Abstract Tu0072: Mechanisms of Inorganic Phosphate-Induced Vascular Calcification in Primary Human Aortic Smooth Muscle Cells

Background: Vascular calcification (VC) is a pathological process associated with aging, atherosclerosis, metabolic syndrome, and chronic kidney disease. It involves vascular smooth muscle cells (VSMCs) transforming into osteoblast-like cells, leading to mineral deposition within the vascular wall. Transcription factors such as runt-related transcription factor 2 (RUNX2) and bone matrix-associated proteins, including sclerostin (SOST), osteoprotegerin, and osteopontin, play crucial roles in this process. Cannabidiol (CBD), a cannabis-derived compound, has demonstrated cardiovascular benefits, but its impact on VC remains unclear. Aims: This study aimed to (1) develop a human aortic smooth muscle cell (HASMC) VC model to investigate the role of bone-related proteins in calcification, (2) analyze protein expression changes in the VC model using proteomic techniques, and (3) evaluate the effects of CBD on the osteogenic transition of HASMCs under calcifying conditions. Methods: HASMCs were cultured under calcifying conditions with inorganic phosphate and calcium to induce VC. Calcification was quantified using Alizarin Red S staining, while Western blotting and enzymatic assays assessed bone-related protein expression, including alkaline phosphatase and RUNX2. Proteomic analysis identified dysregulated pathways and proteins associated with VC. CBD cytotoxicity was evaluated at concentrations up to 8 µM, and apoptosis assays were performed in both calcifying and non-calcifying conditions. Results: Calcifying conditions induced significant phenotypic changes in HASMCs, characterized by reduced smooth muscle marker expression and calcium nodule formation. Enzymatic assays revealed increased alkaline phosphatase activity, while proteomic analysis identified upregulation of cardiovascular-related proteins, including smoothelin, succinate dehydrogenase complex flavoprotein subunit A, and Rho GTPase–activating protein 17. CBD treatment unexpectedly increased calcium deposits and apoptotic activity in calcified HASMCs, suggesting a potentially adverse role in the calcification process. Conclusion: Elevated phosphate levels drive osteogenic changes in HASMCs, contributing to VC progression. While CBD has shown promise in cardiovascular therapy, its exacerbation of calcium deposition in this model highlights the need for caution. These findings underscore the importance of understanding VC mechanisms and the therapeutic implications of CBD in vascular health.