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Y. Lin, C. Luna, P. Gonzalez, K. Franz, D. L. Epstein, P. B. Liton; Role of Intralysosomal Redox-Active Iron in Oxidative Stress-Induced Damage in Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1622.
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© ARVO (1962-2015); The Authors (2016-present)
In vitro studies have demonstrated the accumulation of iron during aging in different tissues. This accumulation of iron is believed to contribute to the age-related oxidative damage and has been associated with several age-related diseases, including Alzheimer’s disease, Parkinson’s disease, type 2 diabetes, cardiovascular diseases, and macular degeneration. Here we investigated the potential role of intralysosomal redox-active iron in oxidative stress-induced damage in trabecular meshwork (TM) cells.
Confluent cultures of porcine TM cells were used for these studies. Chronic oxidative stress was applied using the hyperoxic model (40% O2 versus physiological 5% O2 for two weeks); acute oxidative stress was applied by incubating the cells for three hours with H2O2 (250 µM, 500 µM, 750 µM, 1000 µM). The levels of mRNA for several iron-regulated genes were quantified by real-time PCR using specific primers. Protein levels of ferritin light chain (FTL) were analyzed by western-blot. Cytotoxicity was assayed with the MultiTox-Fluor Multiplex from Promega. Lysosomal labilization was evaluated by fluorometry using lysotracker red.
Exposure of porcine TM primary cultures to chronic oxidative stress increased the mRNA content of several genes involved in iron homeostasis, including transferrin receptor (TFRC, 1.85±0.25 fold), ferritin H (FTH, 2.01±0.16 fold), FTL (2.44±1.01), and metallothionein (MT1A, 6.09±2.00 fold). Similar to what has been reported in the TM from glaucoma donors, the mRNA of ceruloplasmin (CP, 0.47 ±0.1 fold) was downregulated with chronic oxidative stress. Western-blot analysis also demonstrated elevated levels of FTL suggesting an increase in the labile iron pool size in the stressed cultures. Pre-treatment of porcine TM cells with the intralysosomal iron chelator desferrioxamine (dfo) completely protected in a dosage-dependent manner against H2O2-induced cell death and H2O2-induced lysosomal permeabilization.
Our results indicate that chronic exposure of TM cells to oxidative stress induces changes in the expression of iron-regulated genes, which suggests the intracellular accumulation of redox-active iron in the stressed cultures. Due to its potential of generating reactive oxygen species through Fenton reactions, the accumulation of iron in TM cells with age may play a role in the oxidative stress damage observed in the outflow pathway of POAG donors. Iron chelation therefore may represent a novel therapeutic approach to POAG.
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