Oxidative Stress and Lysosomal Function in the Outflow Pathway

描述（由申请人提供）：原发性开角型青光眼（POAG）是一种迟发性疾病，通常伴有眼内压（IOP）升高，这是由于常规流出道无法维持正常水平的房水（AH）流出阻力所致。TM中的细胞通过AH中存在的活性氧（ROS）和正常代谢产生的慢性氧化应激。暴露于ROS被认为有助于衰老和POAG中流出通路的形态学和生理学改变。尽管细胞抗氧化系统功能正常，但持续暴露于氧化应激导致不可降解的交联修饰蛋白质和脂质的积累。这种不可降解的材料被自噬并储存在溶酶体隔室内，导致溶酶体细胞系统的进行性衰竭，负责细胞器和长寿蛋白的周转。溶酶体区室也负责外源性吞噬物质的降解。我们的初步数据显示，在细胞内的活性氧的生产，氧化蛋白质的含量，以及在TM细胞暴露于慢性氧化应激的酸性区室含量普遍增加显着增加。增加的溶酶体质量与增加的溶酶体酶活性无关。然而，我们观察到较高水平的次优半乳糖苷酶活性SA-<$-Gal。我们的实验室最近报道，与正常供体相比，青光眼TM样本流出通路中SA-<$-Gal阳性染色的细胞数量增加。此外，已经描述了在脑水肿流出途径中存在细胞外溶酶体基质囊泡。我们假设TM细胞长期暴露于氧化应激促进了溶酶体酸性区室内不可降解物质的积累，导致吞噬和胞吐细胞功能的失败。我们进一步假设，这种吞噬和排出功能障碍可导致非内化物质的细胞外积累，以及膜结合囊泡内不可降解物质的释放，这可导致在青光眼中观察到的异常ECM沉积。为了验证这一假设，我们将原代培养的TM细胞慢性氧化应激，我们将监测氧化物质的溶酶体内积累，并将这种积累与改变吞噬功能。我们还将通过蛋白质组学方法评估应激TM细胞中释放囊泡的数量和组成的潜在变化。最后，我们将探讨是否存在基质囊泡，ECM沉积，溶酶体功能障碍，并改变流动阻力在眼前节暴露于慢性氧化应激。 公共卫生相关性：这项资助提案的目的是了解随着时间的推移，在流出通道细胞内积累的废物如何对其他基本细胞功能产生负面影响，从而导致原发性开角型青光眼（POAG）的病理学。了解负责废物降解的细胞机制的故障如何可能有助于POAG的病理学将开辟新的治疗靶点策略。

DESCRIPTION (provided by applicant): 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 ¿-galactosidase activity, SA-¿-Gal. An increased number of cells positively stained for SA-¿-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.