Accumulation Of Lipofuscine In Retina Pigment Epithelium Cells Increases Susceptibility To Oxidative Stress

Abstract

Purpose: : Age-related macular degeneration (AMD) is the leading cause of blindness in the developed countries. The etiology is thought to be related to cumulative lipofuscine (LF) and oxidative stress in the retina pigment epithelium (RPE). However, the mechanism remains poorly understood. The purpose of this work is to study whether LF-accumulated and senescent RPE cells will increase susceptibility to reactive oxygen stress (ROS).

Methods: : H₂O₂ -induced oxidative stress were measure in ARPE-19 cells which were cultured with four conditions (young, young with LF accumulation, replicative senescence, and senescence with LF accumulation). Five experiments were carried out and fluorescence intensity was measured by flow cytometry: (1) We treated young and replicative senescent RPE cells by loading photoreceptor outer segments (POS) which were extracted from porcine retinas to induce LF accumulation;(2) RPE cells cocultured with FITC labeled POS for functional analysis; (3) mitochondrial mass was measured by labeling 10-n-nonyl-acridine orange (NAO); (4)Reactive oxygen stress was measured by staining DCFHDA. (5) Cytotoxicity analyses by staining MTT on cells was analyzed by ELISA reader.

Results: : The senescent cells and LF-accumulated senescent cells were found to have higher levels of reactive oxygen stress (ROS) (young: 100±5, senescent with LF accumulate:170±10 (arbitrary unit, au), P<0.05), and mitochondrial mass was greater in the senescent with LF accumulate cells (young: 100±7,:223±8 (au), P<0.05). In Functional analysis these cells showed that the phagocytic ability was reduced in the senescent cells and LF-accumulated senescent cells (young: 100±15, senescent with LF accumulate:10±3 (au), P<0.05). Cytotoxicity analysis showed that the susceptibility to oxidative stress was higher in senescence cells and LF-accumulated senescent cells (young: 1.0±0.05, senescent with LF accumulate:0.5±0.1 (au), P<0.05).

Conclusions: : These results suggest that accumulation of LF in RPE results in reduced phagocytic activity and increased susceptibility to ROS, which might be involved in the etiology of AMD.

Keywords: age-related macular degeneration • ipofuscin • oxidation/oxidative or free radical damage