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C. C. Luna, G. Li, P. B. Liton, P. Challa, D. L. Epstein, P. Gonzalez; Chronic Oxidative Stress Leads to Increased Cellular Production of Reactive Oxygen Species in Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2064.
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© ARVO (1962-2015); The Authors (2016-present)
Trabecular meshwork (TM) cells subjected to chronic oxidative stress undergo changes similar to cellular senescence. The senescent phenotype in several cell types is characterized by an increase in cellular production of reactive oxygen species (ROS). Our objective was to evaluate whether exposure to chronic oxidative stress leads to a similar increase of ROS generation in TM cells, which could contribute to the pathology of the TM in glaucoma.
Porcine TM cells were subjected to chronic oxidative stress either by treatment with 200 microM H2O2 twice a day for 4 days or by incubation in hyperoxic conditions for 15 days. Senescence markers sa-beta-galactosidase (using the fluorogenic substrate C12 FDG) and autofluorescence were measured by FacScan. Production of intracellular ROS was analyzed by FacScan using H2DCFDA. Generation of extracellular superoxide was determined by measuring SOD inhibitable reduction of WST1. The potential role of p53/p21, p38/p16, and PI3K pathways in the acquisition of cellular senescence markers was evaluated using specific inhibitors (Pifithrin alpha, SB203580 and wortmannin, respectively) compared to untreated control cultures.
Chronic oxidative stress resulted in a significant increase in the generation of both extracellular superoxide (2.8 fold, p=0.004) and intracellular ROS (4 fold, p=0.012) concomitant with an increase in the senescence markers sa-beta-galactosidase (68%) and autofluorescence (90%). While inhibition of the p53/p21 DNA damage response pathway resulted in a general decrease in the senescent-associated changes, inhibition of PI3K produced an increase in sa-beta-galactosidase in TM cells.
The acquisition of phenotypic changes characteristic of cellular senescence that result from chronic oxidative stress in TM cells is associated with an increase in the cellular production of ROS. The increase of ROS production by senescent cells in the TM has the potential to cause functional alterations of the TM in aging and glaucoma.
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