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Payam Falatoonzadeh, Shari R. Atilano, Marilyn Chwa, Janelle Pavlis, Anthony B. Nesburn, Nitin Udar, Maria C. Kenney; Accumulation of Reactive Oxygen/Nitrogen Species is a Result of Decreased Expression of Antioxidant Enzymes in Keratoconus Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1108.
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© ARVO (1962-2015); The Authors (2016-present)
Keratoconus (KC), a leading cause of corneal transplantation, is associated with oxidative damage of the cornea. Our study investigates the regulation of reactive oxygen/nitrogen species (ROS/RNS) with regards to the enzymatic production and elimination pathways in KC corneal cells.
Normal and KC cells were cultured in Minimal Essential Media with 10% fetal bovine serum to establish primary stromal fibroblast cultures. RNA extracted from pellets of these Normal (n=5) and KC cells (n=5) was reverse transcribed into cDNA for gene expression analysis. RT-PCR was performed for enzymes associated with ROS/RNS production pathways, including multiple subunits of the NADPH oxidase family and nitric oxide synthase (NOS). Gene expression analyses were also performed for antioxidant enzymes responsible for ROS/RNS elimination, including Superoxide Dismutase and Peroxiredoxin.
Gene expression levels of enzymes responsible for ROS/RNS production, including DUOX1, DUOX2, NOS2, and NAPDH subunits were statistically unchanged, presenting less than two-fold shifts in KC cells when compared to Normal cell levels. There was a significant reduction for five antioxidant enzymes, PRDX3 (1.99 fold, p=0.001), PRDX6 (1.9 fold, p=0.0001), SOD1 (2.56 fold, p=0.0001), SOD2 (1.08 fold, p=0.025) and SOD3 (4.34 fold, p=0.001). There was also a noticable and significant decrease in USP36 (1.92 fold, p<0.0001), a deubiquinating cysteine protease gene which regulates the stability of SOD2.
Our data suggests that the increased oxidative stress present in KC cells is a result of decreased eliminative potential of ROS/RNS by the antioxidant enzymes rather than overproduction. The lower expression of various antioxidant enzymes found within the KC cells, as well as regulatory factors such as the USP36 gene, support that the increased accumulation of ROS/RNS may be due to reduced elimination. Furthermore, there were no differences between Normal and KC cell expression for any of the NADPH oxidase enzyme subunits or NOS, which suggests that the elevated ROS/RNS levels are unlikely due to these production pathways. The abnormal expression of these multiple antioxidant genes may effectively weaken KC cell defenses to oxidative stress.
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