Christine M. B. Skumatz

Loss of melanin from human RPE with aging: possible role of melanin photooxidation

The pigment melanin, which is believed to play a photoprotective role, was quantified here in human RPE cells from donors of different age. Electron spin resonance (ESR) spectroscopy was shown to provide a quantitative measure of melanin and was used as a non-destructive measure of melanin content. Results indicated an age-related melanin loss in RPE cells, with melanin content diminishing 2.5-fold between the first and the ninth decade of life. To determine whether photo-oxidation may contribute to age-related changes in RPE melanin, RPE in human eyecups, isolated human and bovine RPE cells, purified melanin granules, or synthetic dopa melanin were irradiated with various wavelengths and intensities of visible light. Samples were analysed for changes in melanin content by ESR spectroscopy, and by absorption and emission spectrophotometry. The concentration of hydrogen peroxide was measured in some samples, and some human eyecups were examined by transmission electron microscopy. Irradiation of RPE in eyecups with intense visible light was found to produce a time-dependent photobleaching of melanosomes that was accompanied by the formation of hydrogen peroxide. Photobleaching of isolated RPE melanosomes and synthetic dopa melanin resulted in enhanced melanin fluorescence, as previously shown for melanin from aged donors by others, and significantly reduced ESR signal intensity, resembling the changes in melanin with aging observed here. We conclude that the content of melanin in RPE cells undergoes an age-related change to which photo-oxidation may contribute. This observation raises the question of whether age-related changes in melanin reduce the photoprotective role of the pigment in aging RPE cells.

Spectroscopic and morphological studies of human retinal lipofuscin granules

The emission properties of ocular lipofuscin granules isolated from human retinal pigment epithelial cells are examined by using steady-state fluorescence spectroscopy and spectrally resolved confocal microscopy. The shape of the emission spectrum of a thick sample of lipofuscin granules dried on glass varies with excitation energy. The polarization of this emission is wavelength-dependent, exhibiting significant polarization near the excitation wavelength and becoming mostly depolarized over the majority of the emission spectrum. These results show that the yellow-emitting fluorophores [e.g., A2E (2-[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E,7E-octatetraenyl]-1-(2-hydroxyethyl)-4-[4-methyl-6-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E-hexatrienyl]-pyridinium)] are excited as a result of energy transfer within the granules and therefore are not the dominant blue-absorbing chromophores within lipofuscin granules. Atomic force microscopy images show lipofuscin granules to be an aggregated structure. Bulk and in vivo emission measurements must therefore take into account the effect of Raleigh scattering. When corrected for scattering, the emission spectrum of a thick lipofuscin deposit or intracellular lipofuscin resembles that for A2E. The sum of the emission spectra of a collection of individual granules also resembles the emission spectrum of A2E, but the spectrum of individual granules varies significantly. This result suggests that the agreement between the emission spectra of lipofuscin and A2E is fortuitous, and the collective data indicate the presence of several blue-absorbing chromophores in lipofuscin and show A2E is not the dominant yellow-emitting fluorophore in many of the granules studied.

Formate, the toxic metabolite of methanol, in cultured ocular cells

Methanol has neurotoxic actions on the human retina due to its metabolite, formic acid, which is a mitochondrial toxin. In methanol poisoned animals, morphologic changes were seen both in retinal photoreceptors and in cells of the underlying retinal pigment epithelium (RPE). Here the effects of formate exposure on the two retinal cell types were analyzed in more detail in vitro using photoreceptor (661W) and RPE (ARPE-19) cell lines. Cells were exposed for time courses from minutes to days to sodium formate at pH 7.4 or to formic acid at pH 6.8, to simulate the metabolic acidosis that accompanies methanol poisoning. Formate accumulation, cellular ATP, cytotoxicity (lactate dehydrogenase (LDH) release) and cell phenotype were analyzed. Formate accumulated with a similar biphasic pattern in both cell types, and to similar levels whether delivered as sodium formate or as formic acid. ATP changes with sodium formate treatment differed between cell types with only 661W cells showing a rapid (within minutes), transient ATP increase. The subsequent ATP decrease was earlier in 661W cells (6 h) than the ATP decrease in ARPE-19 cells (24 h), and although both cell types showed evidence of cytotoxicity, the effects were greater for 661W cells. Both cell types showed enhanced morphologic and biochemical changes with formic acid treatment including earlier and/or greater effects on ATP depletion and cytotoxicity; again effects were more pronounced in 661W cells. Formate therefore is toxic for both cell lines, with 661W cells exhibiting greater sensitivity. Medium pH also appears to play a significant role in formate toxicity in vitro.

