Melanin protects RPE cells from damage and degeneration induced by photochemical and photosensitized oxidation

Abstract

Purpose : Oxygen pressure is extremely high in the retinal pigment epithelium (RPE) cells. The RPE, as its name implies, is rich in melanin content. Studies showed that an age-related decline occurs in either quantity or quality of melanin in adult RPE, which correlates with metabolic disturbance (impaired lipid metabolism) in the RPE, indicating that melanin might play a critical role in metabolic support for the RPE. We investigate whether melanin protects RPE from photochemical damage using albino and pigmented porcine primary cells.

Methods : Primary RPE cells from pigmented and albino porcine at 100% confluence were incubated with or without melanin (12.5, 25, and 50 µg/cm2 growth area) for 24 hours. The cells were then exposed to three different treatments: (1) photo-oxidation of the bisretinoids all-trans-retinal (which are well-delineated bisretinoid ingredients of RPE lipofuscin) (atRAL, Sigma-Aldrich, R2500) at concentrations of 20, 40, 60, and 80 μM for durations of 6, 12, 24, and 48 hours, (2) H₂O₂ at 50, 250, 500, and 750 µM of various concentrations for 3 hours, and (3) broadband UV(A) radiation (315-400 nm) for 10, 20, and 40 minutes. Eumelanin nanoparticles (which are predominantly present in RPE melanosomes) were synthesized using a dopamine polymerization method (PMID: 36269679). Several in vitro assays were conducted, including confocal microscopy to assess RPE morphology, immunohistochemical analysis to detect the expression of tight junction proteins (using ZO-1 and Na K -ATPase), measurement of mitochondrial membrane potential to evaluate ROS generation (using MitoSOX), MTT assay to assess cell viability, and TUNEL staining to evaluate apoptosis.

Results : Our results demonstrate that pigmented RPE cells exhibit a significantly higher resistance to degeneration induced by photosensitized oxidation and chemical exposure, including bisretinoids lipofuscin, H₂O₂, and UV radiation, in comparison to nonpigmented RPE cells. Treatment with melanin is shown to be significantly effective in preventing degeneration in both pigmented and nonpigmented primary RPE cells caused by photosensitized oxidation and chemical exposure.

Conclusions : Melanin exhibited photoprotective properties in primary RPE cultures by inhibiting photochemical reactions and photosensitized oxidation, leading to the prevention of RPE degeneration.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.