The Inhibitory Effect of Blue Light on Phagocytic Activity by ARPE-19 Cells.

Chronic exposure of the retina to short wavelength visible light is a risk factor in pathogenesis of age-related macular degeneration. The proper functioning and survival of photoreceptors depends on efficient phagocytosis of photoreceptor outer segments (POS) by retinal pigment epithelium. The purpose of this study was to analyze the phagocytic activity of blue light-treated ARPE-19 cells, and to examine whether the observed effects could be related to altered levels of POS phagocytosis receptor proteins and/or to oxidation of cellular proteins and lipids. POS phagocytosis was measured by flow cytometry. Phagocytosis receptor proteins αv and ß5 integrin subunits and Mer tyrosine kinase (MerTK) were quantified by western blotting. The intact functional heterodimer αvß5 was quantified by immunoprecipitation followed by immunoblotting. Cellular protein and lipid hydroperoxides were analyzed by coumarin boronic acid probe and iodometric assay, respectively. Cell irradiation induced reversible inhibition of specific phagocytosis and transient reductions in phagocytosis receptor proteins. Full recovery of functional heterodimer was apparent. Significant photooxidation of cellular proteins and lipids was observed. The results indicate that transient inhibition of specific phagocytosis by blue light could be related to the reduction in phagocytosis receptor proteins. Such changes may arise from oxidative modifications of cell phagocytic machinery components.

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, ß5 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/ß-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.

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 ß5) and ß-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 αvß5 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 ß5 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 RPE's role in photoreceptor turnover, thereby contributing to retinal degenerations.

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.

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.

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 pigment's ability to protect cells against H(2) O(2) , but the function of pigment granules within RPE cells should be extended beyond a role in light irradiation to include participation in iron homeostasis.

Integrating photoacoustic ophthalmoscopy with scanning laser ophthalmoscopy, optical coherence tomography, and fluorescein angiography for a multimodal retinal imaging platform.

Photoacoustic ophthalmoscopy (PAOM) is a newly developed retinal imaging technology that holds promise for both fundamental investigation and clinical diagnosis of several blinding diseases. Hence, integrating PAOM with other existing ophthalmic imaging modalities is important to identify and verify the strengths of PAOM compared with the established technologies and to provide the foundation for more comprehensive multimodal imaging. To this end, we developed a retinal imaging platform integrating PAOM with scanning laser ophthalmoscopy (SLO), spectral-domain optical coherence tomography (SD-OCT), and fluorescein angiography (FA). In the system, all the imaging modalities shared the same optical scanning and delivery mechanisms, which enabled registered retinal imaging from all the modalities. High-resolution PAOM, SD-OCT, SLO, and FA images were acquired in both albino and pigmented rat eyes. The reported in vivo results demonstrate the capability of the integrated system to provide comprehensive anatomic imaging based on multiple optical contrasts.

Near-infrared light photoacoustic ophthalmoscopy.

We achieved photoacoustic ophthalmoscopy (PAOM) imaging of the retina with near-infrared (NIR) light illumination. A PAOM imaging system with dual-wavelength illumination at 1064 nm and 532 nm was built. We compared in vivo imaging results of both albino and pigmented rat eyes at the two wavelengths. The results show that the bulk optical absorption of the retinal pigment epithelium (RPE) is only slightly higher than that of the retinal vessels at 532 nm while it becomes more than an order of magnitude higher than that of the retinal vessels at 1064 nm. These studies suggest that although visible light illumination is suitable for imaging both the retinal vessels and the RPE, NIR light illumination, being more comfortable to the eye, is better suited for RPE melanin related investigations and diagnoses.

Dynamic analyses reveal cytoprotection by RPE melanosomes against non-photic stress.

