Density-dependent expression of FGF-2 in response to oxidative stress in RPE cells in vitro

Mitsumasa Wada, Claire Mazow Gelfman, Hiroshi Matsunaga, Mitra Alizadeh, Lawrence S Morse, James T. Handa, Leonard M Hjelmeland

Research output: Contribution to journalArticle

31 Citations (Scopus)

Abstract

Purpose. The purpose of this study is to demonstrate the effect of culture density on the steady state mRNA levels of fibroblast growth factor-2 (FGF-2) when retinal pigment epithelial (RPE) cells are subjected to oxidative stress in vitro. Methods. Subconfluent and confluent cultures of the established RPE cell line ARPE-19, were treated with increasing concentrations of tert-butyl hydroperoxide (tBH) or hydrogen peroxide (H2O2). Cell viability was measured using the WST-1 assay, and intracellular reactive oxygen intermediate (ROI) production was quantified by dichlorofluoroscein (DCF) fluorescence. Steady state changes in heme oxygenase-1 (HO-1) and FGF-2 mRNAs were measured by Northern blot analysis. Results. Confluent cultures of ARPE-19 cells were less susceptible than subconfluent cultures to the toxic effects of the chemical oxidants. The intracellular reactive oxygen intermediate production was higher in subconfluent than confluent cultres with increasing tBH concentration. At nontoxic concentrations of tBH and H2O2, a dose dependent increase in FGF-2 expression was seen as a function of culture density. FGF-2 mRNA expression was induced after tBH treatment in subconfluent, but not confluent cells. On the other hand, FGF-2 mRNA induction was observed after H2O2 treatment in confluent, but not subconfluent cultures. In contrast, no density dependent induction of HO-1 mRNA was seen after treatment with either tBH or H2O2. Conclusions. These results suggest that care should be taken to control for cell density in similar types of in vitro experiments.

Original languageEnglish (US)
Pages (from-to)226-231
Number of pages6
JournalCurrent Eye Research
Volume23
Issue number3
DOIs
StatePublished - 2001

Fingerprint

tert-Butylhydroperoxide
Retinal Pigments
Fibroblast Growth Factor 2
Oxidative Stress
Epithelial Cells
Messenger RNA
Heme Oxygenase-1
Oxygen
Poisons
Oxidants
Northern Blotting
Hydrogen Peroxide
Cell Survival
Cell Count
Fluorescence
In Vitro Techniques
Cell Line

Keywords

  • Basic fibroblast growth factor
  • Fluorescent probes
  • Free radicals
  • Oxidative damage
  • Retinal pigment epithelium

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems

Cite this

Density-dependent expression of FGF-2 in response to oxidative stress in RPE cells in vitro. / Wada, Mitsumasa; Gelfman, Claire Mazow; Matsunaga, Hiroshi; Alizadeh, Mitra; Morse, Lawrence S; Handa, James T.; Hjelmeland, Leonard M.

In: Current Eye Research, Vol. 23, No. 3, 2001, p. 226-231.

Research output: Contribution to journalArticle

Wada, Mitsumasa ; Gelfman, Claire Mazow ; Matsunaga, Hiroshi ; Alizadeh, Mitra ; Morse, Lawrence S ; Handa, James T. ; Hjelmeland, Leonard M. / Density-dependent expression of FGF-2 in response to oxidative stress in RPE cells in vitro. In: Current Eye Research. 2001 ; Vol. 23, No. 3. pp. 226-231.
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AU - Hjelmeland, Leonard M

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N2 - Purpose. The purpose of this study is to demonstrate the effect of culture density on the steady state mRNA levels of fibroblast growth factor-2 (FGF-2) when retinal pigment epithelial (RPE) cells are subjected to oxidative stress in vitro. Methods. Subconfluent and confluent cultures of the established RPE cell line ARPE-19, were treated with increasing concentrations of tert-butyl hydroperoxide (tBH) or hydrogen peroxide (H2O2). Cell viability was measured using the WST-1 assay, and intracellular reactive oxygen intermediate (ROI) production was quantified by dichlorofluoroscein (DCF) fluorescence. Steady state changes in heme oxygenase-1 (HO-1) and FGF-2 mRNAs were measured by Northern blot analysis. Results. Confluent cultures of ARPE-19 cells were less susceptible than subconfluent cultures to the toxic effects of the chemical oxidants. The intracellular reactive oxygen intermediate production was higher in subconfluent than confluent cultres with increasing tBH concentration. At nontoxic concentrations of tBH and H2O2, a dose dependent increase in FGF-2 expression was seen as a function of culture density. FGF-2 mRNA expression was induced after tBH treatment in subconfluent, but not confluent cells. On the other hand, FGF-2 mRNA induction was observed after H2O2 treatment in confluent, but not subconfluent cultures. In contrast, no density dependent induction of HO-1 mRNA was seen after treatment with either tBH or H2O2. Conclusions. These results suggest that care should be taken to control for cell density in similar types of in vitro experiments.

AB - Purpose. The purpose of this study is to demonstrate the effect of culture density on the steady state mRNA levels of fibroblast growth factor-2 (FGF-2) when retinal pigment epithelial (RPE) cells are subjected to oxidative stress in vitro. Methods. Subconfluent and confluent cultures of the established RPE cell line ARPE-19, were treated with increasing concentrations of tert-butyl hydroperoxide (tBH) or hydrogen peroxide (H2O2). Cell viability was measured using the WST-1 assay, and intracellular reactive oxygen intermediate (ROI) production was quantified by dichlorofluoroscein (DCF) fluorescence. Steady state changes in heme oxygenase-1 (HO-1) and FGF-2 mRNAs were measured by Northern blot analysis. Results. Confluent cultures of ARPE-19 cells were less susceptible than subconfluent cultures to the toxic effects of the chemical oxidants. The intracellular reactive oxygen intermediate production was higher in subconfluent than confluent cultres with increasing tBH concentration. At nontoxic concentrations of tBH and H2O2, a dose dependent increase in FGF-2 expression was seen as a function of culture density. FGF-2 mRNA expression was induced after tBH treatment in subconfluent, but not confluent cells. On the other hand, FGF-2 mRNA induction was observed after H2O2 treatment in confluent, but not subconfluent cultures. In contrast, no density dependent induction of HO-1 mRNA was seen after treatment with either tBH or H2O2. Conclusions. These results suggest that care should be taken to control for cell density in similar types of in vitro experiments.

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