Hypersensitive response to oxidative stress in keratoconus corneal fibroblasts.
OBJECTIVE
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 (H(2)O(2)). 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 (DeltaPsim) 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 DeltaPsim (P = 0.015) and increased activation of caspase-9 (P = 0.013) and -12 (P = 0.01) compared with normal cultures. Changes in DeltaPsim were independent of cathepsin inhibition. The combination of low-pH+H(2)O(2) 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.
Department of Ophthalmology, University of California, Irvine Medical Center, Orange, California 92868, USA.
Marilyn
M
Chwa
Shari R
SR
Atilano
Dieter
D
Hertzog
Hong
H
Zheng
Jonathan
J
Langberg
Dae W
DW
Kim
M Cristina
MC
Kenney
D000328:Adult; D015153:Blotting, Western; D053454:Caspase 12 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D056657:Cathepsin K; D002403:Cathepsins / Q000235:genetics; D003319:Corneal Stroma / Q000473:pathology*; D004272:DNA, Mitochondrial / Q000235:genetics; D003576:Electron Transport Complex IV / Q000235:genetics; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity*; D006863:Hydrogen-Ion Concentration; D007640:Keratoconus / Q000473:pathology*; D053078:Membrane Potential, Mitochondrial; D008875:Middle Aged; D018384:Oxidative Stress; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism
D004272:DNA, Mitochondrial; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D006861:Hydrogen Peroxide; C117108:cytochrome C oxidase subunit II; D003576:Electron Transport Complex IV; D002403:Cathepsins; C506444:CASP12 protein, human; D053454:Caspase 12; D053453:Caspase 9; C534130:CTSK protein, human; D056657:Cathepsin K