Zhong Aileen, Mirzaei Zahra, Simmons Craig A
Translational Biology and Engineering Program, Ted Rogers Centre for Heart Research, 661 University Avenue, Toronto, ON, M5G 1M1, Canada.
Institute of Biomaterials and Biomedical Engineering, University of Toronto, 164 College Street, Toronto, ON, M5S 3G9, Canada.
Cardiovasc Eng Technol. 2018 Jun;9(2):158-167. doi: 10.1007/s13239-018-0363-0. Epub 2018 May 14.
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 (5 kPa) or stiff (50 kPa) 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.
瓣膜硬化是由肌成纤维细胞积累过多细胞外基质引起的主动脉瓣狭窄的一个标志。我们旨在阐明基质硬度是否调节成年瓣膜内皮细胞（VECs）向肌成纤维细胞的内皮-间充质转化（EndMT），以此作为进一步促进瓣膜纤维化的一种机制。此外，我们特别研究了Wnt/β-连环蛋白信号通路在EndMT过程中肌成纤维细胞发育中的作用，因为Wnt/β-连环蛋白信号在心脏发育过程中的EndMT中发挥作用，在瓣膜疾病中被重新激活，并且是瓣膜间质细胞机械调节性肌成纤维细胞生成所必需的。将克隆来源的猪VECs培养在柔软（5kPa）或坚硬（50kPa）的硅胶Sylgard 527底物上，并用转化生长因子（TGF）-β1处理以诱导EndMT。免疫荧光染色显示，TGF-β1优先促进较硬底物上VECs的EndMT，这通过内皮标志物VE-钙黏蛋白的减少和成肌纤维细胞标志物α-平滑肌肌动蛋白（α-SMA）的增加得以证明。通过免疫染色评估，这些变化在体外和体内均伴有β-连环蛋白的核定位。用内皮抑素降解β-连环蛋白可减少VEC的成肌纤维细胞转化，α-SMA纤维表达降低表明了这一点。我们得出结论，TGF-β1诱导的主动脉VECs的EndMT依赖于基质硬度，并且Wnt/β-连环蛋白信号在EndMT过程中促进肌成纤维细胞生成。