PURPOSE: Isolated melanosomes are known to have antioxidant properties but whether the granules perform an antioxidant function within cells is unclear. The aim of this study was to determine whether retinal pigment epithelium (RPE) melanosomes are competent to protect cultured cells against non-photic oxidative stress induced by treatment with H(2)O(2). METHODS: Porcine melanosomes, either untreated or irradiated with visible light to simulate age-related melanin photobleaching, were introduced by phagocytosis into ARPE-19 cells. Cells were treated with H(2)O(2) using two delivery methods: as a pulse, or by continuous generation following addition of glucose oxidase to the medium. Cell survival in melanosome-containing cells was compared to survival in cells containing phagocytosed control latex beads using two real-time cell death assays. RESULTS: Following H(2)O(2) delivery by either method, greater resistance to critical concentrations of H(2)O(2) was seen for cells containing melanosomes than for cells containing beads. Melanosome-mediated protection manifested as a delay in the time of onset of cell death and a slower rate of cell death over time. Photobleaching diminished the stress resistance conferred by the pigment granules. Individual cells in co-cultures were differentially sensitive to oxidative stress depending upon their particle content. Additional features of the time course of the cell death response were revealed by the dynamic analyses conducted over hours post oxidant treatment. CONCLUSIONS: The results show, for the first time, that melanosomes perform a cytoprotective function within cultured cells by acting as an antioxidant. The outcomes imply that melanosomes perform functions within RPE cells aside from those related to light irradiation, and also suggest that susceptibility to ubiquitous pro-oxidizing agents like H(2)O(2) is partly determined by discrete features of individual RPE cells such as their granule content.

Raman microspectroscopy of melanosomes: the effect of long term light irradiation.

Melanosomes are long lived organelles in retinal pigment epithelium cells and are primarily responsible for photoprotection. However, with aging or prolong light radiation, the function of melanosomes diminishes, which may be due to photobleaching of melanin pigments present in melanosomes. In this study, melanosomes were isolated from the retinal pigment epithelium cells and exposed to green light (532 nm), and the chemical changes were monitored using Raman microspectroscopy. Photochemical changes were recorded for different power levels and exposure times. The threshold power and the rate for irreversible photobleaching of melanosomes were calculated by fitting the experimental data with a proposed model.

Dynamics of H2O2 availability to ARPE-19 cultures in models of oxidative stress.

Oxidative injury to cells such as the retinal pigment epithelium (RPE) is often modeled using H(2)O(2)-treated cultures, but H(2)O(2) concentrations are not sustained in culture medium. Here medium levels of H(2)O(2) and cytotoxicity were analyzed in ARPE-19 cultures after H(2)O(2) delivery as a single pulse or with continuous generation using glucose oxidase (GOx). When added as a pulse, H(2)O(2) is rapidly depleted (within 2 h); cytotoxicity at 24 h, determined by the MTT assay for mitochondrial function, is unaffected by medium replacement at 2 h. Continuous generation of H(2)O(2) produces complex outcomes. At low GOx concentrations, H(2)O(2) levels are sustained by conditions under which generation matches depletion, but when GOx concentrations produce cytotoxic levels of H(2)O(2), oxidant depletion accelerates. Acceleration results partly from the release of contents from oxidant-damaged cells as indicated by testing depletion after controlled membrane disruption with detergents. Cytotoxicity analyses show that cells can tolerate short exposure to high H(2)O(2) doses delivered as a pulse but are susceptible to lower chronic doses. The results provide broadly applicable guidance for using GOx to produce sustained H(2)O(2) levels in cultured cells. This approach will be specifically useful for modeling chronic stress relevant to RPE aging and have a wider value for studying cellular effects of sublethal oxidant injury and for evaluating antioxidants that may protect significantly against mild but not lethal stress.

Mitochondria impairment correlates with increased sensitivity of aging RPE cells to oxidative stress.