Photoaging of human retinal pigment epithelium is accompanied by oxidative modifications of its eumelanin

Although photodegradation of the retinal pigment epithelium (RPE) melanin may contribute to the etiology of age‐related macular degeneration, the molecular mechanisms of this phenomenon and the structural changes of the modified melanin remain unknown. Recently, we found that the ratio of pyrrole‐2,3,4,5‐tetracarboxylic acid (PTeCA) to pyrrole‐2,3,5‐tricarboxylic acid (PTCA) is a marker for the heat‐induced cross‐linking of eumelanin. In this study, we examined UVA‐induced changes in synthetic eumelanins to confirm the usefulness of the PTeCA/PTCA ratio as an indicator of photo‐oxidation and compared changes in various melanin markers and their ratios in human melanocytes exposed to UVA, in isolated bovine RPE melanosomes exposed to strong blue light and in human RPE cells from donors of various ages. The results indicate that the PTeCA/PTCA ratio is a sensitive marker for the oxidation of eumelanin exposed to UVA or blue light and that eumelanin and pheomelanin in human RPE cells undergo extensive structural modifications due to the life‐long exposure to blue light.

Melanosome–iron interactions within retinal pigment epithelium-derived cells

Melanosomes were recently shown to protect ARPE‐19 cells, a human retinal pigment epithelium (RPE) cell line, against oxidative stress induced by hydrogen peroxide. One postulated mechanism of antioxidant action of melanin is its ability to bind metal ions. The aim here was to determine whether melanosomes are competent to bind iron within living cells, exhibiting a property previously shown only in model systems. The outcomes indicate retention of prebound iron and accumulation of iron by granules after iron delivery to cells via the culture medium, as determined by both colorimetric and electron spin resonance analyses for bound‐to‐melanosome iron. Manipulation of iron content did not affect the pigments ability to protect cells against H2O2, but the function of pigment granules within RPE cells should be extended beyond a role in light irradiation to include participation in iron homeostasis.

Photic Injury to Cultured RPE Varies Among Individual Cells in Proportion to Their Endogenous Lipofuscin Content as Modulated by Their Melanosome Content

PURPOSE We determined whether photic stress differentially impairs organelle motility of RPE lipofuscin and melanin granules, whether lethal photic stress kills cells in proportion to lipofuscin abundance, and whether killing is modulated by melanosome content. METHODS Motility of endogenous lipofuscin and melanosome granules within the same human RPE cells in primary culture was quantified by real-time imaging during sublethal blue light irradiation. Cell death during lethal irradiation was quantified by dynamic imaging of the onset of nuclear propidium iodide fluorescence. Analyzed were individual cells containing different amounts of autofluorescent lipofuscin, or similar amounts of lipofuscin and a varying content of phagocytized porcine melanosomes, or phagocytized black latex beads (control for light absorbance). RESULTS Lipofuscin granules and melanosomes showed motility slowing with mild irradiation, but slowing was greater for lipofuscin. On lethal irradiation, cell death was earlier in cells with higher lipofuscin content, but delayed by the copresence of melanosomes. Delayed death did not occur with black beads, suggesting that melanosome protection was due to properties of the biological granule, not simple screening. CONCLUSIONS Greater organelle motility slowing of the more photoreactive lipofuscin granule compared to melanosomes suggests that lipofuscin mediates mild photic injury within RPE cells. With lethal light stress endogenous lipofuscin mediates killing, but the effect is cell autonomous and modulated by coincident melanosome content. Developing methods to quantify the frequency of individual cells with combined high lipofuscin and low melanosome content may have value for predicting the photic stress susceptibility of the RPE monolayer in situ.

Photosensitized Oxidative Stress to ARPE-19 Cells Decreases Protein Receptors that Mediate Photoreceptor Outer Segment Phagocytosis

