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Recently, we confirmed that in human aortic valve interstitial cells (HAVICs) isolated from patients with aortic valve stenosis (AVS), calcification is induced in high inorganic phosphate (high-Pi) medium by warfarin (WFN). Because WFN is known as a vitamin K antagonist, reducing the formation of blood clots by vitamin K cycle, we hypothesized that vitamin K regulates WFN-induced HAVIC calcification. Here, we sought to determine whether WFN-induced HAVIC calcification in high-Pi medium is inhibited by menaquinone-4 (MK-4), the most common form of vitamin K 2 in animals. HAVICs obtained from patients with AVS were cultured in α -modified Eagle's medium containing 10% FBS, and when the cells reached 80%-90% confluency, they were further cultured in the presence or absence of MK-4 and WFN for 7 days in high-Pi medium (3.2 mM Pi). Intriguingly, in high-Pi medium, MK-4 dose-dependently accelerated WFN-induced HAVIC calcification and also accelerated the calcification when used alone (at 10 nM). Furthermore, MK-4 enhanced alkaline phosphatase (ALP) activity in HAVICs, and 7 days of MK-4 treatment markedly upregulated the gene expression of the calcification marker bone morphogenetic protein 2 (BMP2). Notably, MK-4-induced calcification was potently suppressed by two pregnane X receptor (PXR) inhibitors, ketoconazole and coumestrol; conversely, PXR activity was weakly increased, but in a statistically significant and dose-dependent manner, by MK-4. Lastly, in physiologic-Pi medium, MK-4 increased BMP2 gene expression and accelerated excess BMP2 (30 ng/ml)-induced HAVIC calcification. These results suggest that MK-4, namely vitamin K 2 , accelerates calcification of HAVICs from patients with AVS like WFN via PXR-BMP2-ALP pathway. SIGNIFICANCE STATEMENT: For aortic valve stenosis (AVS) induced by irreversible valve calcification, the most effective treatment is surgical aortic or transcatheter aortic valve replacement, but ∼20% of patients are deemed unsuitable because of its invasiveness. For effective drug treatment strategies for AVS, the mechanisms underlying aortic valve calcification must be elucidated. Here, we show that menaquinone-4 accelerates warfarin-induced calcification of AVS-patient human aortic valve interstitial cells in high inorganic phosphate medium; this effect is mediated by pregnane X receptor-bone morphogenetic protein 2-alkaline phosphatase signaling, which could be targeted for novel drug development.

Menaquinone-4 Accelerates Calcification of Human Aortic Valve Interstitial Cells in High-Phosphate Medium through PXR