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M.J. Radeke, M.K. Staples, A.L. Lightfoot, D.H. Anderson, L.V. Johnson; Changes in RPE Cell Expression Induced by Tobacco Smoke Constituents . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1719.
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Purpose: The risk of acquiring age-related macular degeneration (AMD) is significantly increased amongst both male and female smokers. The molecular basis of this association is unclear, however it is theorized to be due to the effects of specific molecular constituents of smoke, oxidative stress, and/or tissue hypoxia. In order to gain insight into this pathogenic relationship, we assessed the changes in gene expression induced in retinal pigmented epithelial (RPE) cells in response to tobacco smoke extract and oxidizing agents. Methods: Confluent cultures of primary fetal human RPE cells and ARPE19 cells were exposed to an aqueous extract of tobacco smoke, or the oxidizing agents, hydrogen peroxide or t-butyl hydroperoxide, for varying times (0-24 hours). Changes in gene expression were identified using DNA microarray analysis and quantified using SYBR Green I based real time quantitative PCR. Results: Quantitative PCR identified several genes with expression levels that changed significantly following treatment of RPE cells with oxidizing agents. These genes included the angiogenic factors, angiopoietin 1 and 2; a gene associated with anti-oxidant activity, heme oxygenase 1; cell stress genes, ubiquitin C and alpha B-crystallin; and complement-related genes: C2, C7, C9, and C5a receptor. Preliminary data from cells exposed to smoke extract suggest that the RPE responds similarly by increasing the transcript levels of heme oxygenase 1, ubiquitin C, and complement genes. Microarray analyses of smoke-treated cells showed a 50-fold induction in nuclear respiratory factor 1, a transcription factor that binds to the anti-oxidant response element and regulates numerous genes involved in the cellular response to oxidative and chemical stress. Conclusions: This initial characterization of the response of RPE cells to tobacco smoke extract or oxidizing agents suggests that the positive correlation between smoking and AMD may, in part, be the result of increased oxidative stress and/or exposure to reactive oxygen species. In addition, the finding of increased expression of complement components, ubiquitin C, and alpha B-crystallin, each of which is associated with drusen and/or RPE cells in close proximity to drusen, suggest a potential molecular mechanism for smoking’s link to AMD.
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