Quercetin attenuates warfarin-induced vascular calcification in vitro independently from matrix Gla protein

Abstract

Warfarin can stimulate vascular calcification in vitro via activation of β-catenin signaling and/or inhibition of matrix Gla protein (MGP) carboxylation. Calcification was induced in vascular smooth muscle cells (VSMCs) with therapeutic levels of warfarin in normal calcium and clinically acceptable phosphate levels. Although TGF/BMP and PKA pathways are activated in calcifying VSMCs, pharmacologic analysis reveals that their activation is not contributory. However, β-catenin activity is important because inhibition of β-catenin with shRNA or bioflavonoid quercetin prevents calcification in primary human VSMCs, rodent aortic rings, and rat A10 VSMC line. In the presence of quercetin, reactivation of β-catenin using the glycogen synthase kinase-3β (GSK-3β) inhibitor LiCl restores calcium accumulation, confirming that quercetin mechanism of action hinges on inhibition of the β-catenin pathway. Calcification in VSMCs induced by 10 μm warfarin does not associate with reduced levels of carboxylated MGP, and inhibitory effects of quercetin do not involve induction of MGP carboxylation. Further, down-regulation of MGP by shRNA does not alter the effect of quercetin. These results suggest a new β-catenin-targeting strategy to prevent vascular calcification induced by warfarin and identify quercetin as a potential therapeutic in this pathology.

FIGURE 1.

Attenuation of warfarin-induced VSMC calcification by quercetin. A and B, calcium content in A10 cells (A) or human VSMCs (B) cultured in promineralizing medium containing 1.6 mm P_i and 10 μm warfarin (black bar) supplemented with quercetin (gray bars), as indicated below graphs (n = 3). C, von Kossa stain for mineralized matrix deposition (left) and calcium content in rat aortic rings (right) cultured in promineralizing medium in the absence (black bar) or presence (gray bar) of 100 μm quercetin (random mix of 4–5 2 mm aortic rings from 3 rats used in each condition), *, p < 0.05; **, p < 0.01.

FIGURE 2.

Identification of β-catenin (b-cat), PKA, and TGF/BMP signaling pathways as potential mediators of warfarin-induced calcification in VSMCs. A–C, activity of specified luciferase reporters (above graphs) analyzed in A10 cells induced to calcify for 8 days in promineralizing medium (1.6 mm inorganic phosphate and 10 μm warfarin) with or without 100 μm quercetin (A); in noncalcifying A10 cells cultured for 6 days in the presence of individual supplements (B); and in noncalcifying A10 cells cultured for only 3 days in promineralizing medium with or without 100 μm quercetin (C). *, p < 0.05; **, p < 0.01.

FIGURE 3.

Regulation of intracellular VSMC signaling by either warfarin or quercetin. A and B, activity of indicated luciferase reporters (above graphs) analyzed in A10 cells cultured in 1.6 mm P_i supplemented with 10 μm warfarin (A) or 100 μm quercetin (B). C, luciferase activity for indicated signaling pathways (above graphs) in A10 cells cultured in promineralizing medium with the pharmacologic activators of PKA (forskolin) or β-catenin (b-cat) (Wnt3a) (n = 6). *, p < 0.05; **, p < 0.01.

FIGURE 4.

Role of β-catenin, PKA, and BMP signaling in warfarin-induced vascular calcification. Total calcium levels in A10 cells cultured in promineralizing medium for 8 days to induce calcification were determined. A–C, cells were treated with the PKA inhibitor H-89 (A), with the BMP antagonist Noggin (B), or with lentivirus expressing shRNA for rat β-catenin (bcat-sh) (C) (n = 4). Luciferase activity for indicated signaling pathways is shown in the right bar graphs (n = 6). scr-sh, scrambled shRNA. *, p < 0.05; **, p < 0.01.

FIGURE 5.

LiCl rescues warfarin effects on β-catenin signaling and vascular calcification in the presence of quercetin. A10 cells were cultured for 6 days in of promineralizing medium (1.6 mm P_i and 10 μm warfarin) and 100 μm quercetin (to block calcification), supplemented with increasing concentrations of LiCl from 10–500 nm (hatched bars). A, luciferase assay for β-catenin activity (n = 4). B, matrix calcium levels (n = 4). Bars show -fold induction as compared with cells cultured in 1.6 mm P_i alone. *, p < 0.05; **, p < 0.01.

FIGURE 6.

Quercetin-mediated inhibition of calcification is independent from MGP. A, GlaMGP expression determined by Western blot (upper panel) and calcified matrix deposition (lower panel) in A10 cells cultured in procalcifying medium (+, 10 μm warfarin; ++, 100 μm warfarin) in the presence or absence of quercetin or vitamin K (VitK) as indicated. NS, not significant. B, down-regulation of MGP expression in A10 cells with lentivirus expressing shRNA for rat MGP (MGPsh) as determined by Western blot (upper panel) does not affect the ability of quercetin to attenuate warfarin-induced calcification (lower panel; n = 3). *, p < 0.05; **, p < 0.01.