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Published on: November 20, 2017
Menaquinone-4 Accelerates Calcification of Human Aortic Valve Interstitial Cells in High-Phosphate Medium through PXR
Wei Yang1, Zaiqiang Yu1, Mari Chiyoya1
1Departments of Thoracic and Cardiovascular Surgery (W.Y., Z.Y., M.C., X.L., K.D., I.F.), Vascular Biology (T.I., K.S.), and Pharmacology (S.M., K.-I.F.), Hirosaki University Graduate School Medicine, Hirosaki, Japan; and Institute of Molecular Function, Saitama, Japan (M.T.).
Insights
Vitamin K2 (menaquinone-4) accelerates warfarin-induced aortic valve calcification in human cells. This process involves the pregnane X receptor-bone morphogenetic protein 2-alkaline phosphatase pathway, offering potential drug targets for aortic valve stenosis.
Area of Science:
- Biochemistry
- Cell Biology
- Cardiovascular Research
Background:
- Aortic valve stenosis (AVS) involves irreversible calcification, often treated surgically.
- Understanding the mechanisms of valve calcification is crucial for developing effective drug therapies.
- Warfarin (WFN) induces calcification in human aortic valve interstitial cells (HAVICs) in high inorganic phosphate (high-Pi) medium.
Purpose of the Study:
- To investigate whether menaquinone-4 (MK-4), a form of vitamin K2, inhibits WFN-induced HAVIC calcification.
- To elucidate the molecular pathways involved in MK-4-mediated effects on HAVIC calcification.
Main Methods:
- HAVICs from AVS patients were cultured in high-Pi medium with or without MK-4 and WFN.
- Alkaline phosphatase (ALP) activity and gene expression of bone morphogenetic protein 2 (BMP2) were measured.
- The role of pregnane X receptor (PXR) was assessed using PXR inhibitors and by measuring PXR activity.
Main Results:
- MK-4 dose-dependently accelerated WFN-induced HAVIC calcification and also accelerated calcification alone.
- MK-4 enhanced ALP activity and upregulated BMP2 gene expression in HAVICs.
- MK-4-induced calcification was suppressed by PXR inhibitors, and MK-4 increased PXR activity.
Conclusions:
- MK-4, or vitamin K2, accelerates calcification of HAVICs from AVS patients, similar to WFN.
- The PXR-BMP2-ALP pathway mediates MK-4's pro-calcific effect in HAVICs.
- This pathway represents a potential target for novel drug development for AVS treatment.
Abstract:
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.
