Title of article :
Corneal Oxidative Damage in Keratoconus Cells due to Decreased Oxidant Elimination from Modified Expression Levels of SOD Enzymes, PRDX6, SCARA3, CPSF3, and FOXM1
Author/Authors :
Atilano, Shari R Department of Ophthalmology - Gavin Herbert Eye Institute - University of California Irvine - Irvine, CA, USA , Lee, Daniel H Department of Ophthalmology - Gavin Herbert Eye Institute - University of California Irvine - Irvine, CA, USA , Fukuhara, Paula S Department of Ophthalmology - Gavin Herbert Eye Institute - University of California Irvine - Irvine, CA, USA , Chwa, Marilyn Department of Ophthalmology - Gavin Herbert Eye Institute - University of California Irvine - Irvine, CA, USA , Nesburn, Anthony B Department of Ophthalmology - Gavin Herbert Eye Institute - University of California Irvine - Irvine, CA, USA , Udar, Nitin Department of Ophthalmology - Gavin Herbert Eye Institute - University of California Irvine - Irvine, CA, USA , Kenney, M. Cristina Department of Ophthalmology - Gavin Herbert Eye Institute - University of California Irvine - Irvine, CA, USA
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 H2DCFDA 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.
Keywords :
CPSF3 , FOXM1 , Keratoconus , PRDX6 , SCARA3 , SOD
