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Susanne Wasmuth, Martin Busch, Albrecht Lommatzsch, Daniel Pauleikhoff; Interplay of zinc, complement, and retinal pigment epithelial cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):6558. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Compared to the rest of the body, retina and retinal pigment epithelial (RPE) cells hold strikingly high zinc concentrations that are decreased in age-related macular degeneration (AMD). Genetic alterations point to an over-activation of the complement system in AMD patients and previous studies showed functional impact of complement on RPE cells that undergo cell death during AMD. Therefore, in this in vitro study, the influence of complement on zinc-supplemented RPE cells was examined.
Human ARPE-19 cells as RPE cell model were incubated with increasing concentrations of zinc sulphate and zinc chloride alone and in combination with human complement serum (HCS). Controls included heat-inactivated HCS, C7-deficient control sera, or bovine serum albumin (BSA). Viability was monitored by conversion of thiazolylblue, uptake of propidiumiodide, and Hoechst staining. In the cell culture supernatants the content of reactive oxygen species (ROS) were measured. Further, production of interleukin (IL)-6, IL-8, monocyte chemoattractant protein (MCP)-1, and vascular endothelial growth factor (VEGF) was assayed by sandwich ELISA.
RPE cells incubated with >150 µM of both zinc compounds showed decreased viability after single administration for 24 hours. No obvious toxicity was demonstrated by 25-50 µM zinc for several weeks. All serum formulations including HCS attenuated the toxicity of high dose and short term-added zinc, while BSA was less effective. After 48 hours, the HCS group showed impaired morphology comparable to BSA-treated cells. In contrast, the cells treated with HI-HCS and especially those with C7-deficient serum displayed a better appearance. There was a tendency of higher ROS amounts by zinc-treatment and by HCS-treatment. The production of cytokines and VEGF was enhanced by HCS and unaffected by zinc.
The degeneration of zinc-rich RPE cells may poison the retinal microenvironment during AMD. Unspecific binding of potentially toxic free zinc ions by serum proteins like albumin may provide in part the initially observed protection by HCS. On the other hand negative side-effects like zinc-induced complement-activation may predominate after longer incubation period. Increased ROS suggest a stress response towards zinc and complement.
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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