Deciphering cellular dynamics and crosstalk of trabecular meshwork and Schlemm's canal cells in a bioengineered 3D extracellular matrix hydrogel microenvironment

Abstract

In the conventional outflow pathway, Schlemm's canal (SC) inner wall endothelium interfaces with the trabecular meshwork (TM). Biomechanical changes in this microenvironment contribute to increased resistance to aqueous outflow, a characteristic of ocular hypertensive glaucoma. Notably, TM undergoes fibrotic-like remodeling and stiffening. Existing in vitro TM/SC models fail to accurately replicate native cell-cell and cell-extracellular matrix (ECM) interactions, limiting their use for studying glaucomatous outflow pathobiology. In this dissertation, we utilized a biomimetic ECM hydrogel system made from natural polymers resembling native tissue proteins. This ECM hydrogel can be (i) used to encapsulate donor-derived primary human TM cells or (ii) employed as a substrate for culturing donor-derived primary human SC cells on top. As ECM hydrogels gradually emerge as a preferred model in diverse research laboratories, a standardized fabrication method is essential to improve accessibility and consistency across experimental protocols. Thus, a detailed methodology for producing these ECM hydrogels is provided in Chapter 2. In Chapter 3, using the 3D TM hydrogel system, we demonstrated that simvastatin-mediated inactivation of Yes-associated protein (YAP) and transcriptional coactivator with PDZ binding motif (TAZ) attenuates pathological changes in TM cells. YAP/TAZ are key mechanotransducers involved in glaucoma pathogenesis and are shown to be regulated by the mevalonate pathway. By inhibiting this pathway, we hypothesized that statins could potentially improve TM cell pathobiology by modulating YAP/TAZ activity. Thus, targeting the mevalonate pathway with statins may offer therapeutic potential for glaucoma. Despite significant progress in understanding TM and SC cells individually, the dynamic interactions between them and their role in glaucoma pathogenesis remain poorly understood. These interactions are crucial in the pathogenesis of glaucoma, yet no effective model exists to study them. Therefore, in Chapter 4, we developed a novel co-culture hydrogel system to explore TM-SC interactions and assess how glaucomatous TM cells affect SC behavior. Our findings show that glaucomatous TM cells alone can induce pathological changes in SC cells, underscoring the critical role of cell-cell and cell-ECM interactions in glaucoma progression. Collectively, these biomimetic ECM hydrogels provide a unique platform for investigating glaucomatous outflow mechanisms and offering insights into disease pathogenesis.