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G. B. Walker, T. Lin, K. Kurji, S. S. Prasad, O. L. Moritz, J. Z. Cui, J. A. Matsubara; Altered Cytokine Profiles Secreted by Human Retinal Pigment Epithelium: I. Drusen Components. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6165.
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© ARVO (1962-2015); The Authors (2016-present)
Drusen are hallmark extracellular deposits associated with the dry form of age related macular degeneration (AMD). Stimulation of human retinal pigment epithelial cells (hRPE) with components of drusen, advanced glycation end products (AGEs) or amyloid beta (Aβ) in vitro, result in gene expression changes associated with inflammatory and immune response pathways as identified by microarrays analyses. This study focuses on secreted levels of cytokine and growth factors in culture medium after stimulation with drusen components in human RPE cells in vitro.
We used xMAP technology (BioPlex) and a panel of antibodies against 27 cytokines and 3 growth factors to detect their secreted levels in medium after stimulation with Aβ (0.3 µg/ml, 12 and 24 hrs) or AGE (10µg/ml, 24 hrs) on primary culture of hRPE cells. Experiments were repeated three times and significant changes were considered when p<0.05 and fold changes ≥ 2.0. Standard curves were generated and the absolute values of secreted cytokines reported using a high sensitivity assay with accuracy across a range of 1 to 10,000 pg/ml.
AGE stimulation resulted in the upregulation of 12 genes and downregulation of 5 genes that reached significance. Of the upregulated, secreted genes, 10 (of 27) were cytokines and 2 (of 3) were growth factors. The highest levels of fold changes were observed for IL2, IL17, FGFb, PDGFbb. The five downregulated genes after AGE stimulation were all cytokines. Aβ stimulation resulted in the significant secretion of 5 genes. Two of the 5 were angiogenic factors, bFGF and VEGF. The others included proinflammatory cytokines, IL17 and IL9, and an anti-inflammatory cytokine, IL1ra.
AGE and Aβ stimulation results in a pattern of secreted cytokines and growth factors that are proinflammatory and proangiogenic in hRPE in vitro. Future studies are needed to determine if drusen components promote proinflammatory and proangiogenic processes in RPE cells in vivo.
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