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M.C. Kenney, M. Chwa, H. Zheng, V. Butani, S.R. Atilano; Oxidative Stress Damages mtDNA and Decreases Mitochondrial Membrane Potential in Keratoconus Fibroblast Cultures . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1324.
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To determine if oxidative stress challenges cause mitochondrial DNA (mtDNA) damage or mitochondrial dysfunction in normal and keratoconus (KC) fibroblasts.
KC (n=5) and age–matched normal (n=6) corneal fibroblast cultures were incubated 1 hour at 37oC at either pH 7.0 or pH 5.0 with or without hydrogen peroxide (H2O2). ROS production was assayed using the fluorescent dye 2’,7’–dichlorodihydro–fluorescein diacetate (H2DCFDA). Mitochondrial membrane potential (ΔΨm) measurements were analyzed with the JC–1 detection kit. Total genomic DNA was isolated from cultured cells and the mtDNA was identified by long–extension polymerase chain reaction (LX–PCR). The ratio of mtDNA to nDNA was measured by PCR analysis for mtND2 and r18s. The RNA was extracted from the cultures, reverse transcribed to cDNA and RT–PCR performed for mitochondrial encoded genes (COX II (complex IV), cytochrome b (complex III), mtND6 (complex I), mtND2 (complex I)) and fibrinolysis genes (uPA and uPAR).
Untreated KC fibroblasts had a 1.8–fold increased of ROS production compared to normal fibroblasts (p<0.04). The low pH stressed KC fibroblasts (p<0.04) and H2O2 treated cells (p<0.05) showed a 2.6 fold decrease of ΔΨm compared to normal fibroblasts. The LX–PCR mtDNA was damaged after low pH/H2O2 challenges. The mtND2 gene expression was decreased while uPA was elevated after low pH/H2O2 treatments.
KC fibroblast cultures have an elevated ROS burden and loss of ΔΨm compared to normal fibroblast cultures. The mtDNA damage and altered gene expressions occur in response to oxidative stress. These findings support the hypothesis that increased levels of ROS production and mitochondrial dysfunction occur in oxidatively stressed corneal cells and may play a role in the oxidative damage found in KC corneas.
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