Abstract
Purpose :
The extracellular matrix (ECM) in the glaucomatous trabecular meshwork (GTM) is markedly stiffened. This potent biophysical cue drives progressive cellular dysfunction. Increased Yes-associated protein (YAP) activity is linked to GTM cell pathobiology, and a recent genome-wide study identified YAP1 among novel glaucoma risk loci. Epigenetic modifications such as histone acetylation are further associated with TM failure in glaucoma. Yet, the molecular underpinnings of cellular dysfunction in response to TM stiffening, and the contributions of YAP signaling and epigenetic remodeling remain unclear. Here, we investigate the effects of ECM stiffness modulation on YAP activity and histone acetylation, and how these elements modulate GTM cell dysfunction using ECM hydrogels.
Methods :
ECM hydrogels were made by photocrosslinking functionalized collagen type I, elastin-like polypeptide, and hyaluronic acid. Bioinert alginate was incorporated to facilitate stiffening (via Ca2+ crosslinking) and dynamic softening (via alginate lyase degradation) of ECM-alginate hydrogels. Donor-derived normal TM (NTM)/GTM cells were plated on/encapsulated in hydrogels and stimulated with histone deacetylase inhibitor trichostatin-A (TSA; 150 nM) or histone acetyltransferase inhibitor garcinol (5 µM). Fibrotic marker level expression, YAP activity, cytoskeletal/nuclear organization, and histone acetylation were quantified.
Results :
ECM-alginate hydrogel stiffness was increased ~3-fold upon Ca2+ crosslinking (p<0.001); alginate lyase treatment restored soft baseline levels. ECM stiffening increased F-actin, alpha-smooth muscle actin, nuclear size/tension, YAP nuclear localization, and histone acetylation in NTM and GTM cells (p<0.05). These pathological features were completely reversed in NTM cells by ECM softening, while GTM cells displayed a persistent fibrotic phenotype with elevated histone acetylation. Strikingly, NTM cells in a soft ECM environment treated with TSA showed increased YAP transcriptional activity, nuclear size/tension, and F-actin compared to controls (p<0.001). Likewise, GTM cells treated with garcinol exhibited markedly decreased nuclear YAP and expression of fibrotic markers.
Conclusions :
Our data suggest that targeting ECM stiffening to interrupt aberrant YAP signaling and histone acetylation is emerging as a promising strategy to permanently restore TM tissue function in glaucoma.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.