The combined effect of drusen component amyloid beta and oxidative stress on polarized retinal pigment epithelium

Abstract

Purpose : Multiple risk factors contribute to the development of age-related macular degeneration (AMD), but less is known of the combined effect of these risk factors on the retinal pigment epithelium (RPE), the cell type first injured in the disease process. Here we investigated the combined effect of two risk factors – a drusen component (amyloid beta, Aβ) and oxidative stress on RPE in terms of expression of inflammatory mediators and complement genes using a polarized cell model of ARPE19.

Methods : To measure NF-κB activation, ARPE19/NF-κB-luciferase reporter cells were established and cultured on Transwell inserts to form a polarized monolayer. The cells were exposed on the apical surface to oxidative stressor H₂O₂ and/or basolaterally to oligomeric Aβ_1-40. Gene expression of inflammatory mediators (caspase-1, IL-18, IL-6, IL-8, MCP-1, TNF-α) and complement components (CD46, CD55, CD59, factor B, factor D, factor H, factor I, C3 and C5) was measured by quantitative polymerase chain reaction (PCR). Results were analyzed with Student’s t test and significance was set at p <0.05.

Results : The combined stimulation with H₂O₂ apically and Aβ basolaterally promoted NF-κB activation, upregulated IL-18 and MCP-1 (mean fold change normalized to control ± standard error of mean: 2.7±0.1, 3.8±0.3, and 1.5±0.0, respectively, N=3). Stimulation with Aβ or H₂O₂ alone induced an upregulation of IL-18 (2.4±0.5, 4.3±0.5, N=3) and exposure to H₂O₂ alone also caused NF-κB activation (2.6±0.3, N=3). Single stimulation had no effect on MCP-1 expression. Regarding the complement genes, the combined stimulation upregulated CD46, CD55, and C3 (1.5±0.1, 1.5±0.1, and 1.6±0.4, respectively), while stimulation with Aβ or H₂O₂ alone had no effect on any of complement components studied.

Conclusions : The combined effect of drusen component Aβ and oxidative stressor H₂O₂ achieved a greater effect on RPE compared to either stimulus alone. Our results suggest there may be a differential response to stimuli on the apical and basal surfaces and multiple risk factors may contribute to AMD disease processes through regulation of inflammatory and complement genes. Future studies will allow further insight into the interactions between risk factors to better understand AMD pathogenesis.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.