Department of Thoracic and Cardiovascular Surgery, Hirosaki University Graduate School of Medicine, Hirosaki, Japan.
Department of Vascular Biology, Hirosaki University Graduate School of Medicine, Hirosaki, Japan.
J Bone Miner Metab. 2019 Nov;37(6):944-956. doi: 10.1007/s00774-019-01001-3. Epub 2019 Apr 8.
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.
华法林是一种维生素 K 拮抗剂，是预防与心房颤动或心脏瓣膜手术后相关的血栓栓塞最常用的抗凝剂。尽管多项研究表明，长期使用华法林会加速主动脉瓣钙化和主动脉瓣狭窄（AS）的发展，但这种现象的详细机制仍不清楚。因此，我们的目的有两个：一是用高无机磷（Pi）条件建立华法林诱导人主动脉瓣间质细胞（HAVICs）钙化的条件，并探讨其潜在机制。我们从受钙化性主动脉瓣狭窄（AS 组）影响的主动脉瓣和不受任何钙化迹象影响的主动脉瓣（非 AS 组）中制备并培养 HAVICs。在 Pi 浓度为 3.2 mM 时，华法林显著增加了 AS 组而非非 AS 组 HAVICs 的钙化和碱性磷酸酶（ALP）活性。此外，华法林处理 7 天后，骨形态发生蛋白 2（BMP2）的基因表达，一种成骨标记物，显著增加。3.2 mM Pi 下华法林诱导的 AS 组 HAVICs 钙化被 Smad 抑制剂 dorsomorphin 和孕烷 X 受体（PXR）抑制剂酮康唑和香豆素显著抑制，但不被 NF-κB 抑制剂 SN-50 影响。华法林还能在生理 Pi 浓度（1.0 mM）下增加 BMP2 基因表达。此外，过量的 BMP2（30 ng/mL）促进了华法林诱导的 ALP 上调和 HAVIC 钙化，而 coumestrol 的存在显著降低了这种作用。综上所述，我们的结果表明，华法林通过 PXR-BMP2-ALP 途径加速 AS 患者 HAVICs 的钙化。
