Abstract
Purpose.:
Progressive accumulation of extracellular material at the basolateral side of the retinal pigment epithelium (RPE) is a key event in the pathogenesis of age-related macular degeneration (AMD). The authors previously demonstrated that modifications with lipid peroxidation products, such as 4-hydroxynonenal (HNE) and malondialdehyde (MDA), stabilize photoreceptor outer segment (POS) proteins against lysosomal degradation. Herein, they tested RPE cells for the basolateral release of undegraded modified POS proteins.
Methods.:
Polarized cultures of the human RPE cell line ARPE-19 on permeable membranes were incubated with iodine-125–labeled POS on the apical side. After 24 hours, radioactivity was quantified in apical medium, cell lysates, and basolateral medium after separation of undegraded proteins by precipitation. Protein composition of basolaterally released POS material was analyzed by two-dimensional gel electrophoresis. C3a- and SC5b-9-specific enzyme-linked immunosorbent assays were used to assess complement activation by modified POS.
Results.:
The amount of phagocytic uptake was similar for native and modified POS. Unmodified POS proteins were almost completely (98.1%) degraded, whereas degradation of HNE- and MDA-modified POS proteins was significantly reduced (47.2%; 56.5%). Undegraded POS proteins accumulated intracellularly (14.2%; 12.1%) and were trafficked through the cells to be released into the basolateral medium (38.5%; 31.5%). Protein composition of basolaterally released material matched the original POS preparations. Protein modifications did not confer increased complement-activating capacity to POS material.
Conclusions.:
Inhibition of lysosomal degradation by lipid peroxidation-related protein modifications induces apical-to-basolateral transcytosis of undegraded POS proteins by human RPE cells in vitro. This mechanism may contribute to sub-RPE deposit formation and drusen biogenesis in AMD.
Progressive deposition of extracellular material between the basolateral side of the retinal pigment epithelium (RPE) and adjacent Bruch's membrane is a hallmark of early-stage age-related macular degeneration (AMD).
1,2 Among the various histologically and clinically distinguishable manifestations of sub-RPE deposits, basal linear deposits (BLinD) and soft drusen are recognized as specific for AMD.
3,4 Both BLinD and soft drusen represent accumulations of material described as membranous debris
5 between the RPE basement membrane and the inner collagenous layer of Bruch's membrane and are, therefore, considered diffuse and focal manifestations, respectively, of the same lesion.
1,3,6 The prognostic relevance of drusen size and number for the progression of AMD has been well established.
7–9
When Heinrich Müller initially coined the term
drusen in 1856, he already suggested that material deposition by the retinal pigment epithelium might contribute to their development.
10 Today, more than one and a half centuries later, the mechanisms of sub-RPE deposit formation are still not completely resolved. However, proteomic studies revealed that drusen and aged Bruch's membranes contain proteins that are covalently modified by products of lipid peroxidation, and these modifications were more abundant in AMD eyes than in age-matched control eyes.
11 Lipid peroxidation is a mechanism of oxidative damage that predominantly affects tissues, such as the outer retina, that are exposed to high levels of oxidative or photooxidative stress and are highly enriched in polyunsaturated fatty acids (PUFAs). In lipid peroxidation, oxygen-derived free radicals interact with PUFA double bounds, resulting in cleavage into a variety of highly reactive aldehydes, such as malondialdehyde (MDA) and 4-hydroxynonenal (HNE).
12 Once formed, these lipid peroxidation products rapidly attach covalently to nearby proteins by forming adducts predominantly with cysteine, lysine, and histidine residues, a process that may interfere with protein functionality.
12 Furthermore, modified proteins, also referred to as advanced lipid peroxidation end-products (ALE), are prone to aggregation by cross-linking of the adducts.
12 By these mechanisms, lipid peroxidation products cause severe damage to cellular proteins and other macromolecules and thus impair cellular functions and survival.
We previously detected modifications with MDA and HNE on proteins isolated from RPE-derived lipofuscin, thus demonstrating the occurrence of these substances in the RPE lysosomal compartment and suggesting their contribution to lysosomal dysfunction and lipofuscinogenesis.
13 Indeed, we observed that modifications of photoreceptor outer segment (POS) proteins with MDA and HNE significantly reduced their degradation by RPE cells in vitro.
14 After normal phagocytic uptake, modified POS proteins exhibited increased stability against the proteolytic attack by lysosomal enzymes, resulting in their intracellular accumulation and long-term storage. In the present study, we further analyzed the processing of modified POS by human RPE cells in vitro and tested whether undegradable POS components are disposed into the sub-RPE space, a process that may contribute to local immune processes and sub-RPE deposit formation in vivo.
For transmission electron microscopy (TEM) analysis, cells were fixed with Karnovsky's solution in PBS (pH 7.3), postfixed in 1% OsO4, dehydrated in ethanol, and embedded (Durcupan ACM Fluka; Sigma-Aldrich). Ultrathin sections were stained with uranyl acetate and lead citrate and were examined with a transmission electron microscope (Zeiss EM 109; Carl Zeiss, Oberkochen, Germany). For scanning electron microscopy (SEM) analysis, the specimens were fixed and dehydrated as described and then were treated with hexamethyldisilazane, air dried, sputtered with gold, and examined with a scanning electron microscope (Zeiss DSM 950; Carl Zeiss).
Transcytosis assays were performed exactly as described except that serum content of the culture medium was reduced to 1% during the 24-hour POS incubation period to reduce interference of high-abundance serum proteins with the detection of POS proteins. For isoelectric focusing (IEF), basolateral medium samples resulting from these transcytosis experiments and samples of the original radiolabeled POS preparations were subjected to TCA protein precipitation. Proteins were extracted from the TCA-insoluble fraction in 7 M urea, 2 M thiourea, 4% Triton X-100, 65 mM dithiothreitol, and 0.8% carrier ampholyte (Pharmalyte; Sigma-Aldrich). Equal amounts of protein were separated in an immobilized pH gradient (IPG) strip (Immobiline DryStrip, pH 3–10, 11 cm; Amersham Pharmacia Biotech, Freiburg, Germany) using an IEF system (IPGphor; Amersham Pharmacia Biotech). For subsequent SDS-PAGE, the IPG strip was applied to a precast SDS gradient gel (8–18 ExcelGel; Amersham Pharmacia Biotech), and separation was performed on a horizontal electrophoresis unit (Multiphor II; Amersham Pharmacia Biotech). Phosphor screens were exposed to the dried gels and scanned on a phosphor imaging system (Cyclon Plus Phosphor Imager; PerkinElmer, Rodgau, Germany) using appropriate software (OptiQuant; AcroMetrix, San Francisco, CA).
Human blood was drawn in anticoagulant-free tubes and was sedimented at room temperature for 30 minutes. Serum was separated by centrifugation (2000g, 5 minutes), and 100 μL serum was incubated with 20 μL POS (10 μg total protein) in PBS. In parallel, serum was incubated with 20 μL PBS without POS to serve as vehicle control. After 2 hours at 37°C, complement activation was assessed by detection of C3a and SC5b-9 in 1:104 and 1:10 diluted samples, respectively, using specific enzyme-linked immunosorbent assays (Quidel, San Diego, CA) according to the manufacturer's recommendation. Experiments were performed in triplicate, and results are presented as mean (±SD).