May 2007
Volume 48, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2007
Modification of POS With Lipid Peroxidation Products Results in Transcytosis of Undegraded POS Proteins by RPE Cells
Author Affiliations & Notes
  • T. U. Krohne
    Ophthalmology, University of Bonn, Bonn, Germany
  • N. K. Stratmann
    Ophthalmology, University of Bonn, Bonn, Germany
  • J. Kopitz
    Pathology, University of Heidelberg, Heidelberg, Germany
  • F. G. Holz
    Ophthalmology, University of Bonn, Bonn, Germany
  • Footnotes
    Commercial Relationships T.U. Krohne, None; N.K. Stratmann, None; J. Kopitz, None; F.G. Holz, None.
  • Footnotes
    Support BONFOR grant Kr O-137.0004; DFG grants Ho 1926/2-1 and Ko 1663/2-3; DFG Research Priority Program AMD (SPP 1088)
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 2192. doi:
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      T. U. Krohne, N. K. Stratmann, J. Kopitz, F. G. Holz; Modification of POS With Lipid Peroxidation Products Results in Transcytosis of Undegraded POS Proteins by RPE Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2192.

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Abstract

Purpose:: Progressive formation of sub-RPE deposits in early-stage age-related macular degeneration (AMD) is thought to result from accumulation of incompletely digested material due to impaired lysosomal function of the RPE. We previously demonstrated that products of lipid peroxidation, in particular malondialdehyde (MDA) stabilize photoreceptor outer segment (POS) proteins against degradation by lysosomal proteases. Here we tested whether this reduced degradation results in transcytosis, i.e. basolateral secretion of apically phagocytosed POS proteins by RPE cells.

Methods:: Human RPE cells (ARPE-19) were cultured on laminin-coated permeable membranes (Transwell). Polarized differentiation was confirmed by electron microscopy and transepithelial resistance (TER) measurements. Isolated porcine POS were modified with 20mM MDA and subsequently biotinylated to facilitate quantification by streptavidin ELISA. Cells were incubated with POS in the apical medium for 3 hours and POS protein concentration was assessed after further 24 hours both intracellularly and in the basolateral medium. Undegraded (high-molecular weight) proteins were extracted from the medium by trichloroacetic acid (TCA) precipitation. In control experiments 20mM ammonium chloride was used to completely abolish lysosomal functions.

Results:: Cultured RPE cells exhibited highly polarized morphology and increased TER. Biotin-labelling did not prevent POS phagocytosis and degradation. MDA-modification of POS proteins resulted in a marked reduction of lysosomal degradation. Basolateral secretion of undegraded (TCA-insoluble) POS proteins was significantly increased following MDA-modification of POS. The respective POS protein concentrations in the basolateral medium were 2.5fold (±0.4) higher as compared with control cells incubated with unmodified POS. Similarly ammonium chloride treatment resulted in a 2.0fold (±0.4) increase of basolateral POS protein concentration.

Conclusions:: Modification of POS proteins by MDA stabilizes them against proteolytic degradation within the RPE lysosomal compartment resulting in transcytosis and increased basolateral secretion of incompletely degraded proteins. This mechanism may contribute to the formation of sub-RPE deposits as observed in early-stage AMD.

Keywords: age-related macular degeneration • retinal pigment epithelium • oxidation/oxidative or free radical damage 
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