Abstract
Purpose: :
Oxidative stress from reactive oxygen species (ROS) has been implicated in many diseases, including age–related macular degeneration (AMD), in which the retinal pigment epithelium (RPE) is considered a primary target. The purpose of this study was to determine whether macrophage–induced RPE apoptosis was involved in ROS and caspases.
Methods: :
Mouse RPE cell cultures were established from wild–type (WT) and heterozygous manganese superoxide dismutase (Sod2)–knockout mouse (HET). The intracellular ROS, hydrogen peroxide (H2O2), and superoxide anion (O2–), were measured by using 5–(and 6)–chloromethyl–2`,7`–dichlorodihydrofluorescence diacetate, acetyl ester (CM–H2DCFDA) and dihydroethidium (Het) assays, respectively. Apoptosis was evaluated by Hoechst staining and TUNEL assay. Changes in mitochondrial membrane potential were detected by JC–1 dye. Activated caspases were detected in situ by FITC–VAD–fmk staining.
Results: :
IFN–γ–activated macrophages induced the production of intracellular ROS, decrease in mitochondrial membrane potential, activation of caspases and apoptosis in mRPE cells. The macrophage–induced ROS production, activation of caspases and apoptosis were significantly enhanced in the RPE cells derived from heterozygous Sod2–knockout mice.
Conclusions: :
Our results suggest that activated macrophages may promote RPE cell apoptosis by inducing intracellular ROS production, and disruption of mitochondrial membrane potential and activation of caspases.
Keywords: retinal pigment epithelium • apoptosis/cell death • age-related macular degeneration