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B. Rohrer, K. Kunchithapautham, B. Renner, M. Bandyopadhyay, N. Perron, V. P. Ferreira, M. K. Pangburn, Z. Ablonczy, S. Tomlinson, J. M. Thurman; A Dual-Hit Hypothesis in Retinal Pigmented Epithelial Cell Dysfunction: Oxidative Stress Renders Cells Susceptible to Complement-Mediated Injury. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3521.
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Genetic variations in complement factor H (fH), an inhibitor of the alternative complement pathway (AP), and oxidative stress are associated with all forms of age-related macular degeneration (AMD). Here we hypothesized whether oxidative stress renders the RPE more susceptible to complement activation.
Human embryonic RPE or ARPE-19 were grown as monolayers on transwell plates. Effects on RPE tight junctions were determined by transepithelial electrical resistance (TER) measurements and immunohistochemistry. Endogenous complement inhibitors were analyzed by flow-cytometry and immunohistochemistry, and VEGF secretion by ELISA. Analysis of long-term effects on RPE cell physiology included analysis of energy metabolism, ROS uptake and cell death assays.
(1) At low H2O2 levels or when exposed to serum alone, RPE monolayers have a stable TER. TER deteriorated rapidly in H2O2-exposed monolayers upon adding serum as a source of complement, which was concomitant with a disruption of junctional proteins. The effect was dependent upon sublytic activation of the membrane attack complex. (2) H2O2 reduced levels of CD55 and CD59 cell-surface complement inhibitors and impaired fH activity. (3) Only H2O2 + serum led to C3-deposition and apical VEGF secretion. (4) TER could be stabilized and VEGF secretion blunted in H2O2 + serum treated cells by inhibiting the AP or VEGF-receptor-2. (5) Long-term H2O2 + serum impaired RPE physiology and ATP production, but did not result in apoptosis.
While RPE cells are resistant to low level oxidative stress, oxidative stress sensitizes RPE cells to effects of a misregulated complement system, resulting in VEGF secretion and disruption of RPE barrier integrity. Long-term oxidative stress and complement exposure impairs RPE physiology and energy metabolism. In summary, we have developed a model whereby oxidative stress in RPE can be linked to the molecular events involved in AMD, including activation of the AP and local production of VEGF.
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