Primary open angle glaucoma (POAG) is a late onset disease usually accompanied by elevated intraocular pressure (IOP) that results from the failure of the conventional outflow pathway to maintain normal levels of aqueous humor (AH) outflow resistance. Cells in the TM are subjected to chronic oxidative stress through reactive oxygen species (ROS) present in the AH and generated by the normal metabolism. Exposure to ROS is thought to contribute to the morphological and physiological alterations of the outflow pathway in aging and POAG. Despite the normal function of the cellular antioxidant system, continuous exposure to oxidative stress results in the accumulation of non-degradable cross-linked modified proteins and lipids. This non-degradable material is autophagocytosed and stored within lysosomal compartments, leading to the progressive failure of the lysosomal cellular system, responsible for the turnover of cellular organelles and long-lived proteins. The lysosomal compartment is also responsible for the degradation of exogenous phagocytosed material. Our preliminary data show a significant increased in intracellular ROS production, oxidized proteins content, as well as a generalized increased in the acidic compartment content in TM cells exposed to chronic oxidative stress. The augmented lysosomal mass did not correlate with increased lysosomal enzyme activity. We observed, however, higher levels of the suboptimal [unreadable]-galactosidase activity, SA-[unreadable]-Gal. An increased number of cells positively stained for SA-[unreadable]-Gal in the outflow pathway from glaucoma TM samples compared to normal donors was recently reported by our laboratory. In addition, it has been described the presence of extracellular lysosomal matrix vesicles in the glaucomatous outflow pathway. We hypothesize that chronic exposure of TM cells to oxidative stress promotes the accumulation of non-degradable material within the lysosomal acidic compartment leading to the failure of the phagocytic and exocytic cellular function. We further hypothesize that such phagocytic and exocytic dysfunction can result in the extracellular accumulation of non-internalized material, as well as in the release of non-degradable material within membrane-bound vesicles, which can contribute to the abnormal ECM deposition observed in glaucoma. To test this hypothesis, we will subject primary cultures of TM cells to chronic oxidative stress, and we will monitor the intra-lysosomal accumulation of oxidized material and correlate such accumulation with altered phagocytic function. We will also evaluate potential alterations in the quantity and composition of released vesicles in stressed TM cells by proteomic approaches. Finally, we will explore the presence of matrix vesicles, ECM deposition, lysosomal dysfunction, and altered flow resistance in anterior segments exposed to chronic oxidative stress. PUBLIC HEALTH RELEVANCE: The objective of this grant proposal is to understand how the waste material that accumulates within the cells of the outflow pathway over time may negatively affect other essential cellular functions and thus contribute to the pathology of Primary Open Angle Glaucoma (POAG). Understanding how malfunction of the cellular machinery responsible for the degradation of waste products may contribute to the pathology of POAG will open new therapeutic target strategies.