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David M. Rabin, Sally Temple, Jeffrey H. Stern; Chronic Oxidative Stress Up-regulates Drusen-related Protein Synthesis in Primary Human RPE Cells: A Novel Culture Model for the Study of Age-Related Macular Degeneration. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3912.
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To examine changes in human retinal pigment epithelium (RPE) in response to chronic stress in a novel culture model for Age-Related Macular Degeneration (AMD) and, specifically, to determine if chronic oxidative stress induces the RPE to up-regulate drusen-related protein expression.
Primary human RPE cells isolated from the eyes of adult donors with and without AMD and human fetal RPE were cultured on Costar transwells with placental extracellular matrix for 3 weeks until a confluent polarized monolayer developed. RPE were exposed to sublethal doses (250-1000uM) tert-butylhydroperoxide (TBHP) or vehicle in media for 1 hour/day for 5 days followed by two days in normal media. Following stress treatment, all media and cells were collected for drusen-related protein and mRNA analysis.
In this model of chronic stress in the cultured RPE, a significant up-regulation in drusen-related proteins occurred in a dose- and time-dependent manner. RPE cells exposed to 5 days of oxidative stress demonstrated an up-regulation in crystallin proteins and other molecular chaperones, pro-angiogenic factors, and other drusen-related proteins. These proteins found to be up-regulated in the in vitro model are up-regulated in RPE of human AMD eyes and serve as molecular markers for AMD.
The in vitro RPE model of chronic oxidative stress demonstrates that reactive oxygen species alone are capable of inducing the RPE to up-regulate expression of drusen-related proteins. This AMD model demonstrates that cells change significantly when exposed to chronic stress conditions (>48 hours) and that acute stress conditions (<48 hours) in culture may be less optimal for modeling of diseases where chronic stress is implicated. The results of this study suggest that the initial formation of drusen in dry AMD could be in large part initiated by chronic oxidative stress. The results of this study confirm this long held hypothesis about the pathophysiologic mechanism of drusen formation and suggest specific targets involved in the oxidative stress response as new targets for the development of therapeutics for dry AMD.
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