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Impressum
aB-Crystallin in Retinal Pigment Epithelium is Inducible by Oxidative Stress and TGF-ß
Alge C., Welge-Lüßen U.
Augenklinik der Ludwig-Maximilians-Universität, München
Purpose: Age related macular degeneration (AMD) is the main cause
of blindness in western countries. Oxidative stress and certain growth
factors (TGF-ß, VEGF, FGF) appear to play a contributive role in
the pathogenesis of this degenerative disorder. To this date the influence
of these conditions on the expression of stress proteins in retinal pigment
epithelium (RPE) has not been investigated. It was the aim of this study
to investigate the expression of the small heat shock protein aBCrystallin,
which is expressed at elevated levels in various neurodegenerative disorders,
in response to oxidative stress and growth factors.
Materials and Methods: RPE derived from 6 normal human donor eyes
was analyzed for aB-Crystallin expression by RT-PCR and western-blot
analysis. Monolayer cultures of RPE cells from 5 donors were treated with
1,0 ng/ml TGF-ß, 200 pg/ml bFGF and 10 ng/ml VEGF for 12-72 hours.
Oxidative stress was induced by exposure of the cells to H2O2
(2 µMol) for 30 minutes. Cells were harvested 24 -72 hours after
treatment. Induction of aB-Crystallin was assessed by western- and northern-blot
analysis.
Results: aB-Crystallin is constitutively being expressed
in RPE under in vivo and in vitro conditions. Treatment of cultured RPE
cells with TGF-ß and H2O2 significantly increased
aB-Crystallin mRNA levels (6-10x), whereas the effect on the protein
level was only small (2-3x). The presence of Actinomycin, an inhibitor
of mRNA synthesis, markedly inhibited aB-Crystallin induction on
mRNA and protein level. The other growth factors showed no stimulatory
effect on aB-Crystallin expression.
Conclusion: In the present study we demonstrate for the first time
that in RPE aBCrystallin is inducible by oxidative stress and TGF-ß
under in vitro conditions. Blocking the effect by Actinomycin indicates
that the observed aB-Crystallin induction is effected at the level
of transcription. These findings suggest that aBCrystallin, which
is involved in the pathogenesis of neurodegenerative disorders, may also
play a role in the pathogenesis of AMD.
Grant Support: Forschungsförderung der DOG, DFG WE 2577/2-1 Department
of Ophthalmology, Ludwig-Maximilians University, Mathildenstr. 8, D- 80336
München