Download Neil Venardos, Michael J. Weyant, Thomas B. Reece, Xianzhong

Warfarin Induces Calcification of the Aortic Valve via ERK1/2
Neil Venardos, Michael J. Weyant, Thomas B. Reece, Xianzhong Meng, David A. Fullerton
Department of Surgery, Division of Cardiothoracic Surgery, University of Colorado
INTRODUCTION
RESULTS
• Approximately 3 million people in the United States currently take warfarin
• Warfarin has been identified as an instigator of calcification in blood vessels
• Long term use of this drug is associated with aortic valve calcification
• Aortic valve interstitial cells (AVICs) have been implicated in the
pathogenesis of aortic stenosis
• In response to pro-inflammatory stimuli, AVICs adopt an inflammatory
phenotype, characterized by the production of bone-forming proteins such as
runt-related transcription factor-2 (Runx-2) and Osterix (Osx)
HYPOTHESES
•
Warfarin, but not heparin or dabigatran, induces an osteogenic phenotypic change
in both normal and diseased human AVICs
Warfarin is non-toxic to human AVICs at these doses
SUMMARY AND CONCLUSIONS
Warfarin induces an osteogenic phenotypic change in human AVICs
PURPOSES
1. Warfarin induces an osteogenic phenotypic
transformation in isolated human AVICs. These
osteogenic changes were not observed with other
anticoagulants such as heparin or dabigatran
• 1) To determine the effect of warfarin on AVIC osteogenic protein expression
• 2) To identify the signaling pathway by which this effect is mediated
METHODS
• Normal AVICs were isolated from normal aortic valves excised from the
explanted hearts of four patients undergoing cardiac transplantation
Warfarin induces β-catenin expression and nuclear translocation in diseased, but not
normal human AVICs
• Diseased AVICs were isolated from excised aortic valve leaflets from four
patients undergoing aortic valve replacement
• Cells were treated with warfarin (10µM), heparin (0.1, 1.0, 10U/mL), and
dabigatran (10, 500, 10000ng/mL) for 48 hours
• After these treatments, cell lysates were analyzed for levels of Runx-2, Osx,
and β-catenin using immunoblotting. ALP and calcium staining were
performed as well, and immunofluorescence was used to assess β-catenin
nuclear translocation/activation
• ERK1/2 and β-catenin pathways (lipoprotein receptor-related protein-6;
LRP6) were inhibited using inhibitors PD98059 and Dkk1, respectively.
Western blotting was used to analyze runx-2 expression.
• LDH assay was performed to assess warfarin toxicity in human AVICs
2. This pro-osteogenic effect is mediated by ERK1/2
signaling in both normal and diseased human
AVICs, while β-catenin pathway signaling mediates
warfarin’s pro-osteogenic effects only in diseased
human AVICs.
These results implicate warfarin in aortic valve
calcification and highlight a potential mechanism for
warfarin-induced aortic stenosis.
Acknowledgement
ERK1/2 inhibition prevents warfarin-induced runx-2 expression in both normal and
diseased AVICs, while β-catenin pathway inhibition prevents this response only in
diseased AVICs
We would like to acknowledge LiHua Ao for her assistance
with the generation of immunofluorescent images used in
this project.