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.
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. Previous work has demonstrated that substrate stiffness influences VIC activation dramatically, therefore culture on polystyrene tissue culture plates is not ideal. We have modeled VIC activation through a tunable hydrogel in vitro culture system, where VICs upregulate ASMA on stiff substrates (30kPa) but remain quiescent on soft substrates (1-5kPa). To test the effect of VEC derived EV on VICs we have isolated VECs and VICs from pig hearts (due to their availability) and we co-culture VICs and EV on stiff substrates for 3 days to measure ASMA expression. Furthermore, we collected EV from male and female VEC and VIC to investigate their miRNA content as it is reported to regulate endocytosis and intracellular processes, therefore it is pivotal for AVS progression. Uncovering previously unknown miRNA sequences regulating fibrosis gives us useful information to apply the investigation of EV mediated paracrine effects in human fibrotic tissues. Using cutting edge imaging techniques (Photo expansion microscopy/ insitu hybridization) we colocalize and visualize EV related miRNA with fibrosis markers, to correlate their expression in human tissue samples. Finally, by understanding the intracellular signaling pathways that mediate the paracrine effects between VICs and VECs we can discover pharmacological targets for small molecules and inhibitors or EV oriented therapies against AVS progression.
Finally, I am planning to briefly discuss my research plan as a future faculty member, in studying the effects of biomaterials on EV secretion and their potential as a personalized therapeutic for aortic valve stenosis and neurodegenerative disorders. Implementing quantitative biological techniques and engineering innovative biomaterial platforms offers the opportunity to elucidate on the EV driven transcellular communication in healthy and diseased cell states. Specifically, studying local membrane stress taking advantage of stress relaxing hydrogels and expansion microscopy, I plan to investigate stress driven EV-cell membrane interactions, as a function of their genetic and protein content at the nanoscale, an area that currently remains largely understudied.