An in vitro System for Studying Osteochondrogenic Differentiation of Smooth Muscle Cells and Modeling Intimal Vascular Calcification

Objective: Smooth muscle cells (SMCs) undergo phenotypic transitions during atherosclerosis, including towards a chondromyocyte (CMC) state associated with intimal calcification. Although standard in vitro calcification assays robustly reproduce mineral deposition, it remains unclear how well they recapitulate these disease-associated SMC states. We sought to define the CMC transcriptional phenotype in atherosclerosis and develop an in vitro system that faithfully reproduces it. Approach and Results: We firstly identified a CMC transcriptional signature in murine and human atherosclerotic plaque through single-cell RNA-sequencing, and spatial transcriptomics. CMCs showed a conserved osteochondrogenic program which localized within plaques and adjacent to calcified regions. We then developed an osteochondrogenic differentiation (OCD) assay by combining well-established calcification components with a high-density SMC micromass culture and TGF-{beta}1 supplementation and benchmarked it against a standard calcification (SC) assay using calcium quantification and bulk RNA-sequencing. Despite comparable calcification, OCD and SC resulted in distinct transcriptional states, with OCD showing preferential upregulation of osteochondrogenic programs, and a higher CMC signature score. Additionally, OCD upregulated genes with a stronger enrichment near coronary artery disease (CAD)-associated loci. These responses were reproducible across several primary human SMC lines. Timecourse analysis also showed that chondrogenic programs preceded calcification and showed directional concordance with the inferred in vivo SMC-to-CMC trajectory. To interrogate regulatory pathways controlling this process, we overexpressed the chondrogenic regulator SOX9, which enhanced cartilage and extracellular matrix programs while repressing inflammatory pathways. Finally, we examined 552 CAD-associated genes nominated across five genome-wide association studies. Of these, 240 were differentially expressed by day 12, and included established SMC regulators as well as a number of candidates not previously characterized in osteochondrogenic SMC transition. Conclusions: The OCD assay results in a strong calcification phenotype together with a disease-associated CMC-like transcriptional state, providing a reliable in vitro model for mechanistic investigation of SMC phenotypic transition and prioritization of candidate regulators.