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S.Z. Amin, T.A. Bailey, M.E. Cheetham, J. Greenwood, V. Chong, P.J. Luthert; Modulation of Sub-Retinal Pigment Epithelial Deposits in vitro . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4228.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: In donor eyes of age related macular degeneration (AMD) patients, deposits under the retinal pigment epithelium (RPE) are a frequent finding. The role of sub-RPE deposits in the pathogenesis of AMD is unknown. They have, however, been implicated as either the cause or the consequence of AMD. Previously, we have developed an in vitro model of sub-RPE deposit formation (IOVS 2001;42:4:pp S222, abstract number 1196) where the deposits resemble those seen in vivo. In the current study, we tested the hypothesis that deposit formation in vitro could be modified by the application of the pro-inflammatory cytokine tumour necrosis factor (TNF) α or matrix metalloproteinase (MMP) 2. Methods: ARPE-19 cells were grown on collagen type I coated membrane supports for 5, 7 and 11 weeks. Subsequently, they were challenged with porcine retinal homogenate for 5 days, which increases phagocytosis and promotes increased deposit formation. The cells were then exposed to either TNF-α (10 and 40 ng/ ml) or MMP-2 (1 and 70 ng/ ml) in serum free media or serum free medium alone for 48 hours and processed for electron microscopy. Sub-RPE deposits were assessed ultrastructurally using a point-counting technique. The supernatants from the cultures were analysed for MMP activity by zymography. Results: Cells treated with TNF-α or MMP-2, showed a dramatic reduction in the amount of sub-RPE deposit (ANOVA; p = 0.004 and p = 0.035 respectively). There was an apparent dose response relationship with TNF-α. Low doses of MMP-2 (1ng/ ml) reduced sub-RPE deposit in 7 and 11-week old cultures but not in 5-week cultures. Zymography demonstrated that unchallenged cells produced predominantly MMP-2. The addition of TNF-α stimulated MMP-9 production alone. The amount of MMP-9 produced was the same at 40 ng/ ml or 10 ng/ ml. MMP-2 addition increased the amount of active MMP-2 in the system with no effect on MMP-9 production. Conclusions: These results demonstrate that TNF-α and MMP-2 promote net degradation of sub-RPE deposits in vitro. It is unlikely that this modulation is entirely due to MMP-9 production as the lower dose of TNF-α, which was less effective than the higher dose, induced similar MMP-9 activity. These results show that sub-RPE deposits can be formed and modified in vitro. Further investigation of these phenomena may lead to novel strategies for intervention in AMD.
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