Warfarin Induces Calcification of the Aortic Valve Through Extracellular Signal-regulated Kinase 1/2 and ß-catenin Signaling.

ABSTRACT

BACKGROUND: Recent clinical evidence suggests an association between warfarin use and calcification of the aortic valve. We sought to determine the effect of warfarin on aortic valve interstitial cell (AVIC) osteogenic protein expression and the signaling pathways by which this effect is mediated. METHODS: Human AVICs were isolated from normal aortic valves of patients undergoing cardiac transplantation, whereas diseased AVICs were isolated from patients undergoing aortic valve replacement for aortic stenosis. AVICs were treated with various anticoagulants, and osteogenic protein expression was evaluated using immunoblotting. Phosphorylation of lipoprotein receptor-related protein 6 (LRP6) and extracellular signal-regulated kinase 1/2 (ERK1/2) was evaluated after treatment with warfarin. AVICs were pretreated with LRP6 inhibitor dkk1 and ERK1/2 inhibitor PD98059 followed by treatment with warfarin, and osteogenic protein expression was evaluated. RESULTS: Warfarin, but not heparin or dabigatran, significantly increased Runx-2 and Osx expression in both normal and diseased human AVICs. Upregulation of ß-catenin protein expression and nuclear translocation occurred in diseased AVICs but not normal AVICs after warfarin treatment. Warfarin induced phosphorylation of LRP6 in diseased AVICs only and phosphorylation of ERK1/2 in both normal and diseased AVICs. LRP6 inhibition attenuated warfarin-induced Runx-2 expression in diseased AVICs. ERK1/2 inhibition attenuated warfarin-induced Runx-2 expression in both normal and diseased AVICs. CONCLUSIONS: Warfarin induces osteogenic activity in normal and diseased isolated human AVICs. This effect is mediated by ERK1/2 in both diseased and normal AVICs, but in diseased AVICs ß-catenin signaling also plays a role. These results implicate the role of warfarin in aortic valve calcification and highlight potential mechanisms for warfarin-induced aortic stenosis.