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Background: Aortic valve stenosis is a sexually dimorphic disease, with women often presenting with sustained fibrosis and men with more extensive calcification. However, the intracellular molecular mechanisms that drive these clinically important sex differences remain underexplored. Methods: Hydrogel biomaterials were designed to recapitulate key aspects of the valve tissue microenvironment and to serve as a culture platform for sex-specific valvular interstitial cells (VICs; precursors to profibrotic myofibroblasts). The hydrogel culture system was used to interrogate intracellular pathways involved in sex-dependent VIC-to-myofibroblast activation and deactivation. RNA sequencing was used to define pathways involved in driving sex-dependent activation. Interventions with small molecule inhibitors and siRNA transfections were performed to provide mechanistic insight into sex-specific cellular responses to microenvironmental cues, including matrix stiffness and exogenously delivered biochemical factors. Results: In both healthy porcine and human aortic valves, female leaflets had higher baseline activation of the myofibroblast marker α-smooth muscle actin compared with male leaflets. When isolated and cultured, female porcine and human VICs had higher levels of basal α-smooth muscle actin stress fibers that further increased in response to the hydrogel matrix stiffness, both of which were higher than in male VICs. A transcriptomic analysis of male and female porcine VICs revealed Rho-associated protein kinase signaling as a potential driver of this sex-dependent myofibroblast activation. Furthermore, we found that genes that escape X-chromosome inactivation such as BMX and STS (encoding for Bmx nonreceptor tyrosine kinase and steroid sulfatase, respectively) partially regulate the elevated female myofibroblast activation through Rho-associated protein kinase signaling. This finding was confirmed by treating male and female VICs with endothelin-1 and plasminogen activator inhibitor-1, factors that are secreted by endothelial cells and known to drive myofibroblast activation through Rho-associated protein kinase signaling. Conclusions: Together, in vivo and in vitro results confirm sex dependencies in myofibroblast activation pathways and implicate genes that escape X-chromosome inactivation in regulating sex differences in myofibroblast activation and subsequent aortic valve stenosis progression. Our results underscore the importance of considering sex as a biological variable to understand the molecular mechanisms of aortic valve stenosis and to help guide sex-based precision therapies.

As aortic valve stenosis develops, valve tissue becomes stiffer. In response to this change in environmental mechanical stiffness, valvular interstitial cells (VICs) activate into myofibroblasts. We aimed to investigate the role of mechanosensitive calcium channel Transient Receptor Potential Vanilloid type 4 (TRPV4) in stiffness induced myofibroblast activation. We verified TRPV4 functionality in VICs using live calcium imaging during application of small molecule modulators of TRPV4 activity. We designed hydrogel biomaterials that mimic mechanical features of healthy or diseased valve tissue microenvironments, respectively, to investigate the role of TRPV4 in myofibroblast activation and proliferation. Our results show that TRPV4 regulates VIC proliferation in a microenvironment stiffness-independent manner. While there was a trend toward inhibiting myofibroblast activation on soft microenvironments during TRPV4 inhibition, we observed near complete deactivation of myofibroblasts on stiff microenvironments. We further identified Yes-activated protein (YAP) as a downstream target for TRPV4 activity on stiff microenvironments. Mechanosensitive TRPV4 channels regulate VIC myofibroblast activation, whereas proliferation regulation is independent of the microenvironmental stiffness. Collectively, the data suggests differential regulation of stiffness-induced proliferation and myofibroblast activation. Our data further suggest a regulatory role for TRPV4 regarding YAP nuclear localization. TRPV4 is an important regulator for VIC myofibroblast activation, which is linked to the initiation of valve fibrosis. Although more validation studies are necessary, we suggest TRPV4 as a promising pharmaceutical target to slow aortic valve stenosis progression.