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J. A. Matsubara, K. Kurji, J. Cui, T. K. Lin, G. Walker, S. Prasad; Amyloid Beta (Aβ) Stimulation Alters Inflammatory Gene Expression in Cultured Human Retinal Pigment Epithelial Cells (RPE). Invest. Ophthalmol. Vis. Sci. 2009;50(13):710.
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© ARVO (1962-2015); The Authors (2016-present)
Age related macular degeneration (AMD) is the most common cause of irreversible vision loss in the elderly. Two important factors associated with AMD disease progression are oxidative stress and retinal inflammation. Extensive proteomic analyses of drusen identified many oxidative modified proteins, advanced glycation end products (AGEs), amyloid beta (Aβ), apolipoproteins B/E, and activated components/regulators of the complement system. It is hypothesized that constituents of drusen may trigger inflammation and complement activation. The current study investigates the effects of one drusen component, Aβ, on human RPE in vitro.
Cell viability assays were performed in serum-free DMEM containing concentrations of oligomeric 1-40 Aβ ranging from 0.01 to 1.0 µM. Cell survival was determined 24 hours later using MTT assay (3-[4, 5-dimethyl-thiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide). Genome-wide changes in gene expression of RPE stimulated with Aβ (0.3µM) were studied with Agilent Oligo microarrays and verified by quantitative RT-PCR. ELISA analysis was performed to correlate gene expression and secreted protein levels.
MTT assay demonstrated a dose dependent cytotoxicity of Aβ on RPE. Aβ stimulation at 0.01µM, 0.3µM and 1.0µM resulted in 82%, 60%, and 44% RPE cell viability, respectively. RPE cells stimulated with 0.3 µM Aβ resulted in 93 up- and 34 down-regulated genes that were greater than the 1.5 fold threshold used in this study. The bulk of these genes were inflammatory genes, including interleukin IL-1b (+4.8 fold), IL-8 (+2.8 fold), interferon, alpha-inducible protein 27 (IFI 27, +1.81 fold), chemokine (C-X-C motif) ligand 2 (CXCL2, +1.68) and complement factor I (CFI, +1.54 fold). qRT-PCR verified the direction and magnitude of the expression levels of these genes. Other genes represented apoptotic, angiogenic, extracellular matrix, and transcription factor related functional groups. ELISA confirmed secreted level of IL8 (190 pg/ml) was 2 fold higher than control.
Aβ, a component of drusen, promotes expression of inflammatory cytokines, ROS production and complement inhibitors in RPE cells, in vitro. Our results reveal a correlative link between Aβ and proinflammatory events in RPE that may play a role in chronic inflammation and complement activation in the retina of AMD eyes. While our in vitro studies demonstrate proinflammatory events triggered by Aβ, future studies are needed to identify the mechanism and timecourse of Aβ deposition in drusen in AMD eyes.
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