Poster CC1 - Secreted Cytokines from Macrophages Drive Sex Differences in Valvular Myofibroblasts

Introduction: Aortic valve stenosis (AVS) is a sexually dimorphic disease, where male leaflets stiffen due to calcification and female leaflets stiffen due to fibrosis. Inflammation is a hallmark of AVS and secreted cytokines from macrophages are thought to contribute to valve fibro-calcification by driving the activation of valvular interstitial cells (VICs) to myofibroblasts. The molecular mechanisms by which inflammatory cytokines differentially regulate myofibroblast activation as a function of biological sex are poorly characterized. Here, using engineered poly(ethylene glycol) (PEG) hydrogels that recapitulate aortic valve tissue stiffness, we investigated the effect of secreted cytokines from pro-inflammatory M1 macrophages on sex-specific myofibroblast activation of VICs. Due to the observed presence of M1 macrophages in calcified valve leaflets, we hypothesized that M1 secreted cytokines drive osteoblast-like differentiation in VICs via epigenetic alterations that allow increased expression of osteoblast-like markers like runt-related transcription factor 2 (RUNX2).

Materials and

Methods: PEG norbornene (PEG-Nb) hydrogels functionalized with RGD were synthesized and crosslinked with a PEG-dithiol crosslinker via click chemistry reaction. M1 conditioned media (CM) was generated by activating male human adult monocytes (ATCC THP-1) and differentiating them into M1 macrophages by adding lipopolysaccharides and interferon-γ. Secreted cytokines from macrophage cultures were collected. VICs were seeded on top of PEG-Nb hydrogels and cultured with macrophage CM for 3 days. Gels were fixed and stained for α-smooth muscle actin (α-SMA), a marker for myofibroblast activation, and RUNX2, a marker of early osteoblast-like differentiation, for confocal imaging. To determine changes in chromatin structure, chromatin condensation parameter (CCP) was quantified via Sobel Edge Detection algorithm using 4',6-diamidino-2-phenylindole stain.

Results, Conclusions, and Discussions: We found that after 3 days in culture, M1 CM significantly decreased activation in both male and female VICs relative to control (Fig. 1A). Female VICs exhibited increased activation relative to males across all conditions (Fig. 1A). We also observed female-specific osteoblast-like differentiation after M1 treatment, noted by increased levels of RUNX2 nuclear translocation relative to same-sex control (Fig. 1B). Decreased CCP levels on M1-treated females suggests that M1 cytokines push female VICs towards an osteoblast-like phenotype (Fig. 1C). Cytokine array of the M1 CM revealed high levels of tumor necrosis-alpha (TNF-alpha), which has been previously implicated in regulating valve fibro-calcification. After treating the VICs with TNF-alpha, we observed deactivation across sex (Fig. 2B), but osteoblast-like differentiation as a function of TNF-alpha treatment was unique to only female VICs, relative to female untreated control (Fig. 2C).

Our female-specific in vitro cell culture platform recapitulates increased myofibroblast activation observed in female tissues from AVS patients. Our results serve as a stepping-stone toward understanding how female-specific inflammation may contribute to valve fibrosis. Ongoing work is testing the hypothesis that TNF-alpha is a central driver of inflammation-associated valve calcification in females via changes in chromatin state of valvular myofibroblasts. Additionally, we will investigate the MAPK/ERK pathway associated with sex-specific osteoblast-like differentiation of female valvular myofibroblasts via TNF-alpha activity.

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