A tissue-engineered human trabecular meshwork hydrogel for advanced glaucoma disease modeling.

Abnormal human trabecular meshwork (HTM) cell function and extracellular matrix (ECM) remodeling contribute to HTM stiffening in primary open-angle glaucoma (POAG). Most current cellular HTM model systems do not sufficiently replicate the complex native three dimensional (3D) cell-ECM interface, limiting their use for investigating POAG pathology. Tissue-engineered hydrogels are ideally positioned to overcome shortcomings of current models. Here, we report a novel biomimetic HTM hydrogel and test its utility as a POAG disease model. HTM hydrogels were engineered by mixing normal donor-derived HTM cells with collagen type I, elastin-like polypeptide and hyaluronic acid, each containing photoactive functional groups, followed by UV crosslinking. Glaucomatous conditions were induced with dexamethasone (DEX), and effects of the Rho-associated kinase (ROCK) inhibitor Y27632 on cytoskeletal organization and tissue-level function, contingent on HTM cell-ECM interactions, were assessed. DEX exposure increased HTM hydrogel contractility, f-actin and alpha smooth muscle actin abundance and rearrangement, ECM remodeling, and fibronectin deposition - all contributing to HTM hydrogel condensation and stiffening consistent with glaucomatous HTM tissue behavior. Y27632 treatment produced precisely the opposite effects and attenuated the DEX-induced pathologic changes, resulting in HTM hydrogel relaxation and softening. For model validation, confirmed glaucomatous HTM (GTM) cells were encapsulated; GTM hydrogels showed increased contractility, fibronectin deposition, and stiffening vs. normal HTM hydrogels despite reduced GTM cell proliferation. We have developed a biomimetic HTM hydrogel model for detailed investigation of 3D cell-ECM interactions under normal and simulated glaucomatous conditions. Its bidirectional responsiveness to pharmacological challenge and rescue suggests promising potential to serve as screening platform for new POAG treatments with focus on HTM biomechanics.

Comparison of the extracellular vesicle proteome between glaucoma and non-glaucoma trabecular meshwork cells.

Introduction: Extracellular matrix (ECM) materials accumulate in the trabecular meshwork (TM) tissue of patients with glaucoma, which is associated with a decrease in aqueous humor outflow and therefore an increase in intraocular pressure. To explore a potential mechanism for ECM regulation in the TM, we purified extracellular vesicles (EVs) from conditioned media of differentiated TM cells in culture isolated from non-glaucomatous and glaucomatous human donor eyes. Methods: EVs were purified using the double cushion ultracentrifugation gradient method. Fractions containing EV markers CD9 and TSG101 were analyzed using nanoparticle tracking analysis to determine their size and concentration. We then determined their proteomic cargo by mass spectrometry and compared protein profiles of EVs between normal and glaucomatous TM cells using PANTHER. Key protein components from EV preparations were validated with Western blotting. Results: Results showed changes in the percentage of ECM proteins associated with EVs from glaucomatous TM cells compared to non-glaucomatous TM cells (5.7% vs 13.1% respectively). Correspondingly, we found that two ECM-related cargo proteins found across all samples, fibronectin and EDIL3 were significantly less abundant in glaucomatous EVs (<0.3 fold change across all groups) compared to non-glaucomatous EVs. Discussion: Overall, these data establish that ECM materials are prominent proteomic cargo in EVs from TM cells, and their binding to EVs is diminished in glaucoma.