The Role Of Fhl2 In Mechanically Regulated Valve Interstitial Cell Osteogenic Differentiation

Introduction: Lesions in calcific aortic valve disease (CAVD) often include osteoblasts and occur preferentially in the natively stiffer fibrosa side of the valve, suggesting roles for osteogenesis and matrix stiffness in CAVD. Valve interstitial cells (VICs) have osteogenic potential that is modulated by matrix stiffness in vitro . Osteogenesis of mesenchymal stem cells is mediated by FHL2 and modulated by matrix stiffness via RhoA. However, the connection between FHL2 and RhoA and their roles in mechanically-regulated osteogenic differentiation of VICs and CAVD have not been established. Hypothesis: Matrix stiffness regulates RhoA-dependent FHL2 expression to direct VIC osteogenesis. Methods/Results: Primary porcine aortic VICs were grown on collagen-coated polyacrylamide gels of varying elastic moduli in vitro . With increasing substrate stiffness, VICs demonstrated increased RhoA activation by ELISA and increased FHL2 nuclear translocation by immunofluorescence and western blotting. Notably, significant increases were seen on 22 kPa substrates, a stiffness unique to the disease-prone fibrosa, compared to 11 kPa substrates (p<0.05). We investigated the regulatory relationship between RhoA and FHL2 in VICs by controlling RhoA levels with adenoviruses. With constitutively active RhoA, FHL2 nuclear translocation increased (p<0.06); in contrast, with dominant negative RhoA, FHL2 nuclear translocation decreased (p<0.05). The effect of RhoA levels on stiffness-dependent FHL2 activity was verified by knocking down RhoA levels pharmacologically (C3 toxin) which abrogated stiffness-dependent FHL2 nuclear translocation. Alkaline phosphatase activity as a marker of osteogenesis was highest on substrates with fibrosa-like stiffness and was reduced by FHL2 siRNA knockdown. Conclusions: Together, these results support the hypothesis that mechanically-regulated VIC osteogenic differentiation in vitro is mediated by FHL2 operating downstream of RhoA. New insights into the mechanotransduction mechanisms that regulate VIC osteogenesis may lead to novel strategies for treating CAVD.