Glaucoma is a major cause of blindness. A major risk factor for glaucoma, and the only treatable risk factor, is elevated intraocular pressure (IOP). IOP is largely determined by the function of the tissues of the conventional outflow pathway, specifically the trabecular meshwork and the inner wall of Schlemm's canal (SC), and increased fluid flow resistance in these tissues causes elevated IOP. The inner wall of SC has been largely ignored as a target for IOP-lowering therapies, in part because our understanding of this tissue's function has been poor. However, recent research findings have greatly improved our understanding and motivate this proposal. The goal of this R21 is to optimize, characterize and validate a rapid and accurate assay system that interrogates the fluid conductivity of cultured SC cells, motivated by the knowledge that increasing SC cellular conductivity will lower IOP. The long-term vision is to use this assay system to serve as a pre-clinical screening tool for drug discovery and thereby accelerate discovery of a novel class of “SC-active” compounds for treating ocular hypertension in glaucoma patients. We expect such SC-active compounds to have fewer side effects compared to other IOP-lowering compounds, including avoidance of conjunctival hyperemia. Because the hydraulic conductivity of SC cells is normally controlled by the formation of membrane-delimited fluid pathways (“pores”), the assay system specifically focuses on detection of such pores in a high-content manner. More particularly, the focus is on intracellular pores (I pores), thought to form an under-exploited secondary aqueous outflow pathway. The key elements of this assay system include: (1) primary SC cell isolation from non-glaucomatous and glaucomatous human eyes; (2) substrate micropatterning to control cultured cell spread/height and enhance assay rigor; (3) delivery of focal mechanical stretch to the apical surface of cultured SC cells using superparamagnetic microspheres and external magnets; (4) a novel fluorescent assay that, together with automated light microscopy and image processing tools, allows the rapid detection of pores that form in SC cells in a manner that minimizes introduction of inadvertent bias; and (5) a high-content format in which cells are cultured in 96 well plates, allowing relatively rapid testing of the effects of agents on SC cells. There is a high degree of rigor in the proposal, which is underpinned by a significant body of peer-reviewed research and strong preliminary data. The applicants have extensive experience in culturing and characterizing human primary SC cells, and the proposal includes a variety of positive and negative controls to validate the assay system. Further, the multi-well assay format has the advantage of naturally allowing multiple technical and biological replicates on each 96 well plate, enabling characterization of repeatability and good statistical rigor. It is expected, as suggested by the proposa...