Abstract

SO.08.07

Phagocytosis of oxidatively damaged POS by human RPE cells induces lipofuscinogenesis and basolateral secretion of undegraded POS proteins (transcytosis)

Krohne T. U.¹, Stratmann N. K.¹, Kopitz J.², Holz F. G.^1
1University Eye Hospital, Bonn, ²Department of Molecular Pathology, University of Heidelberg

Objective: We previously demonstrated that modification of photoreceptor outer segments (POS) by lipidperoxidation products like 4-hydroxynonenal (HNE) and malondialdehyde (MDA) results in reduced lysosomal degradation in the retinal pigment epithelium (RPE). Here we tested for a potential role of this mechanism in the development of intracellular lipofuscin and subcellular deposits (drusen) that represent the clinical hallmarks of early-stage age-related macular degeneration (AMD).
Methods: Isolated porcine POS were modified with either HNE or MDA. Human RPE cells (ARPE-19) were cultured on laminin-coated permeable membranes (Transwell) und incubated daily from the apical side with POS. The development of autofluorescent lipofuscin granules was documented by fluorescence microscopy and quantified by flow cytometry (FACS). For analysis of basolateral secretion of POS proteins cells were incubated with ¹²⁵iodide-labelled POS in the apical medium. Subsequently radioactivity was measured in the high-molecular weight (TCA-insoluble) protein fraction of the basolateral medium.
Results: In RPE cells incubated with HNE/MDA-modified POS the lysosomal degradation of POS was strongly inhibited. Non-degraded POS accumulated intracellularly and formed autofluorescent lipofuscin granules. Mean cellular autofluorescence increased within only 7 days 3.2fold (±0.1) as compared with control cells incubated with untreated POS. In addition it was found that modified POS proteins were guided through the RPE monolayer without being degraded (transcytosis). Following 24h of incubation with modified POS in the medium on the apical side of the RPE monolayer 19.4% (±0.6) of POS protein was detectable undegraded in the medium on the basolateral side as compared with 0.8% (±0.1) in controls with untreated POS.
Conclusions: Modification of POS with lipidperoxidation products inhibits lysosomal functions of the RPE and hence induced lipofuscinogenesis and transcytosis of indegraded POS proteins through the RPE monolayer. This mechanism may be involved in the pathogenesis of typical RPE changes in early-stage AMD like lipofuscin accumulation and drusen formation.

Copyright: Porstmann Kongresse GmbH. All rights reserved. Impressum, Disclaimer.