Impairment of mitochondria function and cellular antioxidant systems are linked to aging and neurodegenerative diseases. In the eye, the retinal pigment epithelium (RPE) is exposed to a highly oxidative environment that contributes to age-related visual dysfunction. Here, we examined changes in mitochondrial function in human RPE cells and sensitivity to oxidative stress with increased chronological age. Primary RPE cells from young (9-20)-, mid-age (48-60)-, and >60 (62-76)-year-old donors were grown to confluency and examined by electron microscopy and flow cytometry using several mitochondrial functional assessment tools. Susceptibility of RPE cells to H(2)O(2) toxicity was determined by lactate dehydrogenase and cytochrome c release, as well as propidium iodide staining. Reactive oxygen species, cytoplasmic Ca(2+) [Ca(2+)](c), and mitochondrial Ca(2+) [Ca(2+)](m) levels were measured using 2',7'-dichlorodihydrofluorescein diacetate, fluo-3/AM, and Rhod-2/AM, respectively, adenosine triphosphate (ATP) levels were measured by a luciferin/luciferase-based assay and mitochondrial membrane potential (ΔΨm) estimated using 5,5',6,6'-tetrachloro 1,1'3,3'-tetraethylbenzimid azolocarbocyanine iodide. Expression of mitochondrial and antioxidant genes was determined by real-time polymerase chain reaction. RPE cells show greater sensitivity to oxidative stress, reduction in expression of mitochondrial heat shock protein 70, uncoupling protein 2, and superoxide dismutase 3, and greater expression of superoxide dismutase 2 levels with increased chronological age. Changes in mitochondrial number, size, shape, matrix density, cristae architecture, and membrane integrity were more prominent in samples obtained from >60 years old compared to mid-age and younger donors. These mitochondria abnormalities correlated with lower ATP levels, reduced ΔΨm, decreased [Ca(2+)](c), and increased sequestration of [Ca(2+)](m) in cells with advanced aging. Our study provides evidence for mitochondrial decay, bioenergetic deficiency, weakened antioxidant defenses, and increased sensitivity of RPE cells to oxidative stress with advanced aging. Our findings suggest that with increased severity of mitochondrial decay and oxidative stress, RPE function may be altered in some individuals in a way that makes the retina more susceptible to age-related injury.

Effect of untreated and photobleached bovine RPE melanosomes on the photoinduced peroxidation of lipids.

Melanin is usually considered a photoprotective pigment and antioxidant agent, but it is unclear how melanosomes protect pigmented cells against oxidative stress induced by light and whether aging modulates its photoprotective function, particularly in long-lived post-mitotic cells such as the retinal pigment epithelium (RPE). To address these issues, we analyzed the effects of untreated and experimentally photobleached melanosomes from cow RPE on the peroxidation of liposomal lipids induced by irradiation with intense visible light or by a rose Bengal photosensitized reaction. Photobleached melanosomes were used as a model of photo-aged pigment granules, and the progress of lipid peroxidation was monitored by electron spin resonance (EPR) oximetry and the iodometric determination of lipid hydroperoxides. We observed that while untreated melanosomes inhibited the rose Bengal induced peroxidation of lipids only moderately, partially photobleached melanosomes had very little effect on this process. Untreated melanosomes also inhibited peroxidation of liposomal lipids induced by intense visible light; however, the inhibitory effect markedly changed with the irradiation time. On the other hand, partially photobleached pigment granules accelerated the photoinduced peroxidation of lipids. The observed effects illustrate the limited efficiency of melanin within granules to scavenge and quench reactive oxygen species randomly generated by photosensitized reactions. The photosensitizing ability of photobleached melanosomes may arise from their capacity to photogenerate hydrogen peroxide. Collectively, our data indicate that natural melanin is only a moderately efficient photoprotective pigment, which upon photoaging may lose its antioxidant efficiency and even become a photosensitizer.

Sublethal photic stress and the motility of RPE phagosomes and melanosomes.

