PROJECT SUMMARY Elevated intraocular pressure (IOP) caused by impaired aqueous humor (AH) drainage through the trabecular meshwork (TM) is recognized to hasten optic nerve atrophy and retinal ganglion cell death in glaucoma patients. Neither the external factors nor intracellular mechanisms regulating AH drainage through the TM have been fully defined in normal or ocular hypertensive eyes. Our recent studies revealed that Growth Differentiation Factor-15 (GDF-15), a distant member of the TGF-β superfamily of cytokines, is a regular constituent of the extracellular matrix (ECM) secreted by human TM cells, exhibits robust upregulation in response to various IOP elevating agents, and induces cellular contractility, α- smooth muscle actin expression, ECM accumulation and SMAD activation in TM cells. Furthermore, AH derived from primary open-angle glaucoma (POAG) human patients exhibited a significant increase (~6 fold) in GDF-15 levels relative to control AH samples from age-matched cataract subjects. Interestingly, transgenic mice overexpressing human GDF-15 exhibited chronically elevated IOP, and short-term perfusion of enucleated mouse eyes with recombinant rGDF-15 resulted in significant AH outflow changes. These promising and novel preliminary observations led us to conclude a crucial role for GDF-15 in homeostasis of AH outflow and IOP, and to hypothesize that alterations in GDF-15 levels disrupt key cellular events involved in AH drainage through the TM, thereby impacting IOP and participating in the etiology of POAG, which is considered one of the leading causes of blindness globally. To establish a mechanistic and deeper understanding of how GDF-15 regulates AH outflow and IOP, investigations will be carried out under three specific aims to determine: 1). The receptors and downstream signaling pathways and the cellular characteristics regulated by GDF-15 in human TM cells, 2). GDF-15- mediated regulation of AH outflow and IOP using gene targeted and transgenic mice and organ cultured human eyes, and 3). Regulation of bio-availability of active GDF-15 from stromal stores in TM cells, and feasibility assessment of circulating GDF-15 as a biomarker of significance in glaucoma patients. To the best of our knowledge, this is the first in-depth study on GDF-15, a stress response cytokine, in TM and glaucoma, the completion of which (cell-based and in vivo rodent and human studies) should demonstrate not only the definitive role of GDF-15 in AH outflow and IOP, but also generate impactful insights into the etiology of ocular hypertension and enable identification of innovative treatments for glaucoma.