Roles of Matrix Mechanics in Regulating Aortic Valve Interstitial Cell Pathological Differentiation

Calcific aortic valve disease (CAVD) is associated with increased presence of myofibroblasts, osteoblastic cells and, occasionally, adipocytes and chondrocytes in lesions. The ectopic cell types in diseased valves may be elaborated by an unidentified multipotent progenitor subpopulation within the valve interstitial cells (VICs) that populate the valve interstitium. Notably, lesions form preferentially in the fibrosa layer, the stiffer layer of the valve leaflet. It has been shown that differentiation of VICs to myofibroblasts and osteoblasts is modulated by matrix stiffness. However, the molecular mechanisms involved in mediating stiffness-dependent mechanotransduction remain obscure. The objectives of this thesis were: (1) to determine whether VICs contain a subpopulation of multipotent mesenchymal progenitor cells and to measure the frequencies of the mesenchymal progenitors and osteoprogenitors; (2) to determine the role of β-catenin and matrix stiffness in transforming growth factor-β1 (TGF-β1)-induced myofibroblast differentiation of VICs; and (3) to preliminarily investigate the involvement of four and a half LIM domains protein 2 (FHL2) in CAVD and stiffness-dependent mechanotransduction downstream of RhoA in VICs. Firstly, VICs were found to contain a subpopulation of mesenchymal progenitors that are inducible to osteogenic, myofibroblastic, adipogenic, and chondrogenic lineages. The frequencies of mesenchymal progenitors and osteoprogenitors were significantly higher than other reported sources. Secondly, it was demonstrated that β-catenin is required in TGF-β1-induced, matrix stiffness-regulated myofibroblast differentiation. Notably, TGF-β1 was only able to induce β-catenin nuclear translocation and myofibroblast differentiation on matrices with fibrosa-like stiffness, but not on matrices with ventricularis-like stiffness. Thirdly, FHL2 was found to be upregulated and colocalized with runt-related transcriptional factor 2 (Runx2) in lesions in the fibrosa layer of diseased valves, suggesting its role in osteogenic processes in CAVD. Notably, increasing matrix stiffness increased FHL2 nuclear translocation and RhoA activity in VICs. Preliminary data showed that matrix stiffness regulates FHL2 nuclear translocation via RhoA activity. These results suggest that differentiation of the rich valve progenitor subpopulation, regulated by both mechanical and biochemical cues, may contribute to the preferential occurrence of ectopic cell types in the fibrosa in CAVD. More broadly, these results highlight the critical role of mechanical environment in modulating cellular biochemical signaling.Ph