Most research on valve calcification focuses on the early initiating molecular and cellular events. How cells in the valve interact with already existing calcification however is not well understood. Our lab discovered a direct in vivo correlation between long bone loss and accumulation of mineral in the aortic valve via inflammation. We identified that mechanical stress is required for osteogenic differentiation and matrix calcification in 3D cultured VIC, but is inhibited by co-culture with VEC. We discovered that healthy VEC actively maintain the quiescent phenotype of VIC in vivo and in vitro through the secretion of nitric oxide (NO), the first such homeostatic function identified in valves. Diseased VEC (via inflammation and/or oxidative stress) produce less NO, which when combined with inflammatory stimulus results in osteogenic differentiation of VIC and the deposition of calcified matrix. Calcific lesions in the valve contain hydroxyapatite, a mineral found in bone but in valves has distinctly different crystal structure. We have identified a novel correlation between the hydroxyapatite (HA) mineral crystallinity in progressively calcified human valves. We have replicated these different levels of HA crystallinity via engineered HA nanoparticles. We have further innovated a 3D culture approach that creates clinically relevant thickened 3D calcific lesions via cyclic stretch. This provides a unique platform to elucidate molecular and cellular mechanisms in advanced stages of CAVD. We are testing how HA presence and crystallinity independently affect VEC-VIC interactions towards CAVD. We then identify molecular intervention strategies that specifically target these later stage events to delay calcific progression using our established in vitro and in vivo approaches.