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K. M. Porter, Y. Lin, D. L. Epstein, P. B. Liton; Chronic Oxidative Stress Impairs Phagocytic Degradative Capacity in Cultured Trabecular Meshwork (TM) Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3225.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the effects of chronic oxidative stress on phagocytic function in cultured TM cells.
Porcine and human TM cells were subjected to chronic oxidative stress using the hyperoxic model and then challenged to phagocytic ligands (FITC- and pHRodo-labeled E. coli, and porcine iris pigment). Phagocytosed material was quantified by flow cytometry. Phagosome maturation was monitored by confocal microscopy, using Lysotracker, and by electron microscopy using horseradish peroxidase. Degradation of endocytosed material was quantified by flow cytometry using DQ BSA. Cathepsin expression levels were quantified by WB and qPCR analyses. Cathepsin activities were assayed using fluorogenic substrates. Gene expression profile analysis was performed using Affymetrix U133 plus 2.0 array and analyzed with Genespring GX and Metacore Softwares. Cytochalasin D, Bafilomycin A1 and Leupeptine/Pepstatin were used to block internalization or degradation of engulfed material
Porcine TM cells grown at 40% O2 displayed increased levels of pHRodo fluorescence compared to cells grown at 5% O2 (250.4 ± 44.9%, p=0.02). Such increase was not observed in the presence of lysosomal proteolysis inhibitors. Pulse-chase experiments showed that while pHRodo fluorescence rapidly decreased overtime in control cells, it progressively increased during the chase period in the cells at 40% O2. Oxidatively stressed cells also displayed diminished proteolysis of endocytosed DQ-BSA (21.2 ± 3.5%, p=0.043). Confirming our previous data, we observed a generalized decrease in cathepsin activity in the stressed cultures. No significant changes were detected following phagocytic challenge. A decrease in cathepsin protein levels with phagocytosis was observed in cells grown at 40% O2. Gene expression profile analysis identified a number of genes differentially expressed in response to phagocytic challenge. Among them, RANKL and insulin-like growth factor, which have been reported to regulate phagosome maturation, were also found to be differentially expressed in the cells at 40% O2.
Our data indicate that while chronic oxidative stress do not seem to negatively affect phagocytic uptake capacity in TM cells, it may impair the TM phagocytic degradative capacity. The intracellular presence of this undigested material might further compromise lysosomal function and contribute to the pathogenesis of primary open angle glaucoma.
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