Characterization of Retinal Pigment Epithelial Melanin and Degraded Synthetic Melanin Using Mass Spectrometry and In Vitro Biochemical Diagnostics.

With increasing age, there is an observable loss of melanin in retinal pigment epithelial (RPE) cells. It is possible that degradation of the pigment contributes to the pathogenesis of retinal disease, as the cellular antioxidant material is depleted. Functionally, intact melanin maintains protective qualities, while oxidative degradation of melanin promotes reactive oxygen species (ROS) generation and formation of metabolic byproducts, such as melanolipofuscin. Understanding the structural and functional changes to RPE melanin with increasing age may contribute to a better understanding of disease progression and risk factors for conditions such as age-related macular degeneration (AMD). In this study, human donor RPE melanin is characterized using MALDI mass spectrometry to follow melanin degradation trends. In vitro models using ARPE-19 cells are used to assess photo-reactivity in repigmented cells. Significant protection against intracellular ROS produced by blue light is observed in calf melanin-pigmented cells versus unpigmented and black latex bead controls (P < 0.0001). UV-B exposure to aged human melanin-pigmented cells results in a significant increase in nitric oxide production versus control cells (P < 0.001). Peroxide-treated synthetic melanin is characterized to elucidate degradation products that may contribute to RPE cell damage.

Calf melanin immunomodulates RPE cell attachment to extracellular matrix protein.

PURPOSE: It is widely accepted that RPE melanin has a protective effect against oxidative damage in RPE cells. It is possible that an additional protective characteristic of melanin is the ability to modulate RPE cell immune response. In this study, in vitro modeling was used to probe the relationship between RPE pigmentation and immune response by monitoring IL-6 expression and secretion in calf melanin pigmented ARPE-19 cells seeded onto glycated extracellular matrix as a stressor. METHODS: ARPE-19 cells were left unpigmented or were pigmented with either calf melanin or latex beads, and were then seeded onto RPE-derived extracellular matrix (ECM) or tissue culture-treated plates (no ECM). ECMs were modified by glycation. IL-6 expression was measured using qPCR and IL-6 secretion was determined using an ELISA, both at 30 min and 24 h after seeding. MTT assay was used to quantify cell attachment to glycated matrices 30 min after seeding. In unpigmented ARPE-19 cells, rate of cell attachment to substrate was monitored for 60 min after seeding using a hemacytometer to count unattached cells. Additionally, cell viability was evaluated using the Neutral Red assay 24 h after seeding. RESULTS: A significant increase in IL-6 expression was observed in calf melanin pigmented cells versus latex bead and unpigmented controls (p < 0.0001) 30 min after seeding onto ECM. Twenty-four hours after seeding, a significant decrease in IL-6 expression was observed in calf melanin pigmented cells (p < 0.0001) versus controls, implicating down-regulation of the cytokine. Additionally, calf melanin pigmented cell populations showed significant increase in attachment compared to unpigmented controls on either no ECM or unmodified ECM. CONCLUSIONS: Pigmentation of RPE cells with calf melanin resulted in significant changes in IL-6 expression regardless of ECM modification, in vitro. These findings suggest that melanin in the RPE may participate in immune response modulation in the retina with particular regard to cell attachment to protein substrates. The results of this study further implicate the role of chemical changes to melanin in regulating inflammation in retinal disease.

Nitrite ion modifies tyrosine and lysine residues of extracellular matrix proteins.

Age-related macular degeneration (AMD) is a disease characterized by degenerative changes in the retinal pigment epithelium and Bruch's membrane. Inflammation is considered a major risk factor for the development and progression of AMD. Nitrite is a potent byproduct of inflammation and has been detected at elevated concentrations in AMD donor tissue. We hypothesize that nitrite chemically modifies the extracellular matrix (ECM) of Bruch's membrane as an initial step to degenerative changes observed in AMD. Non-enzymatically nitrated synthetic ECM peptides, fibronectin and laminin, were used as model systems for inflammation. Using LC/MS, we identified that nitration preferentially occurred on tyrosine and deamination of lysine under the studied conditions. At tyrosine residues, 3-nitrotyrosine was produced and shifted the total mass by the addition of 45 amu. Deamination of lysine occurred and resulted in the formation of either an alkene or alcohol group. The alkene group was observed with a loss of 17 amu. An addition of 1 amu was observed with alcohol formation. We hypothesize that these initial chemical modifications to the structure of ECM proteins may be the responsible for altering the structure and consequent function of Bruch's membrane.

The Anthocyanins, Oenin and Callistephin, Protect RPE Cells Against Oxidative Stress.

The retinal pigment epithelium (RPE) is a highly metabolic layer of postmitotic cells lining Bruch's membrane in the retina. While these cells contain endogenous photosensitizers that mediate blue light-induced damage, it has also been shown that blue light exposure damages mitochondrial DNA in RPE cells resulting in mitochondrial dysfunction and unregulated generation of reactive oxygen species (ROS). As RPE cells are postmitotic, it is imperative to decrease oxidative stress to these cells and preserve function. Dietary plant-derived antioxidants such as anthocyanins offer a simple and accessible solution for decreasing oxidative stress. The anthocyanins malvidin-3-O-glucoside (oenin) and pelargonidin-3-O-glucoside (callistephin) were tested for their ability and efficacy in decreasing ROS generation and preserving mitochondrial redox activity in blue light-irradiated ARPE-19 cells. A significant decrease in intracellular ROS with concurrent increase in mitochondrial redox activity was observed for tested concentrations of oenin, while callistephin was beneficial to stressed cells at higher concentrations. These findings suggest anthocyanins are effective antioxidants in blue light-stressed RPE cells in vitro. Additionally, oxidation products of these anthocyanins were examined using LC/MS and findings suggest the possibility of multiple oxidation sites for these compounds.