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Gla rich protein (GRP) is a calcification inhibitor involved in most of these processes. However, the molecular mechanism of GRP in VC and the specific characteristics, cargo, and functionality of calcifying EVs require further elucidation. Here, we use a combination of human ex vivo aortic fragments and primary vascular smooth muscle cell (VSMC) models to obtain new information on GRP function in VC and EVs released by VSMCs. We demonstrate that GRP inhibits VSMC osteogenic differentiation through downregulation of bone-related proteins and upregulation of mineralization inhibitors, with decreased mineral crystallinity in EVs deposited into the tissue extracellular matrix (ECM). EVs isolated by ultracentrifugation at 30K and 100K from the cell media (CM) and deposited in the ECM from control (CTR) and mineralizing (MM) VSMCs were biochemically, physically, and proteomically characterized. Four different EV populations were identified with shared markers commonly present in all EVs but with unique protein cargo and specific molecular profiles. Comparative proteomics identified several regulated proteins specifically loaded into MM EV populations associated with multiple processes involved in VC. Functional analysis demonstrated that 30K and 100K ECM-MM EVs with higher calcium and lower GRP levels induced macrophage inflammation. Our findings reinforce the functional relevance of GRP in multiple VC processes and suggest that ECM EVs released under calcification stress function as a new signaling axis on the calcification\u2013inflammation cycle.<\/jats:p>","DOI":"10.3390\/ijms252212406","type":"journal-article","created":{"date-parts":[[2024,11,19]],"date-time":"2024-11-19T04:53:05Z","timestamp":1731991985000},"page":"12406","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["Gla Rich Protein (GRP) Mediates Vascular Smooth Muscle Cell (VSMC) Osteogenic Differentiation, Extracellular Vesicle (EV) Calcification Propensity, and Immunomodulatory Properties"],"prefix":"10.3390","volume":"25","author":[{"given":"Carla","family":"Viegas","sequence":"first","affiliation":[{"name":"Centre of Marine Sciences (CCMAR\/CIMAR LA), University of Algarve, 8005-139 Faro, Portugal"},{"name":"GenoGla Diagnostics, Centre of Marine Sciences (CCMAR), University of Algarve, 8005-139 Faro, Portugal"}]},{"given":"Joana","family":"Carreira","sequence":"additional","affiliation":[{"name":"Centre of Marine Sciences (CCMAR\/CIMAR LA), University of Algarve, 8005-139 Faro, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0038-9629","authenticated-orcid":false,"given":"Teresa M.","family":"Maia","sequence":"additional","affiliation":[{"name":"VIB Center for Medical Biotechnology, Technologiepark-Zwijnaarde 75, 9052 Ghent, Belgium"},{"name":"Department of Biomolecular Medicine, Ghent University, Technologiepark-Zwijnaarde 75, 9052 Ghent, Belgium"},{"name":"VIB Proteomics Core, 9052 Ghent, Belgium"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2613-4838","authenticated-orcid":false,"given":"Anjos L.","family":"Macedo","sequence":"additional","affiliation":[{"name":"UCIBIO, Department of Chemistry, and Associate Laboratory i4HB\u2014Institute for Health and Bioeconomy, NOVA School of Science and Technology, Universidade NOVA de Lisboa, 2829-516 Caparica, Portugal"}]},{"given":"Ant\u00f3nio P.","family":"Matos","sequence":"additional","affiliation":[{"name":"Egas Moniz Center for Interdisciplinary Research (CiiEM), Egas Moniz School of Health & Science, 2829-511 Caparica, Portugal"}]},{"given":"Jos\u00e9","family":"Neves","sequence":"additional","affiliation":[{"name":"Service of Cardiothoracic Surgery, Santa Cruz Hospital, Centro Hospitalar de Lisboa Ocidental, 2790-134 Carnaxide, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5145-4753","authenticated-orcid":false,"given":"Dina","family":"Simes","sequence":"additional","affiliation":[{"name":"Centre of Marine Sciences (CCMAR\/CIMAR LA), University of Algarve, 8005-139 Faro, Portugal"},{"name":"GenoGla Diagnostics, Centre of Marine Sciences (CCMAR), University of Algarve, 8005-139 Faro, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2024,11,19]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Marreiros, C., Viegas, C., and Simes, D. 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