The use of hyperoxia to induce chronic mild oxidative stress in RPE cells in vitro.
OBJECTIVE
To establish a model of mild and chronic oxidative stress using hyperoxia for retinal pigment epithelial (RPE) cells in vitro.


METHODS
RPE340 cells and WI38 lung fibroblasts were grown in normal oxygen (20% O2) and hyperoxia (40% O2 or 60% O2). After cell viability was examined, the levels of reactive oxygen intermediates (ROI) by flow cytometry and heme oxygenase-1 (HO-1) mRNA by northern analysis were measured as markers of oxidative stress in both cell types. Proliferative ability and gene expression pattern of growth factors were studied to demonstrate the phenotypic changes induced by mild oxidative stress upon these cells.


RESULTS
While decreased by 60% O2, 40% O2 did not affect viability in both cell types, ROI production and HO-1 mRNA expression were elevated in hyperoxia compared to controls, but were inhibited with the antioxidant dehydro-ascorbic acid (DHA). The proliferation of cells by hyperoxia was inhibited in both cell types. The expression of growth factors induced by hyperoxia was cell type dependent. Fibroblast growth factor-2 mRNA was unchanged in RPE cells, but was increased in fibroblasts. Transforming growth factor-beta2 was decreased in RPE cells, but unchanged in fibroblasts. Vascular endothelial growth factor was downregulated in RPE cells, while upregulated in fibroblasts. Connective tissue growth factor was decreased in RPE cells, but was unchanged in fibroblasts.


CONCLUSIONS
The results demonstrate that hyperoxia induces mild oxidative stress which alters the phenotype of cells in a cell type specific manner.
Department of Ophthalmology, University of California, Davis, CA 95616-8794, USA.
S
S
Honda
L M
LM
Hjelmeland
J T
JT
Handa
D015152:Blotting, Northern; D002455:Cell Division; D002460:Cell Line; D002470:Cell Survival; D002908:Chronic Disease; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005434:Flow Cytometry; D015870:Gene Expression; D006419:Heme Oxygenase (Decyclizing) / Q000235:genetics; D051547:Heme Oxygenase-1; D006801:Humans; D018496:Hyperoxia / Q000378:metabolism*; D007223:Infant; D008168:Lung / Q000166:cytology / Q000378:metabolism; D008565:Membrane Proteins; D018384:Oxidative Stress; D010641:Phenotype; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D017382:Reactive Oxygen Species
D008565:Membrane Proteins; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; C496541:HMOX1 protein, human; D006419:Heme Oxygenase (Decyclizing); D051547:Heme Oxygenase-1