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

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 K2 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 K2, 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.