Endothelial cell-derived tetrahydrobiopterin prevents aortic valve calcification.

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 ^−/− Gch1 ^fl/fl Tie2 ^Cre) 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 ^−/− Gch1 ^fl/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. [ABSTRACT FROM AUTHOR]