Warfarin calcifies human aortic valve interstitial cells at high-phosphate conditions via pregnane X receptor

Abstract

Warfarin, a vitamin K antagonist, is the most common anticoagulant used to prevent thromboembolisms associated with atrial fibrillation or following valvular surgery. Although several studies have revealed that long-term warfarin use accelerates aortic valve calcification and the development of aortic stenosis (AS), the detailed mechanism for this phenomenon remains unclear. Therefore, our aim was twofold: to establish the conditions for warfarin-induced calcification of human aortic valve interstitial cells (HAVICs) using high-inorganic phosphate (Pi) conditions and to investigate the underlying mechanism. We prepared and cultured HAVICs from aortic valves affected by calcific aortic valve stenosis (AS group) and aortic valves affected by aortic regurgitation but without any signs of calcification (non-AS group). Under Pi concentrations of 3.2 mM, warfarin significantly increased the calcification and alkaline phosphatase (ALP) activity of AS but not non-AS group HAVICs. Furthermore, gene expression of bone morphogenetic protein 2 (BMP2), a calcigenic marker, was significantly increased following 7 days of warfarin treatment. Warfarin-induced calcification of AS group HAVICs at 3.2 mM Pi was significantly inhibited by dorsomorphin, a Smad inhibitor, and the pregnane X receptor (PXR) inhibitors, ketoconazole and coumestrol, but was unaffected by SN-50, an NF-κB inhibitor. Warfarin was also able to increase BMP2 gene expression at a physiological Pi concentration (1.0 mM). Furthermore, excess BMP2 (30 ng/mL) facilitated warfarin-induced ALP upregulation and HAVIC calcification, an effect which was significantly reduced in the presence of coumestrol. Together, our results suggest that warfarin accelerates calcification of HAVICs from AS patients via the PXR–BMP2–ALP pathway.

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Change history

  • 17 February 2020

    In the original publication of the article, part of Fig.��1 was published incorrectly.

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Acknowledgements

We are grateful to Prof. Yasuyuki Ishibashi of Hirosaki University, for his consistent help, support, and encouragement. We thank Emma Andrew, PhD, from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.

Funding

This work was supported by grants from JSPS KAKENHI (Grant numbers 24590310, 16K10619, 16K10449, and 17K10916); grants-in-aid of The Cardiovascular Research Fund, Tokyo, Japan; and the Hirosaki University Educational Improvement and Promotional Aid, Hirosaki, Japan.

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Supplemental Figure 1. PXR and CYP3A4 gene expression on warfarin-induced calcification of HAVICs.

HAVICs were cultured in α-MEM containing 10% fetal bovine serum (FBS). After reaching 90% confluency (Day 0), HAVICs were further cultured in α-MEM in the absence or presence of inorganic phosphate (Pi) concentrations (3.2 mM) for seven days (Day 7). A: PXR gene expression in AS group HAVICs were measured on day 7. All ratios were calculated versus the control group on day 0. Relative gene expression levels were determined by normalizing measured values to those obtained for the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (G3PDH). White bar: untreated cells; blue bar: cells treated with high (3.2mM) Pi (+); black bar: cells treated with 1 µM WFN (+); red bars: cells treated with 1 µM WFN (+) and high (3.2mM) Pi (+). Bars represent the mean ± SEM (n = 3). B: PXR mRNA expression was compared between AS and non-AS group HAVICs cultured for three days at physiological Pi concentrations (1.0 mM) in the absence or presence of warfarin. All ratios were calculated versus the control group on day 0. Relative gene expression levels were determined by normalizing measured values to those obtained for the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (G3PDH). White bar: untreated cells, black bar: cells treated with 1 µM warfarin (WFN, (+)) only. Bars represent the mean ± SEM (n = 3). C: CYP3A4 gene expression in AS group HAVICs were measured on day 7. All ratios were calculated versus the control group on day 0. Relative gene expression levels were determined by normalizing measured values to those obtained for the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (G3PDH). White bar: untreated cells; blue bar: cells treated with high (3.2mM) Pi (+); black bar: cells treated with 1 µM WFN (+); red bars: cells treated with 1 µM WFN (+) and high (3.2mM) Pi (+). Bars represent the mean ± SEM (n = 3). D: CYP3A4 mRNA expression was compared between AS and non-AS group HAVICs cultured for three days at physiological Pi concentrations (1.0 mM) in the absence or presence of warfarin. All ratios were calculated versus the control group on day 0. Relative gene expression levels were determined by normalizing measured values to those obtained for the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (G3PDH). White bar: untreated cells, black bar: cells treated with 1 µM warfarin (WFN, (+)) only. Bars represent the mean ± SEM (n = 3). A significant difference (P < 0.05) when compared with untreated control AS cells is indicated with ‘#’

Supplemental Figure 2. SR12813-induced calcification of HAVICs from AS patients at high Pi (3.2 mM).

HAVICs were cultured in α-MEM containing 10% fetal bovine serum (FBS). After reaching 90% confluency (Day 0), HAVICs were further cultured in medium containing inorganic phosphate (Pi) concentrations (3.2 mM) for seven days. A: Typical images of Alizarin Red S staining of HAVICs obtained from AS patients in the presence or absence of 3 µM SR-12813 in high Pi medium. B: BMP2 gene expression in AS group HAVICs were measured on day 7. All ratios were calculated versus the control group on day 0. Relative gene expression levels were determined by normalizing measured values to those obtained for the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (G3PDH). White bar: untreated cells; blue bar: cells treated with high (3.2mM) Pi (+); black bar: cells treated with 3 µM SR-12813 (+); red bars: cells treated with 3 µM SR-12813 (+) and high (3.2mM) Pi (+). Bars represent the mean ± SEM (n = 3). A significant difference (P < 0.05) when compared with untreated control AS group cells, AS group cells treated with high Pi (3.2 mM) only, and AS group cells treated with SR-12813 (3 µM) only on day 7 is denoted by ‘#’. C: CYP3A4 gene expression in AS group HAVICs were measured on day 7. All ratios were calculated versus the control group on day 0. Relative gene expression levels were determined by normalizing measured values to those obtained for the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (G3PDH). White bar: untreated cells; blue bar: cells treated with high (3.2mM) Pi (+); black bar: cells treated with 3 µM SR-12813 (+); red bars: cells treated with 3 µM SR-12813 (+) and high (3.2mM) Pi (+). Bars represent the mean ± SEM (n = 3)

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Yu, Z., Seya, K., Chiyoya, M. et al. Warfarin calcifies human aortic valve interstitial cells at high-phosphate conditions via pregnane X receptor. J Bone Miner Metab 37, 944–956 (2019). https://doi.org/10.1007/s00774-019-01001-3

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Keywords

  • Aortic valve stenosis
  • Pregnane X receptor
  • Calcification
  • Warfarin
  • Bone morphogenetic protein 2