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AIMS Tetrahydrobiopterin (BH4) is a critical determinant of the biological function of endothelial nitric oxide synthase. The present study was to investigate the role of valvular endothelial cell (VEC)-derived BH4 in aortic valve calcification. METHODS AND RESULTS Plasma and aortic valve BH4 concentrations and the BH4:BH2 ratio were significantly lower in calcific aortic valve disease patients than in controls. There was a significant decrease of the two key enzymes of BH4 biosynthesis, guanosine 5'-triphosphate cyclohydrolase I (GCH1) and dihydrofolate reductase (DHFR), in calcified aortic valves compared with the normal ones. Endothelial cell-specific deficiency of Gch1 in Apoe-/- (Apoe-/-Gch1fl/flTie2Cre) mice showed a marked increase in transvalvular peak jet velocity, calcium deposition, runt-related transcription factor 2 (Runx2), dihydroethidium (DHE), and 3-nitrotyrosine (3-NT) levels in aortic valve leaflets compared with Apoe-/-Gch1fl/fl mice after a 24-week western diet (WD) challenge. Oxidized LDL (ox-LDL) induced osteoblastic differentiation of valvular interstitial cells (VICs) co-cultured with either si-GCH1- or si-DHFR-transfected VECs, while the effects could be abolished by BH4 supplementation. Deficiency of BH4 in VECs caused peroxynitrite formation increase and 3-NT protein increase under ox-LDL stimulation in VICs. SIN-1, the peroxynitrite generator, significantly up-regulated alkaline phosphatase (ALP) and Runx2 expression in VICs via tyrosine nitration of dynamin-related protein 1 (DRP1) at Y628. Finally, folic acid (FA) significantly attenuated aortic valve calcification in WD-fed Apoe-/- mice through increasing DHFR and salvaging BH4 biosynthesis. CONCLUSION The reduction in endothelial-dependent BH4 levels promoted peroxynitrite formation, which subsequently resulted in DRP1 tyrosine nitration and osteoblastic differentiation of VICs, thereby leading to aortic valve calcification. Supplementation of FA in diet attenuated hypercholesterolaemia-induced aortic valve calcification by salvaging BH4 bioavailability. KEY QUESTION Tetrahydrobiopterin (BH4) insufficiency-dependent endothelial nitric oxide synthase dysfunction mediates endothelial dysfunction and leads to vascular diseases. The role of BH4 in aortic valve calcification has not been explored. KEY FINDING Tetrahydrobiopterin and guanosine 5'-triphosphate cyclohydrolase I are reduced in calcified aortic valves. Endothelial cell-specific deficiency of Gch1 promotes murine aortic valve calcification. Tetrahydrobiopterin reduction in valvular endothelial cells promotes valvular interstitial cells (VICs) osteogenesis via ONOO- formation. Folic acid suppresses murine aortic valve calcification by salvaging BH4 bioavailability. TAKE-HOME MESSAGE Tetrahydrobiopterin is closely associated with the development of aortic valve calcification. Valvular endothelial cells dysfunction leads to ONOO- formation, resulting in osteoblastic differentiation of VICs. Salvaging BH4 bioavailability may be a potential therapeutic strategy for aortic valve calcification.