PURPOSE: To determine whether sublethal oxidative stress to the retinal pigment epithelium by visible light treatment affects the translocation of organelles, notably phagosomes and melanosomes. METHODS: Isolated porcine melanosomes were phagocytized by ARPE-19 cells, then cultures were treated with blue light to generate reactive oxygen intermediates (ROIs) by endogenous retinal pigment epithelial (RPE) chromophores throughout the cytoplasm. Other melanosomes were preloaded with a photosensitizer before phagocytosis, and cells were light treated to generate ROIs specifically at the granule surface. Phagosome movement was analyzed by live cell imaging. Also analyzed were phagocytized black latex beads, phagocytized melanosomes pretreated to simulate age-related melanin photobleaching, and endogenous RPE melanosomes in primary cultures of porcine retinal pigment epithelium. RESULTS: Sublethal blue light treatment slowed the movement of some, but not all, phagocytized melanosomes. All phagosomes slowed when ROIs were generated near the organelles through a photosensitized reaction. Melanosome photobleaching, which makes granules more photoreactive, increased the effects of blue light. Blue light treatment also slowed the motility of phagosomes containing latex beads and endogenous pigment granules. CONCLUSIONS: Blue light-induced stress impairs phagosome motility in RPE cells but affects individual organelles differently, suggesting that the effects of mild oxidative injury vary with subcellular location. The mechanisms underlying slowed motility are at least partially local because slowing can be induced by a photosensitized reaction in the subdomain of the organelle and the magnitude of the slowing is greater when the phagosome contents are photoreactive. Photic stress may impair the movement and positioning of RPE organelles, which would have widespread consequences for maintaining a functionally efficient subcellular organization.

Epithelial phenotype and the RPE: is the answer blowing in the Wnt?

Cells of the human retinal pigment epithelium (RPE) have a regular epithelial cell shape within the tissue in situ, but for reasons that remain elusive the RPE shows an incomplete and variable ability to re-develop an epithelial phenotype after propagation in vitro. In other epithelial cell cultures, formation of an adherens junction (AJ) composed of E-cadherin plays an important early inductive role in epithelial morphogenesis, but E-cadherin is largely absent from the RPE. In this review, the contribution of cadherins, both minor (E-cadherin) and major (N-cadherin), to RPE phenotype development is discussed. Emphasis is placed on the importance for future studies of actin cytoskeletal remodeling during assembly of the AJ, which in epithelial cells results in an actin organization that is characteristically zonular. Other markers of RPE phenotype that are used to gauge the maturation state of RPE cultures including tissue-specific protein expression, protein polarity, and pigmentation are described. An argument is made that RPE epithelial phenotype, cadherin-based cell-cell adhesion and melanization are linked by a common signaling pathway: the Wnt/beta-catenin pathway. Analyzing this pathway and its intersecting signaling networks is suggested as a useful framework for dissecting the steps in RPE morphogenesis. Also discussed is the effect of aging on RPE phenotype. Preliminary evidence is provided to suggest that light-induced sub-lethal oxidative stress to cultured ARPE-19 cells impairs organelle motility. Organelle translocation, which is mediated by stress-susceptible cytoskeletal scaffolds, is an essential process in cell phenotype development and retention. The observation of impaired organelle motility therefore raises the possibility that low levels of stress, which are believed to accompany RPE aging, may produce subtle disruptions of cell phenotype. Over time these would be expected to diminish the support functions performed by the RPE on behalf of photoreceptors, theoretically contributing to aging retinal disease such as age-related macular degeneration (AMD). Analyzing sub-lethal stress that produces declines in RPE functional efficiency rather than overt cell death is suggested as a useful future direction for understanding the effects of age on RPE organization and physiology. As for phenotype and pigmentation, a role for the Wnt/beta-catenin pathway is also suggested in regulating the RPE response to oxidative stress. Exploration of this pathway in the RPE therefore may provide a unifying strategy for advancing our understanding of both RPE phenotype and the consequences of mild oxidative stress on RPE structure and function.

Human RPE melanosomes protect from photosensitized and iron-mediated oxidation but become pro-oxidant in the presence of iron upon photodegradation.

