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Martin Busch, Susanne Wasmuth, Albrecht Lommatzsch, Solon Thanos, Daniel Pauleikhoff; Proteomic changes in ARPE-19 cells stimulated with complement serum and UV-irradiated photoreceptor outer segments (UV-POS). Invest. Ophthalmol. Vis. Sci. 2016;57(12):6045.
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© ARVO (1962-2015); The Authors (2016-present)
ARPE-19 cells as a model for retinal pigment epithelial (RPE) cells were shown to undergo proinflammatory and proangiogenic changes upon complement stimulation and UV-POS treatment suggesting a role in AMD pathology. The aim of our study was to reveal potential intracellular signaling pathways mediating these functional changes of RPE cells using proteomics and stimulation with complement serum and UV-POS.
For complement stimulation, ARPE-19 cells were incubated in DMEM/F12 medium supplemented with 5 % human complement serum (HCS) for 24 hours. Medium alone or supplemented with either 5 % heat-inactivated HCS or C7-deficient serum were used as controls. Further groups of cells were pre-treated every other day with 10 µg/ml UV-POS for one week to a total of three pre-treatments. Afterwards the cells were washed and incubated in medium containing 5 % HCS or medium alone. Proteins were separated by 2D-gel electrophoresis and differentially occurring protein spots were peptide mapped with MALDI-TOF mass spectrometry.
Some protein spots were differentially expressed in the various treatment groups and differed between two independent experimental sets. From the two experimental sets, a total of 27 protein spots were selected and processed for MALDI-TOF analysis that identified a total of 56 different proteins, which could be grouped into 7 functional entities including metabolic (12), structural (16), globular (3), cell-cell and cell-matrix interaction-associated proteins (2), gene expression (4), proteins related to inflammatory processes and angiogenesis (7), and proteins involved in signal transduction (10). 60 percent of the identified proteins participating in signal transduction are associated with cellular and oxidative stress response. Out of two cases, expression of these proteins was observed in response to HCS, UV-POS, or their combination.
The results show that complement stimulation and UV-POS uptake regulate the cell proteome. Further experiments are required to clarify how far the identified differentially expressed proteins associated with signal transduction are involved in the mediation of the functional changes of RPE cells in response to complement stimulation and UV-POS.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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