Purchase this article with an account.
E. Chaum, H. Yang, X. Yang, J.C. Lang; Molecular Responses to Oxidative and Non-Oxidative Stress in the Retinal Pigment Epithelium . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1626.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: The focus of these studies is to characterize the molecular responses to oxidative and non-oxidative stress in the human retinal pigment epithelium (RPE). Methods: We analyzed changes in the expression of a panel of genes following oxidative stress (OS) and heat shock stress (HS) in vitro, including: Lens epithelial-derived growth factor (LEDGF), Heat shock protein 27 (Hsp27), Alpha B-crystallin (CRYAB), and Nuclear factor kappa-B (NF-kB). Confluent cultures of explanted RPE cells were treated with either 200µM H2O2 (37°C, 1 hour) or heat shock (42°C, 20 minutes). Total RNA was isolated from replicate cultures immediately following treatment, and after 2, 4, 6, and 24 hours. RT-PCR was performed with 1 µg of total cellular RNA, followed by standard amplification using gene-specific primers. Semi-quantitative analysis of the amplification products was performed on digitized images normalized to ß-actin levels and compared to controls. Results: LEDGF transcription decreased by 50% after both stressors and remained at similarly low levels over the first six hours. After 24 hours, LEDGF transcription returned to baseline levels in cells exposed to OS but increased 3-fold in cells exposed to HS. CRYAB transcription increased by 40% over the first 2 hours after HS, but decreased 40% following OS. Both treatments resulted in an eventual decline in expression to 40% of baseline over 24 hours. Hsp27 transcription increased by 40% in the first 2 hours after treatment by both stressors. Transcription remained at a steady state and elevated level in HS treated cells, but declined to baseline levels after 24 hours following OS. NF-kB transcription increased 2-fold and 5-fold following OS and HS, respectively. Transcription remained elevated after HS but declined to baseline levels after 24 hours following oxidative stress. Conclusions: The human RPE demonstrates characteristic and quantifiable molecular responses to induced sublethal stress in vitro. These studies suggest that the degree and timing of stress-specific responses correlate with specific changes in gene expression in the RPE in response to both oxidative and non-oxidative stressors.
This PDF is available to Subscribers Only