Abstract
Purpose :
In the conventional outflow pathway, the Schlemm’s canal (SC) inner wall endothelium interfaces with the trabecular meshwork (TM). Crosstalk between SC and TM cells, and their extracellular matrix (ECM), is critical for maintaining homeostatic outflow function. In glaucoma, the SC/TM interface undergoes progressive fibrotic-like remodeling. Yet, the mechanistic details and temporal sequence underlying outflow tissue failure is not well understood. This is, in part, due to the inability of most in vitro models to accurately simulate the interactive nature of the SC/TM/ECM microenvironment. Here, we investigate the effects of glaucomatous TM cells on normal SC cells in a tissue-like 3D ECM hydrogel co-culture model.
Methods :
Donor-derived normal SC (NSC) cells were plated as monolayers on photocrosslinked ECM hydrogels (collagen type I, elastin-like polypeptide, and hyaluronic acid) encapsulated with age-matched normal TM (NTM) or glaucomatous TM (GTM) cells. Glaucomatous SC (GSC) cells grown atop acellular ECM hydrogels served as controls. The GTM cell cytoskeletal/nuclear phenotype was compared to NTM cells using immuno-stained confocal Z-stacks. Similarly, NSC cell F-actin, alpha-smooth muscle actin (α-SMA), and transglutaminase 2 (TGM2) levels were assessed, with the DAPI channel used to quantify nuclear number, size, and shape.
Results :
GTM cells in the 3D hydrogel displayed an inherent pathological, less proliferative phenotype with more disorganized F-actin stress fibers compared to NTM cells, consistent with previous observations. NSC cells atop GTM hydrogels grew at a slower rate than NTM hydrogels, as shown by decreased nuclear number (p<0.05), approximating GSC cell controls. Furthermore, NSC cells displayed more rounded nuclei with higher nuclear shape index (p<0.0001) and decreased nuclear aspect ratio (p<0.0001) on GTM hydrogels compared to NTM hydrogels, remarkably similar to GSC cells. A pattern of markedly increased F-actin stress fibers and TGM2 expression was observed in NSC cells atop GTM hydrogels compared to NTM hydrogels, with an inverse trend in α-SMA requiring further investigation.
Conclusions :
Our data suggest that glaucomatous TM cells in absence of other disease-associated stimuli are sufficient to induce a pathologic phenotypic conversion of normal SC cells via crosstalk, facilitated by the tissue-like SC/TM cell co-culture model.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.