Single-cell transcriptomic analysis identifies vascular smooth muscle cell transcriptional programs associated with...

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.

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.

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 ATF3, JUND, and CREB5 regulon activity in the stress-responsive program and higher TEAD1, KLF2, and XBP1 activity in the DLX-osteomodulated program. Pseudotime analysis described two transcriptional continua with shared canonical contractile, stress-responsive, and extracellular-matrix-associated organization. In GSE260657, 842 of 1,483 VSMC-enriched cells were confidently projected, while 641 remained unassigned. Six of seven symptomatic-versus-asymptomatic program effects had concordant directions across the single-cell datasets, although no GSE260657 module reached a false-discovery rate below 0.05. In four paired plaques, ruptured regions showed lower contractile scores and higher osteogenic, foam-cell-like, and proliferation scores. qPCR analysis of an independent paired plaque cohort provided orthogonal tissue-level RNA support for selected remodeling-associated gene patterns.

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.