Menaquinone-4 Accelerates Calcification of Human Aortic Valve Interstitial Cells in High-Phosphate Medium through PXR
Menaquinone-4 Accelerates Calcification of Human Aortic Valve Interstitial Cells in High-Phosphate Medium through PXR
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DOI：
10.1124/jpet.119.263160
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发表时间：
2019-12
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影响因子：
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通讯作者：
Wei Yang;Zaiqiang Yu;Mari Chiyoya;Xu Liu;K. Daitoku;S. Motomura;T. Imaizumi;I. Fukuda;K. Furukawa;M. Tsuji;K. Seya
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作者：
Wei Yang;Zaiqiang Yu;Mari Chiyoya;Xu Liu;K. Daitoku;S. Motomura;T. Imaizumi;I. Fukuda;K. Furukawa;M. Tsuji;K. Seya
最近，我们证实，在人主动脉瓣间质细胞（HAVICs）分离自主动脉瓣狭窄（AVS）患者，钙化诱导在高无机磷酸盐（高Pi）培养基华法林（WFN）。由于WFN被认为是维生素K拮抗剂，通过维生素K循环减少血凝块的形成，我们假设维生素K调节WFN诱导的HAVIC钙化。在这里，我们试图确定在高Pi培养基中WFN诱导的HAVIC钙化是否被动物中最常见的维生素K2形式甲基萘醌-4（MK-4）抑制。将从患有AVS的患者获得的HAVIC在含有10%FBS的α-改良的Eagle培养基中培养，并且当细胞达到80%-90%汇合时，将它们在存在或不存在MK-4和WFN的情况下在高Pi培养基（3.2mM Pi）中进一步培养7天。有趣的是，在高Pi培养基中，MK-4剂量依赖性地加速了WFN诱导的HAVIC钙化，并且单独使用（10 nM）时也加速了钙化。此外，MK-4增强HAVIC中碱性磷酸酶（ALP）活性，MK-4治疗7天显著上调钙化标志物骨形态发生蛋白2（BMP 2）的基因表达。值得注意的是，MK-4诱导的钙化被两种甾烷X受体（PXR）抑制剂酮康唑和香豆雌酚有效抑制;相反，MK-4使PXR活性微弱增加，但具有统计学显著性和剂量依赖性。最后，在生理Pi培养基中，MK-4增加BMP 2基因表达并加速过量BMP 2（30 ng/ml）诱导的HAVIC钙化。这些结果表明MK-4（即维生素K2）通过PXR-BMP 2-ALP途径加速WFN等AVS患者的HAVIC的钙化。意义声明对于不可逆瓣膜钙化导致的主动脉瓣狭窄（AVS），最有效的治疗方法是外科主动脉瓣或经导管主动脉瓣置换术，但由于其侵入性，约20%的患者被认为不适合。对于有效的药物治疗策略的AVS，主动脉瓣钙化的机制必须阐明。在这里，我们发现甲基萘醌-4加速华法林诱导的AVS患者人主动脉瓣间质细胞在高无机磷酸盐培养基中的钙化;这种作用是由β-内酰胺酶X受体-骨形态发生蛋白2-碱性磷酸酶信号传导介导的，这可能是新药开发的目标。
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.