Abstract
Purpose:To compare the levels of gene expression for enzymes involved in production and elimination of reactive oxygen/nitrogen species (ROS/RNS) in normal human corneal cells (NL cells) with those in human corneal cells with keratoconus (KC cells) in vitro.Methods:Primary NL and KC stromal fibroblast cultures were incubated with apocynin (an inhibitor of NADPH oxidase) or N-nitro-L-arginine (N-LLA; an inhibitor of nitric oxide synthase). ROS/RNS levels were measured using an H2 DCFDA fluorescent assay. The RT2 Profiler™ PCR Array for Oxidative Stress and Antioxidant Defense was used for initial screening of the NL and KC cultures. Transcription levels for genes related to production or elimination of ROS/RNS were analyzed using quantitative PCR. Immunohistochemistry was performed on 10 intact human corneas using antibodies against SCARA3 and CPSF3.Results:Array screening of 84 antioxidant-related genes identified 12 genes that were differentially expressed between NL and KC cultures. Compared with NL cells, quantitative PCR showed that KC cells had decreased expression of antioxidant genes SCARA3 isoform 2 (0.59-fold, P = 0.02) and FOXM1 isoform 1 (0.61-fold, P = 0.03). KC cells also had downregulation of the antioxidant genes SOD1 (0.4-fold, P = 0.0001) and SOD3 (0.37-fold, P = 0.02) but increased expression of SOD2 (3.3-fold, P < 0.0001), PRDX6 (1.47-fold, P = 0.01), and CPSF3 (1.44-fold, P = 0.02).Conclusion:The difference in expression of antioxidant enzymes between KC and NL suggests that the oxidative stress imbalances found in KC are caused by defects in ROS/RNS removal rather than increased ROS/RNS production.
Highlights
Keratoconus (KC) is a progressive corneal thinning disorder that causes irregular astigmatism and reduced vision
Studies were performed to determine if the ROS/RNS levels in NL and KC cultures could be altered by pre‐treatment with apocynin, an inhibitor of NADPH oxidase, or N‐LLA, an inhibitor of NOS
The KC cultures (n = 5) had a 1.8‐fold increase in ROS/RNS levels compared to untreated NL cultures (P = 0.004), which could not be reversed by pre‐treatment with apocynin (P = 0.9) or N‐LLA (P = 0.8; Figure 3). These inhibitor studies indicated that the higher ROS/RNS levels found in KC cultures could not be reversed by blocking the NADPH oxidase or NOS pathways
Summary
Keratoconus (KC) is a progressive corneal thinning disorder that causes irregular astigmatism and reduced vision. It affects approximately 1 in 375 individuals and is an indication for corneal transplant surgery.[1,2,3,4] In about 6%–10% of cases, KC reportedly has an autosomal dominant inheritance pattern with incomplete penetrance.[5,6,7] The mechanism(s) causing KC are not known but are thought to involve a combination of genetic and environmental factors. Corneas with KC have elevated levels of mitochondrial DNA (mtDNA) damage along with decreased levels of mitochondrial‐encoded cytochrome c oxidase (complex IV subunit 1, MTCO1), consistent with mitochondrial dysfunction.[15]
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