Abstract
Purpose :
Dysfunction of the Schlemm’s canal (SC) inner wall endothelium and trabecular meshwork is the principal cause of decreased outflow facility in glaucoma. Extracellular matrix (ECM) stiffening and increased transforming growth factor beta2 (TGFβ2) in the aqueous humor are strongly associated with outflow tissue dysfunction. Yes-associated protein (YAP) has emerged as key contributor to glaucoma pathogenesis; YAP1 was recently identified as potential genetic risk factor. However, the precise role of SC cell YAP signaling in response to known glaucomatous stressors is poorly understood. Here, we investigate how ECM stiffness/composition and TGFβ2 regulate YAP activity in human SC cells using biomimetic hydrogels, and whether pharmacologic YAP inhibition increases ex vivo outflow facility.
Methods :
ECM hydrogels were fabricated by photocrosslinking functionalized collagen type I, elastin-like polypeptide, and hyaluronic acid. Bioinert alginate was added to facilitate Ca2+-mediated hydrogel stiffening (~3-fold) and alginate lyase-mediated softening. Fibronectin (FN) was used to coat ECM hydrogels (10 μg/cm2). Donor-derived SC cells were plated on hydrogels and stimulated with TGFβ2 (2.5 ng/ml), Y27632 (10 μM), or latrunculin B (Lat B; 2 μM). Verteporfin (VP; 0.5 μM) was used for YAP inhibition. YAP transcriptional activity, cytoskeletal organization, fibrotic marker levels, and ECM remodeling were quantified. Outflow facility with perfusion of VP (10 μM) was measured in enucleated eyes from C57BL/6J mice using iPerfusion.
Results :
ECM stiffening increased YAP activity and F-actin levels in SC cells (p<0.001). Nuclear YAP completely translocated to the cytoplasm within 3 h after ECM softening; F-actin levels reached baseline after 24 h (p<0.0001). FN coating enhanced SC cell YAP activity and F-actin/ECM remodeling. TGFβ2 increased nuclear YAP (p<0.001) contingent on cytoskeletal integrity; Y27632 or Lat B co-treatment interrupted aberrant YAP signaling. VP treatment fully blocked TGFβ2-driven YAP activation in SC cells (p<0.0001) and largely restored FN, TGM2, F-actin, αSMA, and pMLC levels (p<0.001). Importantly, 6/10 mouse eyes displayed ~15-80% increased outflow facility with VP perfusion.
Conclusions :
Our data suggest that YAP modulates SC cell dysfunction in response to known glaucomatous stressors, and that pharmacologic YAP inhibition has promising potential to improve outflow tissue dysfunction.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.