IOVS The Oncologist
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Originally published In Press as doi:10.1167/iovs.08-1969 on May 30, 2008
 (Investigative Ophthalmology and Visual Science. 2008;49:4361-4369.)
© 2008 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.08-1969
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Hypersensitive Response to Oxidative Stress in Keratoconus Corneal Fibroblasts

Marilyn Chwa,1 Shari R. Atilano,1 Dieter Hertzog,2 Hong Zheng,3 Jonathan Langberg,4 Dae W. Kim,1 and M. Cristina Kenney1

1From the Department of Ophthalmology, University of California, Irvine Medical Center, Orange, California; the 2Loma Linda Medical School, Loma Linda, California; the 3Western University of Health Sciences, Pomona, California; and the 4CVRI (Cardiovascular Research Institute), University of California, San Francisco, California.

PURPOSE. It is unclear whether the oxidative damage found in keratoconus (KC) corneas results from innate defects of corneal fibroblasts or is due to excessive environmental challenges encountered by the patient with KC. The purpose of this study was to explore whether KC cells have inherent, exaggerated hypersensitivity to oxidative stressors.

METHODS. Normal and KC corneal stromal fibroblasts were incubated in neutral or low-pH conditions, with or without hydrogen peroxide (H2O2). Reactive oxygen/nitrogen species (ROS/RNS) production was measured with 2',7'-dichlorodihydrofluorescein diacetate dye. Caspase-9 and -12 activities were measured by fluorochrome inhibitor to caspase (FLICA) assays. Long-extension polymerase chain reaction (LX-PCR) was used to amplify mtDNA. RNA was extracted, full-length cDNA synthesized, and PCR performed for mitochondria-encoded genes. Mitochondrial membrane potential ({Delta}{Psi}m) was measured by a cationic dye assay.

RESULTS. In neutral pH conditions, KC fibroblasts had increased ROS production (P = 0.047), higher RNA levels for cytochrome c oxidase (complex IV) subunit II (P < 0.05), and decreased cathepsin K RNA (P = 0.04) compared with levels in normal cultures. In low-pH conditions, KC fibroblasts had decreased {Delta}{Psi}m (P = 0.015) and increased activation of caspase-9 (P = 0.013) and -12 (P = 0.01) compared with normal cultures. Changes in {Delta}{Psi}m were independent of cathepsin inhibition. The combination of low-pH+H2O2 treatment degraded intact mtDNA and decreased the mtDNA-to-nuclear DNA ratio.

CONCLUSIONS. Cultured KC fibroblasts have an inherent, hypersensitive response to oxidative stressors that involves mitochondrial dysfunction and mtDNA damage. KC fibroblast hypersensitivity may play a role in the development and progression of keratoconus.






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