Project Summary Primary open-angle glaucoma (POAG), a progressive optic neuropathy, causes irreversible blindness. There are multiple risk factors for this disease, but the only modifiable attribute is elevated intraocular pressure (IOP). Elevated IOP results from a blockage in the aqueous humor drainage pathway in the anterior eye. As pressure builds, stress is exerted on the optic nerve head in the posterior segment, leading to optic nerve damage and a slow and irreversible decline in vision. The trabecular meshwork (TM) is the tissue responsible for regulating IOP homeostasis. In response to sustained elevated pressure, TM cells are stretched and they release enzymes to focally degrade extracellular matrix (ECM), which allows greater aqueous outflow and lowers IOP. Glaucomatous TM has excess amounts of disorganized ECM compared to age-matched tissue, suggesting that dysfunctional ECM contributes to increased resistance to aqueous outflow. However, it is currently unknown which ECM components cause dysfunction. In this application, we focus on one ECM protein called thrombospondin-1 (TSP- 1). We recently reported that a single nucleotide polymorphism, rs2228262, which causes a missense N700S amino acid change in TSP-1, was significantly associated with elevated IOP in a large POAG family. Our preliminary data show that the TSP-1 N700S variant is present in approximately 9.5% of normal and 38% glaucomatous TM cell strains. Thus, we have a unique opportunity to study the function of the naturally occurring N700S TSP-1 variant in a glaucoma-relevant tissue. We postulate that the N700S TSP-1 variant causes changes in ECM organization and TM cell dysfunction, which contribute to development of fibrotic matrices characteristic of the glaucomatous TM in situ. TSP-1 is a potent activator of transforming growth factor-β (TGFβ), which stimulates ECM synthesis and plays major roles in glaucoma pathophysiology. Yet, there are no glaucoma therapies directed at modulating TGFβ levels. In this application, we will investigate the molecular consequences of the N700S variant in normal and glaucomatous TM cells, and use a peptide TSP-1 antagonist to target the TSP-1-TGFβ pathway. Our overall hypothesis is that modifications to TSP-1 molecular function disrupt ECM organization and TGFβ activity in the normal and glaucomatous TM. Three aims are proposed: (1) to analyze ECM composition, ultrastructural organization and TSP-1 protein-protein interactions in wild-type and N700S variant cell strains derived from normal and glaucomatous TM; (2) to measure the impact of the N700S variant on phagocytosis, matrix metalloproteinase enzyme activity, and TGFβ activity using endogenous, over- expressed and CRISPR-edited TM cells; and (3) to use a peptide antagonist, which competitively and specifically blocks TGFβ activation by TSP-1, to investigate its effects on endogenous and CRISPR-edited N700S TM cells, as well as to determine its effects on outflow rates in an ex vivo m...