Inflammatory serum factors from aortic valve stenosis patients modulate sex differences in valvular myofibroblast activation and osteoblast-like differentiation.

Aortic valve stenosis (AVS) is a sexually dimorphic cardiovascular disease that is driven by fibrosis and calcification of the aortic valve leaflets. Circulating inflammatory factors present in serum from AVS patients contribute to sex differences in valve fibro-calcification by driving the activation of valvular interstitial cells (VICs) to myofibroblasts and/or osteoblast-like cells. However, the molecular mechanisms by which inflammatory factors contribute to sex-specific valve fibro-calcification remain largely unknown. In this study, we identified inflammatory factors present in serum samples from AVS patients that regulate sex-specific myofibroblast activation and osteoblast-like differentiation. After correlating serum proteomic datasets with clinical and in vitro myofibroblast datasets, we identified annexin A2 and cystatin C as candidate inflammatory factors that correlate with both AVS patient severity and myofibroblast activation measurements in vitro. Validation experiments utilizing hydrogel biomaterials as cell culture platforms that mimic the valve extracellular matrix confirmed that annexin A2 and cystatin C promote sex-specific VIC activation to myofibroblasts via p38 MAPK signaling. Additionally, annexin A2 and cystatin C increase osteoblast-like differentiation primarily in male VICs. Our results implicate serum inflammatory factors as potential AVS biomarkers that also contribute to sexually dimorphic AVS progression by driving VIC myofibroblast activation and/or osteoblast-like differentiation. Collectively, the results herein further our overall understanding as to how biological sex may impact inflammation-driven AVS and may lead to the development of sex-specific drug treatment strategies.

Correction: Inflammatory serum factors from aortic valve stenosis patients modulate sex differences in valvular myofibroblast activation and osteoblast-like differentiation.

Correction for 'Inflammatory serum factors from aortic valve stenosis patients modulate sex differences in valvular myofibroblast activation and osteoblast-like differentiation' by Brandon J. Vogt et al., Biomater. Sci., 2022, https://doi.org/10.1039/d2bm00844k.

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

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.

Secreted Factors From Proinflammatory Macrophages Promote an Osteoblast-Like Phenotype in Valvular Interstitial Cells.

OBJECTIVE: Resident valvular interstitial cells (VICs) activate to myofibroblasts during aortic valve stenosis progression, which further promotes fibrosis or even differentiate into osteoblast-like cells that can lead to calcification of valve tissue. Inflammation is a hallmark of aortic valve stenosis, so we aimed to determine proinflammatory cytokines secreted from M1 macrophages that give rise to a transient VIC phenotype that leads to calcification of valve tissue. Approach and Results: We designed hydrogel biomaterials as valve extracellular matrix mimics enabling the culture of VICs in either their quiescent fibroblast or activated myofibroblast phenotype in response to the local matrix stiffness. When VIC fibroblasts and myofibroblasts were treated with conditioned media from THP-1-derived M1 macrophages, we observed robust reduction of αSMA (alpha smooth muscle actin) expression, reduced stress fiber formation, and increased proliferation, suggesting a potent antifibrotic effect. We further identified TNF (tumor necrosis factor)-α and IL (interleukin)-1ß as 2 cytokines in M1 media that cause the observed antifibrotic effect. After 7 days of culture in M1 conditioned media, VICs began differentiating into osteoblast-like cells, as measured by increased expression of RUNX2 (runt-related transcription factor 2) and osteopontin. We also identified and validated IL-6 as a critical mediator of the observed pro-osteogenic effect. CONCLUSIONS: Proinflammatory cytokines in M1 conditioned media inhibit myofibroblast activation in VICs (eg, TNF-α and IL-1ß) and promote their osteogenic differentiation (eg, IL-6). Together, our work suggests inflammatory M1 macrophages may drive a myofibroblast-to-osteogenic intermediate VIC phenotype, which may mediate the switch from fibrosis to calcification during aortic valve stenosis progression.