PURPOSE To determine whether previously shown photodynamic (PD)-induced inhibition of specific photoreceptor outer segment (POS) phagocytosis by ARPE-19 cells is associated with reductions in receptor proteins mediating POS phagocytosis, and if PD treatment with merocyanine-540 (MC-540) produces additional effects leading to its inhibition of nonspecific phagocytosis. METHODS ARPE-19 cells preloaded with MC-540 or rose bengal (RB) were sublethally irradiated with green light. Phagocytosis of POS was measured by flow cytometry and POS receptor proteins (Mer tyrosine kinase receptor [MerTK] and integrin subunits αv and β5) and β-actin were quantified by Western blotting at 0.5 and 24 hours after irradiation, with comparison to samples from nonsensitized control cultures. The intact integrin heterodimer αvβ5 was quantified by immunoprecipitation followed by blotting. The distribution of N-cadherin, ZO-1, and F-actin was visualized by fluorescence microscopy. RESULTS Mild PD stress mediated by both photosensitizers that elicits no significant morphologic changes produces transient and recoverable reductions in MerTK. The individual αv and β5 integrin subunits are also reduced but only partially recover. However, there is sufficient recovery to support full recovery of the functional heterodimer. Light stress mediated by MC-540 also reduced levels of actin, which is known to participate in the internalization of particles regardless of type. CONCLUSIONS After PD treatment POS receptor protein abundance and phagocytosis show a coincident in time reduction then recovery suggesting that diminution in receptor proteins contributes to the phagocytic defect. The additional inhibition of nonspecific phagocytosis by MC-540-mediated stress may result from more widespread effects on cytosolic proteins. The data imply that phagocytosis receptors in RPE cells are sensitive to oxidative modification, raising the possibility that chronic oxidative stress in situ may reduce the efficiency of the RPEs role in photoreceptor turnover, thereby contributing to retinal degenerations.

Zeaxanthin and α-tocopherol reduce the inhibitory effects of photodynamic stress on phagocytosis by ARPE-19 cells.

Zeaxanthin and α-tocopherol have been previously shown to efficiently protect liposomal membrane lipids against photosensitized peroxidation, and to protect cultured RPE cells against photodynamic killing. Here the protective action of combined zeaxanthin and α-tocopherol was analyzed in ARPE-19 cells subjected to photodynamic (PD) stress mediated by rose Bengal (RB) or merocyanine-540 (MC-540) at sub-lethal levels. Stress-induced cytotoxicity was analyzed by the MTT assay. The peroxidation of membrane lipids was determined by HPLC-EC (Hg) measurements of cholesterol hydroperoxides using cholesterol as a mechanistic reporter molecule. The specific phagocytosis of FITC-labeled photoreceptor outer segments (POS) isolated from bovine retinas was measured by flow cytometry, and the levels of phagocytosis receptor proteins αv integrin subunit, β5 integrin subunit and MerTK were quantified by Western blot analysis. Cytotoxicity measures confirmed that PD stress levels used for phagocytosis analysis were sub-lethal and that antioxidant supplementation protected against higher, lethal PD doses. Sub-lethal PD stress mediated by both photosensitizers induced the accumulation of 5α-OOH and 7α/β-OOH cholesterol hydroperoxides and the addition of the antioxidants substantially inhibited their accumulation. Antioxidant delivery prior to PD stress also reduced the inhibitory effect of stress on POS phagocytosis and partially reduced the stress-induced diminution of phagocytosis receptor proteins. The use of a novel model system where oxidative stress was induced at sub-lethal levels enable observations that would not be detectable using lethal stress models. Moreover, novel observations about the protective effects of zeaxanthin and α-tocopherol on photodynamic damage to ARPE-19 cell membranes and against reductions in the abundance of receptor proteins involved in POS phagocytosis, a process essential for photoreceptor survival, supports the importance of the antioxidants in protecting of the retina against photooxidative injury.

Oxidative stress increases HO-1 expression in ARPE-19 cells, but melanosomes suppress the increase when light is the stressor.

PURPOSE Phagocytized melanosomes in ARPE-19 cells were previously shown to decrease susceptibility to oxidative stress induced by hydrogen peroxide treatment and increase stress due to light irradiation relative to cells containing control black latex beads. Here we asked whether differential expression of antioxidant enzymes in cells containing pigment granules could explain the outcomes. METHODS ARPE-19 cells were loaded by phagocytosis with porcine RPE melanosomes or black latex beads (control particles). Heme oxygenase-1 (HO-1), HO-2, glutathione peroxidase (GPx), and catalase were quantified by Western blot analysis before and after treatment with sublethal hydrogen peroxide or blue light (400-450 nm). The stress was confirmed as sublethal by cell survival analysis using real-time quantification of propidium iodide fluorescence. RESULTS Phagocytosis itself produced transient changes in protein levels of some antioxidant enzymes, but steady-state levels (7 days after phagocytosis) did not differ in cells containing melanosomes versus beads. Sublethal stress, induced by either hydrogen peroxide or light, had no effect on catalase or HO-2 in either particle-free or particle-loaded cells. In contrast, HO-1 protein was upregulated by treatment with both hydrogen peroxide and light. Particle content did not affect the HO-1 increase induced by hydrogen peroxide, but the increase induced by blue light irradiation was partially blocked in cells containing black beads and blocked even more in cells containing melanosomes. CONCLUSIONS The results do not implicate differential antioxidant enzyme levels in stress protection by melanosomes against hydrogen peroxide, but they suggest a multifaceted role for melanosomes in regulating light stress susceptibility in RPE cells.