{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,9,13]],"date-time":"2026-09-13T10:04:46Z","timestamp":1789293886251,"version":"build-2803163510"},"posted":{"date-parts":[[2026,9,10]]},"group-title":"Cell Biology","reference-count":36,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,9,10]],"date-time":"2026-09-10T00:00:00Z","timestamp":1788998400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,9,10]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:sec>\n                  <jats:title>Objective<\/jats:title>\n                  <jats:p>\n                    Smooth muscle cells (SMCs) undergo phenotypic transitions during atherosclerosis, including towards a chondromyocyte (CMC) state associated with intimal calcification. Although standard\n                    <jats:italic>in vitro<\/jats:italic>\n                    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\n                    <jats:italic>in vitro<\/jats:italic>\n                    system that faithfully reproduces it.\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Approach and Results<\/jats:title>\n                  <jats:p>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.<\/jats:p>\n                  <jats:p>\n                    We then developed an osteochondrogenic differentiation (OCD) assay by combining well-established calcification components with a high-density SMC micromass culture and TGF-\u03b21 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\n                    <jats:italic>in vivo<\/jats:italic>\n                    SMC-to-CMC trajectory.\n                  <\/jats:p>\n                  <jats:p>To interrogate regulatory pathways controlling this process, we overexpressed the chondrogenic regulator SOX9, which enhanced cartilage and extracellular matrix programs while repressing inflammatory pathways.<\/jats:p>\n                  <jats:p>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.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions<\/jats:title>\n                  <jats:p>\n                    The OCD assay results in a strong calcification phenotype together with a disease-associated CMC-like transcriptional state, providing a reliable\n                    <jats:italic>in vitro<\/jats:italic>\n                    model for mechanistic investigation of SMC phenotypic transition and prioritization of candidate regulators.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.09.05.749615","type":"posted-content","created":{"date-parts":[[2026,9,10]],"date-time":"2026-09-10T20:35:13Z","timestamp":1789072513000},"source":"Crossref","is-referenced-by-count":0,"title":["An\n                  <i>in vitro<\/i>\n                  System for Studying Osteochondrogenic Differentiation of Smooth Muscle Cells and Modeling Intimal Vascular Calcification"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6481-6875","authenticated-orcid":false,"given":"Jo\u00e3o P.","family":"Monteiro","sequence":"first","affiliation":[{"name":"Department of Medicine, Division of Cardiovascular Medicine, Stanford University, Stanford, CA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4391-5377","authenticated-orcid":false,"given":"Matthew D.","family":"Worssam","sequence":"additional","affiliation":[{"name":"Department of Medicine, Division of Cardiovascular Medicine, Stanford University, Stanford, CA, 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