The Roles of Matrix Stiffness and ß-Catenin Signaling in Endothelial-to-Mesenchymal Transition of Aortic Valve Endothelial Cells.

Abstract

Valve stiffening is a hallmark of aortic valve stenosis caused by excess extracellular matrix accumulation by myofibroblasts. We aimed to elucidate whether matrix stiffness regulates endothelial-to-mesenchymal transition (EndMT) of adult valvular endothelial cells (VECs) to myofibroblasts as a mechanism to further promote valve fibrosis. In addition, we specifically examined the role of the Wnt/β-catenin signaling pathway in the development of myofibroblasts during EndMT, as Wnt/β-catenin signaling has been implicated in EndMT during heart development, is reactivated in valve disease, and is required for mechanically-regulated myofibrogenesis of valve interstitial cells. Clonally derived porcine VECs were cultured on soft (5kPa) or stiff (50kPa) silicone Sylgard 527 substrates and treated with transforming growth factor (TGF)-β1 to induce EndMT. Immunofluorescent staining revealed that TGF-β1 preferentially promoted EndMT in VECs on stiffer substrates, evidenced by a decrease in the endothelial marker VE-cadherin and an increase in the myofibroblast marker α-smooth muscle actin (α-SMA). These changes were accompanied by β-catenin nuclear localization both in vitro and in vivo, assessed by immunostaining. Degradation of β-catenin with endostatin reduced VEC myofibroblast transition, as indicated by decreased α-SMA fiber expression. We conclude that TGF-β1-induced EndMT in aortic VECs is dependent on matrix stiffness and Wnt/β-catenin signaling promotes myofibrogenesis during EndMT.