Melanin Protects Cultured Retinal Pigment Epithelial (RPE) Cells From Oxidative Stress

Abstract

Abstract: : Purpose: Early age-related macular degeneration (AMD) is characterized by loss of melanin and accumulation of extracellular deposits, so called drusen. Oxidative stress has been implicated to play an important role in AMD pathogenesis. This study was designed to investigate the possible role of melanin in prevention of retinal pigment epithelial (RPE) cell damage. Influence of RPE-melanin content on expression of the stress protein alpha B-crystallin as a measure for cellular stress and the extracellular matrix (ECM) component fibronectin in response to oxidative stress has been investigated. Methods: Monolayer cultures of primary human RPE cells were re-melanized using synthetic melanin (50µg/ml) for 48 h. Oxidative stress of pigmented and non pigmented cells was accomplished by exposure to 300 µMol H₂O₂ for 1 h. Cells were harvested 2 to 24 h after oxidative stress. Induction of alpha B-crystallin and fibronectin mRNA was assessed by northern blot analysis. The house-keeping gene GAPDH was used as internal standard. Results: Exposure of RPE cells to 300 µMol H₂O₂ for 1 h substantially increased alpha B-crystallin expression four hours after treatment (3-4 fold). Re-melanization of RPE cells prior to oxidative stress markedly attenuated induction of the stress protein alpha B-crystallin. Accordingly, H₂O₂-treatment of non-pigmented cells lead to an increase of fibronectin mRNA (3 fold) after 6 hours. This effect was not observed in re-melanized RPE cells. GAPDH expression was independent of re-melanization. Conclusion: In re-melanized RPE cells oxidative injury failed to elicit a stress response and did not induce ECM protein synthesis. These in vitro data indicate that melanin may be a potent antioxidant in RPE cells at the molecular level. Thus melanin may make RPE cells less susceptible to the alterations induced by oxidative stress. We therefore speculate that loss of melanin with age may alter the gene expression pattern of the RPE, finally contributing to development of AMD.

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