GSE174817
Genes that escape X chromosome inactivation modulate sex differences in valve myofibroblasts
Aortic valve stenosis (AVS) 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 under explored. Hydrogel scaffolds were designed to recapitulate key aspects of the valve tissue microenvironment and serve as a platform for culture of sex-specific valvular interstitial cells (VICs; precursors to pro-fibrotic myofibroblasts). The hydrogel culture system was used to interrogate intracellular pathways involved in sex-dependent VIC-to-myofibroblast activation and deactivation. RNA-seq was used to define pathways likely to be involved in driving sex-dependent activation. Interventions using small molecule inhibitors were performed to provide mechanistic insight into sex-specific cellular responses to microenvironmental cues, including matrix stiffness and exogenously delivered biochemical factors. In both healthy porcine and human aortic valves, female leaflets had higher baseline activation of the myofibroblast marker, a-smooth muscle actin (a-SMA), compared to male leaflets. When isolated and cultured, female VICs had higher levels of basal a-SMA stress fibers that further increased in response to the hydrogel matrix stiffness, both of which were higher than male VICs. A transcriptomic analysis of male and female porcine VICs revealed Rho-associated protein kinase (RhoA/ROCK) signaling as a potential driver of this sex-dependent myofibroblast activation. Further, we found that genes that escape X-chromosome inactivation, such as BMX and STS (encoding for Bmx non-receptor tyrosine kinase and steroid sulfatase, respectively) partially regulate the elevated female myofibroblast activation via RhoA/ROCK 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 via RhoA/ROCK signaling. Together, in vivo and in vitro results confirm sex-dependencies in myofibroblast activation pathways, and transcriptome analyses and small molecule interventions implicate genes that escape X-chromosome inactivation in regulating sex differences in AVS progression. Our results underscore the importance of considering sex as a biological variable to understand the molecular mechanisms of AVS and help guide sex-based precision therapies. Overall design: Male and female porcine VICs were isolated, seeded on PEG-norbornene hydrogels (elastic modulus ~5.8 kPa) or tissue culture plastic, and cultured in Media 199 (Life Technologies) supplemented with 1% fetal bovine serum, 50 U/mL penicillin, 50 µg/mL streptomycin, and 1 µg/mL amphotericin B for 48 hours. 4 male and 4 female biological replicates are provided, resulting in 16 total samples. VICs cultured on hydrogels were considered to be quiescent fibroblasts, and VICs cultured on tissue culture plastic were considered to be myofibroblasts.