Role of the Rho pathway in regulating valvular interstitial cell phenotype and nodule formation

The differentiation of valvular interstitial cells (VICs) to a myofibroblastic or osteoblast-like phenotype is commonly found in calcific valvular stenosis, although the molecular-level mechanisms of this process remain poorly understood. Due to the role of the Rho pathway in various vascular diseases and in the expression of a myofibroblast phenotype, the present study was inspired by the hypothesis that Rho activation is involved in regulating cellular processes related to valve calcification. It was found that increased RhoA and Rho kinase (ROCK) activity was associated with increased nodule formation in VIC cultures in vitro, and intentional induction of RhoA activity led to a further increase in nodules and expression of α-smooth muscle actin. VICs treated with ROCK inhibitors were also examined for nodule formation, proliferation, apoptosis, and expression of myofibroblastic or osteoblastic markers. ROCK inhibition dramatically reduced myofibroblast-regulated nodule formation in VIC cultures, as evidenced by a decrease in nodule number, total nodule area, α-smooth muscle actin-positive stress fibers, apoptosis, and gene expression of myofibroblast-related phenotypic markers. Meanwhile, ROCK inhibition was less effective at reducing nodule formation associated with osteogenic activity. In fact, ROCK inhibition increased the expression of alkaline phosphatase and effected only a modest decrease in nodule number when applied to VIC cultures with higher osteogenic activity. Thus, the Rho pathway possesses a complex role in regulating the VIC phenotype and nodule formation, and it is hoped that further elucidation of these molecular-level events will lead to an improved understanding of valvular disease and identification of potential treatments.