PROJECT SUMMARY Glaucoma is a leading cause of irreversible blindness worldwide. While the etiology of the disease is complex, it is typically associated with elevated intraocular pressure (IOP) due to increased resistance to aqueous humor outflow through the trabecular meshwork (TM). The human TM is approximately 20 fold stiffer in glaucoma, suggesting a prominent role of TM mechanobiology. Although current outflow pathway models estimate that the juxtacanalicular region of the TM contributes to 90-95% of the outflow resistance, it is widely accepted that outflow itself is not uniform around the circumference of the TM, but is highly segmental with regions of relatively high flow (HF), and low flow (LF). Although this has been recognized previously, nearly all studies over the past few decades have essentially ignored this fact. Whether there are inherent differences in TM cells of HF and LF regions and between non-glaucomatous and glaucomatous individuals remains unclear. Preliminary data in support of this proposal shows that, with glaucoma tissues, there are more LF regions, they are stiffer, and are associated with elevated matrix crosslinking enzyme activity. Conversely, HF regions are softer, fewer, and have lower levels of crosslinking activity. Based on these and other observations, we have hypothesized that there are innate differences in cells between the segmental flow regions, and these directly regulate extracellular matrix (ECM) turnover, crosslinking, and outflow. The precise mechanism that underlies the relative shift to increased LF regions is unclear. In order to mechanistically understand the regulatory link between matrix biomechanics, composition, and segmental outflow, we will use two general experimental approaches, (A) using perfused human anterior segment organ culture, we will compare biomechanical and biochemical properties of HF and LF regions, measure crosslinking, and, isolate cells from these; and (B) use cell derived matrices to determine cell-matrix interactions. Specifically, in Aim 1, we will isolate TM cells from different flow regions of glaucomatous and non-glaucomatous TM, characterize cell surface receptor distribution, and investigate their mechanotransduction response to biophysical stimuli. We will also obtain and characterize cell derived ECM, and determine the effect that these ECM have on cellular behavior. In Aim 2, we will ascertain and quantify the nature of ECM crosslinks, document differences in crosslinking enzyme activity, and determine if inhibiting crosslinks changes the biomechanics and composition of segmental regions in both normal and glaucomatous eyes. We will also determine if substratum biomechanics modulates crosslinking in segmental flow cells. Finally, in Aim 3, we will use a targeted approach to identify regulators of the homeostatic response and manipulate outflow regions. Particularly we will target the specific role that ECM binding integrin α7β1 has in mediating outflow, ECM r...