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-β1 (TGF-β1) and Wnt/β-catenin pathways are observed in the fibrosa, the stiffer layer of the leaflet, but their association is unknown. We elucidated the roles of β-catenin and extracellular matrix stiffness in TGF-β1-induced myofibroblast differentiation of valve interstitial cells (VICs). METHODS AND RESULTS: TGF-β1 induced rapid β-catenin nuclear translocation in primary porcine aortic VICs in vitro through TGF-β receptor I kinase. Degrading β-catenin pharmacologically or silencing it with small interfering RNA inhibited TGF-β1-induced myofibroblast differentiation without altering Smad2/3 activity. Conversely, increasing β-catenin availability with Wnt3A alone did not induce differentiation. However, combining TGF-β1 and Wnt3A caused greater myofibroblast differentiation than TGF-β1 treatment alone. Notably, in VICs grown on collagen-coated PA gels with physiological stiffnesses, TGF-β1-induced β-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, β-catenin, TGF-β1, and phosphorylated Smad2/3 in the fibrosa. CONCLUSIONS: Myofibroblast differentiation of VICs involves matrix stiffness-dependent crosstalk between TGF-β1 and Wnt signaling pathways and may explain in part why the stiffer fibrosa is more susceptible to disease.