Background. The molecular mechanisms underlying aortic valve calcification are poorly understood. Here, we aimed to identify the master regulators of calcification by comparison of genes in valve interstitial cells (VICs) with calcified and noncalcified aortic valves.Methods. Calcified aortic valves were surgically excised from patients with aortic valve stenosis who required aortic valve replacements. Noncalcified and calcified sections were obtained from aortic valve leaflets. Collagenase-digested tissues were seeded into dishes, and VICs adhering to the dishes were cultured for 3 weeks, followed by comprehensive gene expression analysis. Functional analyses of identified proteins were performed by in vitro calcification assays. Tissue localization was determined by immunohistochemical staining for normal (n = 11) and stenotic valves (n = 30).Results. We found 87 genes showing greater than a twofold change in calcified tissues. Among these genes, 68 were downregulated and 19 were upregulated. Cyclooxygenase-1 (COX1) messenger RNA and protein levels were upregulated in VICs from calcified tissues. The COX1 messenger RNA and protein levels in VICs were also strongly increased by stimulation with osteoblast differentiation medium. These were VIC-specific phenotypes and were not observed in other cell types. Immunohistochemical staining revealed that COX1-positive VICs were specifically localized in the calcified area of aortic valve tissues.Conclusions. The VIC-specific COX1 overexpression played a crucial role in calcification by promoting osteoblast differentiation in aortic valve tissues. (Ann Thorac Surg 2020;110:40-9) (C) 2020 by The Society of Thoracic Surgeons