Hydrogels provide highly tunable microenvironments that can be adjusted to recapitulate the stiffness of the valve matrix at different stages of fibrosis progression and enable researchers to study VIC mechanosensing in a controlled manner. In this work, the crosslinking density of poly(ethylene glycol) (PEG) hydrogels was tailored to control the matrix mechanical properties, while the fibronectin-derived adhesive peptide, RDGS, was used to promote VIC-matrix interactions. When cultured on soft PEG hydrogels (~1-5 kPa), VICs maintained a quiescent fibroblast phenotype, but transitioned to an activated myofibroblast phenotype on stiffer PEG matrices (E’~13-30kPa). Thus, these biomaterial systems provided a reliable in vitro platform for VIC cultures in healthy and disease scenarios.
In recent years, the tumor suppressor gene phosphatase and tensin homolog (PTEN) has emerged as an important player in the regulation of fibrosis in various tissues (e.g., lung, skin), which motivated us to investigate PTEN as a potential protective factor against matrix-induced myofibroblast activation in VICs. In aortic valve samples from humans, we found high levels of PTEN in healthy tissue and low levels of PTEN in diseased tissue. Then, using pharmacological inducers to treat VIC cultures, we observed PTEN overexpression prevented stiffness-induced myofibroblast activation, whereas genetic and pharmacological inhibition of PTEN further activated myofibroblasts. We also observed increased nuclear PTEN localization in VICs cultured on stiff matrices, and nuclear PTEN also correlated with smaller nuclei, altered expression of histones and a quiescent fibroblast phenotype. Together, these results suggest that PTEN not only suppresses VIC activation, but functions to promote quiescence, and could serve as a potential pharmacological target for the treatment of AVS.
Furthermore, this study investigates the paracrine effects mediated by extracellular vesicles (EV) and their modulation of VIC activation to myofibroblasts under the prism of AVS sexual dimorphism. We investigate the role of EV mediated VIC differentiation in sexually dimorphic valve disease, with the goal of identifying sex-specific AVS therapies. We developed sex-specific in vitro models of fibro-calcification using porcine-derived VICs (due to their wide availability) to mimic diseased valve tissue and, subsequently, investigated the role of VEC derived EV to sex-specific AVS progression both in vitro and in human valve tissue samples. Understanding the sex specific VEC-VIC paracrine interactions and intracellular signaling can lead to the long-term goal of developing non-invasive inhibitors or small molecules to hinder VEC mediated AVS progression or EV therapies revolving around the release of vesicles from degradable scaffolds.