Single-cell transcriptomic analysis identifies vascular smooth muscle cell transcriptional programs associated with human carotid plaque remodeling.

Background: Vascular smooth muscle cells (VSMCs) contribute to plaque structure and exhibit diverse transcriptional programs in human atherosclerosis. Although human plaque single-cell studies have established substantial VSMC heterogeneity, an important unresolved question is whether discovery-defined programs remain recognizable across independent patients and sequencing platforms without forced integration, and whether those programs retain associations with plaque instability. We therefore focused on program-level reproducibility, inferred regulatory activity, continuous expression organization, and rupture-associated variation. Methods: We used three public human carotid plaque transcriptomic datasets in nonredundant analytical roles rather than merging all single-cell cohorts into a single atlas. GSE253903, a 10x Genomics single-cell cohort, was used for broad plaque mapping and focused VSMC characterization. SCENIC and transcriptional-continuum analyses were restricted to finalized VSMCs. Discovery signatures and assignment rules were frozen before independent assessment in the Smart-seq2 dataset GSE260657, with uncertain cells permitted to remain unassigned. GSE120521 supported exploratory paired comparison between relatively stable and visibly ruptured plaque regions. Results: Quality control retained 46,209 cells from 12 patients in GSE253903, including 3,784 primary VSMCs after removal of immune-like contaminants. Five descriptive VSMC programs were identified: canonical contractile, extracellular-matrix-associated/fibromyocyte-like, DLX-osteomodulated, stress-responsive, and interferon-associated. VSMC-restricted SCENIC analysis showed higher inferred Conclusion: Human carotid plaque VSMCs display heterogeneous transcriptional programs with program-associated inferred regulon activity and continuous expression organization. An uncertainty-aware, cross-platform assessment supported partial conservation of these programs and linked selected remodeling patterns to plaque instability. This framework is intended to complement, rather than replace, spatial and mechanistic studies by identifying reproducible program-level features that can be prioritized for localization and functional testing.