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Eszter Emri, Sascha Dammeier, Franziska Klose, Lajos Csincsik, Marius Ueffing, Imre Lengyel; Precision medicine using primary human foetal retinal pigment epithelial cells: effects of zinc supplementation. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3989.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal pigment epithelial (RPE) cells can be used as a model system to study sub-RPE deposit formation, the hallmark of age-related macular degeneration (AMD). In AMD, cellular zinc levels decline. Zinc supplementation can attenuate the progression to late AMD, potentially at least in part, via inhibiting complement catabolism. However, the molecular pathways involved in the effect of zinc are not fully explored. In this study, we examined the effect of changing apical or basal zinc in our primary RPE cell model.
We cultured primary human foetal RPE cells from 3 individuals on transwell inserts (Corning), coated with Geltrex. After one week, the apical or basal compartments were replaced with medium containing 125 uM added zinc or left untreated and cultured for a further 4 weeks. Thereafter, apical and basal secretomes and the RPE cells were harvested for genotyping, RNAseq, lipidomics and proteomics analysis or fixed for fluorescence and electron microscopy (EM).
All 3 cell cultures showed pigmentation, high transepithelial resistance (TER), ZO-1 staining and accumulation of sub-RPE deposit on EM. TER values varied in between individual cultures with 1114, 477 or 244 Ohm*cm2. Qualitative differences were detected in cell pigmentation, showing individual differences between the cell lines. When cells were treated with zinc apically or basally, an increase in TER values could be measured in all 3 treated cultures (in average 728±337 and 662±245 Ohm*cm2, respectively vs. 611±260 in controls). In depth analysis of RNAseq data support the phenotypic differences. Based on principal component analysis the 3 cell cultures feature 3 clusters. Comparing zinc treatment to untreated controls show statistically significant differences, such as for fibrillin-1 (FBN1), where transcript levels were increased by apical and basal zinc treatment (p=0.0313; p=0.0625, respectively), as well as at protein level (p<0.05).
Our results indicate that primary human RPE cells can serve as an ex vivo model system to investigate and eventually understand complex pathophysiological processes such as sub-RPE deposit formation as well as protective mechanisms in AMD.Acknowledgement:This work was supported by COST TD 1304 ‘The Network for the Biology of Zinc’ and EYE-RISK European Union’s Horizon 2020 Research and Innovation Programme under Grant Agreement No 634479.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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