Single-cell spatial transcriptomics reveals clonal smooth muscle cell heterogeneity and programs associated with atherosclerotic plaque calcification.

Osteochondrogenic transdifferentiation of vascular smooth muscle cells (SMCs) is a key driver of atherosclerotic calcification. Although SMC phenotypic plasticity has been well described, how clonally related SMCs interact with spatially defined microenvironments to drive calcification remains incompletely understood. We integrated single-cell RNA sequencing, mitochondrial mutation-based lineage tracing, and high-resolution spatial transcriptomics to construct a spatially resolved transcriptomic atlas of human carotid atherosclerotic plaques across AHA stages III-V. Plaque SMCs originated from multiple distinct clones, each capable of differentiating into diverse phenotypes. Pseudotime analysis revealed that fibroblast-like SMCs (FLSMCs), which arose from clones with higher mitochondrial mutation burdens, could transition into multiple SMC subtypes, including osteogenic-like SMCs (OLSMCs). In AHA stage IV-V plaques, OLSMCs were markedly enriched compared with stage III, particularly within peri-calcification zones located 0.16–0.32 mm from calcified regions. Within these niches, OLSMCs and foam macrophages (MPs) showed pronounced spatial enrichment (80–160 µm) and significant co-localization. Mechanistically, SPP1/FN1 signaling from foam MPs activated CD44/integrin pathways in OLSMCs, promoting calcification. These findings indicate that mitochondrial mutation-linked clonal expansion drives osteogenic differentiation within peri-calcification niches. Our data also suggest that the SPP1/FN1-CD44/integrin axis mediates inflammation-driven crosstalk between foam MPs and OLSMCs, providing a foundation for future studies of plaque calcification.