Genes That Escape X Chromosome Inactivation Modulate Sex Differences in Valve Myofibroblasts

Abstract

Background: 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. Methods: Hydrogel scaffolds were designed to recapitulate key aspects of the valve tissue microenvironment and serve as a culture platform for 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-sequencing was used to define pathways involved in driving sex-dependent activation. Interventions using small molecule inhibitors and small interfering RNA (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, alpha-smooth muscle actin (α-SMA), compared to male leaflets. When isolated and cultured, female porcine and human VICs had higher levels of basal α-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. 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 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.

Hydrogel cultures reveal Transient Receptor Potential Vanilloid 4 regulation of myofibroblast activation and proliferation in valvular interstitial cells

“…Further, the fibronectin-derived peptide sequence (RGD) was conjugated to the material through an azide functionality at 2 mM to promote VIC-matrix interactions. [34][35][36]42 Using this material system, we first sought to determine if VIC mechanosensitivity was consistent with previous observations using materials synthesize from alternative chemistries. 28,35,43 To establish VIC phenotype response to crosslinking density changes in the DBCO-azide PEG hydrogel system, we cultured VICs on both soft and stiffened hydrogels and measured the relative mRNA expression levels of various fibrotic factors (Figure 1C).…”

“… 30 In this work, the elastic moduli of the soft and stiff hydrogels ranged from 1.5 kPa initially to 15 kPa after stiffening (Figure 2B). Further, the fibronectin‐derived peptide sequence (RGD) was conjugated to the material through an azide functionality at 2 mM to promote VIC‐matrix interactions 34–36,42 …”

“…A range from 0.5 mM (low) to 15 mM RGD (high) concentrations are typically used to ensure cell adhesion and mechanosensing of the material 33 . Our lab has shown that a concentration between 2 and 5 mM works best to ensure VIC response to hydrogels 34–36 …”

“…33 Our lab has shown that a concentration between 2 and 5 mM works best to ensure VIC response to hydrogels. [34][35][36] To produce soft hydrogels, gel solution was immediately sandwiched between an azide-functionalized coverslip and a Sigmacote (Sigma) treated glass slide. To produce stiff hydrogels, pre-formed soft hydrogels formed were soaked in a 2 mM photo-initiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) solution in phosphate buffered saline (PBS) for 20 min at 37°C.…”

Chemical and molecular tools to probe biological sex differences at multiple length scales

“…Since valve fibrosis is sexually dimorphic, FRET-based calcium sensors may be particularly useful to investigate sex dimorphisms in calcium shuttling in contractile cardiomyocytes and other cardiac cells. 19,20…”