast-like VSMCs significantly enriched in stable plaques (p = 1.83 × 10⁻201), while inflammatory (synthetic), proliferative, and mesenchymal-like phenotypes were predominant in unstable plaques (p = 1.59 × 10⁻90). In line with these findings, GSEA revealed that VSMCs in stable plaques were associated with contractile function and pro-fibrotic pathways, whereas those in unstable plaques were enriched in several cytotoxic and inflammatory processes. CellChat analysis further demonstrated that VSMCs exhibited the highest degree of cellular interactions within the plaque microenvironment. Notably, interactions between VSMCs and cytotoxic T-lymphocytes were significantly increased in unstable plaques, suggesting an immune-VSMC crosstalk driving plaque instability.
Conclusion: This study highlights VSMC phenotypic switching as a key driver of carotid plaque instability, with unstable plaques favoring inflammatory and proliferative states while stable plaques retain contractile, fibroblast, and myofibroblast-like phenotypes. Increased interactions between VSMCs and cytotoxic T-lymphocytes were associated with instability, underscoring an immune-VSMC crosstalk in plaque vulnerability. These findings provide novel insights into VSMC plasticity and potential therapeutic targets for plaque stabilization.
