β-Catenin Mediates Mechanically Regulated, Transforming Growth Factor-β1-Induced Myofibroblast Differentiation of Aortic Valve Interstitial Cells

Objective-In calcific aortic valve disease, myofibroblasts and activation of the transforming growth factor-beta 1 (TGF-beta 1) and Wnt/beta-catenin pathways are observed in the fibrosa, the stiffer layer of the leaflet, but their association is unknown. We elucidated the roles of beta-catenin and extracellular matrix stiffness in TGF-beta 1-induced myofibroblast differentiation of valve interstitial cells (VICs). Methods and Results-TGF-beta 1 induced rapid beta-catenin nuclear translocation in primary porcine aortic VICs in vitro through TGF-beta receptor I kinase. Degrading beta-catenin pharmacologically or silencing it with small interfering RNA inhibited TGF-beta 1-induced myofibroblast differentiation without altering Smad2/3 activity. Conversely, increasing beta-catenin availability with Wnt3A alone did not induce differentiation. However, combining TGF-beta 1 and Wnt3A caused greater myofibroblast differentiation than TGF-beta 1 treatment alone. Notably, in VICs grown on collagen-coated PA gels with physiological stiffnesses, TGF-beta 1-induced beta-catenin nuclear translocation and myofibroblast differentiation occurred only on matrices with fibrosa-like stiffness, but not ventricularis-like stiffness. In diseased aortic valves from pigs fed an atherogenic diet, myofibroblasts colocalized with increased protein expression of Wnt3A, beta-catenin, TGF-beta 1, and phosphorylated Smad2/3 in the fibrosa. Conclusion-Myofibroblast differentiation of VICs involves matrix stiffness-dependent crosstalk between TGF-beta 1 and Wnt signaling pathways and may explain in part why the stiffer fibrosa is more susceptible to disease. (Arterioscler Thromb Vasc Biol. 2011;31:590-597.)