Differentiation of valve interstitial cells (VICs) into pro-calcific cells is one of the central events in calcific aortic valve (AoV) disease (CAVD). While the paracrine pathways and the responsivity of VICs to mechanical compliance of the surrounding environment are well characterized, the molecular programming related to variations in local stiffness, and its link to cytoskeleton dynamics, is less consolidated. By using a simple method to produce 2D poly-acrylamide gels with stiffness controlled with atomic force microscopy (AFM), we manufactured adhesion substrates onto which human VICs from stenotic valves were plated, and subsequently investigated for cytoskeleton dynamics and activation of the mechanosensing-related transcription factor YAP. As a comparison, we employed VICs from patients undergoing valve substitution for valve insufficiency, a non-calcific AoV disease, which does not involve extensive inflammation. While the two VICs types did not differ for basic responses onto substrates with different stiffness values (e.g. adhesion and proliferation), they were subject to a different dynamics of stiffness-dependent YAP nuclear shuttling, revealing for the first time an intracellular force transduction mechanism distinctive for calcific aortic valve disease. In VICs from stenotic valves, YAP nuclear translocation occurred in concert with an increase in cytoskeleton tensioning and loading of the myofibroblast-specific protein αSMA onto the F-actin cytoskeleton. AFM force mapping performed along radial sections of human calcific valve leaflets identified, finally, areas with high and low levels of rigidity within a similar range to those controlling YAP nuclear translocation in vitro. Since VICs juxtaposed to these areas exhibited nuclear localized YAP, we conclude that subtle variations in matrix stiffness are involved in mechanosensing-dependent VICs activation and pathological differentiation in CAVD.