PURPOSE: To determine the effects of human retinal pigment epithelial (RPE) cell pigment granules on photosensitized and iron ion-mediated oxidation and the effect of the photodegradation of melanosomes on their antioxidant properties. METHODS: RPE cells were isolated from human and bovine eyes; pigmented and nonpigmented bovine retinal pigment epithelia were isolated separately. Melanosomes, melanolipofuscin, and lipofuscin granules were isolated from human RPE donors older than 60. Melanosomes were photodegraded by exposure to blue light. Oxidation of RPE cells or of linoleate was induced by iron/ascorbate in the presence and absence of pigment granules. The photosensitized oxidation of histidine was induced by blue light irradiation of cationic porphyrin. The progress of oxidation was monitored by electron spin resonance oximetry. RESULTS: Iron/ascorbate induced rapid oxidation in suspensions of nonpigmented bovine RPE cells. The rates of oxidation were diminished approximately four times in suspensions of pigmented bovine RPE cells. Adding bovine melanosomes or synthetic melanin to nonpigmented bovine RPE cells resulted in a concentration-dependent decrease in the rate of oxidation to levels similar to those of pigmented bovine retinal pigment epithelium. Human melanosomes exerted a concentration-dependent inhibitory effect on photosensitized and iron-mediated oxidation. Photodegradation of human melanosomes led to loss of the inhibitory effect on iron-mediated oxidation, whereas their ability to inhibit photosensitized oxidation was enhanced. CONCLUSIONS: Human melanosomes act as effective antioxidants by preventing iron ion-induced oxidation. Photodegradation of melanosomes results in the loss of these antioxidant properties while it preserves their ability to deactivate cationic photosensitizers.

Photobleaching of melanosomes from retinal pigment epithelium: II. Effects on the response of living cells to photic stress.

Melanosomes of the retinal pigment epithelium (RPE) are long lived organelles that may undergo photobleaching with aging, which can diminish the antioxidant efficiency of melanin. Here, isolated porcine RPE melanosomes were experimentally photobleached with visible light to simulate aging and compared with untreated granules or control particles (black latex beads) for their effects on the survival of photically stressed ARPE-19 cultures. Particles were delivered to cultures for uptake by phagocytosis then cells were exposed to violet light and analyzed by a new live cell imaging method to identify the time of apoptotic blebbing as a dynamic measure of reduced cell survival. Results indicated that untreated melanosomes did not decrease photic injury to ARPE-19 cells when compared with cells lacking particles or with cells containing control particles, as might be expected if melanin performed an antioxidant function. Instead cells with untreated melanosomes showed reduced survival indicated by an earlier onset of blebbing and a lower fraction of surviving cells after photic stress. Cell survival was reduced even further in stressed cells containing melanosomes that were photobleached, and survival decreased with increasing photobleaching time. Photobleaching of RPE melanosomes therefore makes cells containing them more sensitive to light-induced cytotoxicity. This observation raises the possibility that aged melanosomes increase RPE cell photic stress in situ, perhaps contributing to reduced tissue function and to degeneration of the adjacent retina that the RPE supports. How melanosomes (photobleached or not) interact with their local subcellular environment to modify RPE cell survival is poorly understood and is likely determined by the physicochemical state of the granule and its constituent melanin. The live cell imaging method introduced here, which permitted detection of a graded effect of photobleaching, provides a sensitive bioassay for probing the effects of melanosome modifications.

Photobleaching of melanosomes from retinal pigment epithelium: I. Effects on protein oxidation.

Melanin in the long-lived melanosomes of the retinal pigment epithelium (RPE) may undergo photobleaching with aging, which appears to diminish the antioxidant function of melanin and could make photobleached melanosomes less efficient in protecting biomolecules from oxidative modification. Here we analyzed whether photobleaching of melanosomes affects their ability to modify the oxidation state of nearby protein. As conventional methods developed to study soluble antioxidants are not well suited for analysis of granules such as melanosomes, we developed a new analytic method to focus on particle surfaces that involves experimentally coating granules with the cytoskeletal protein beta-actin to serve as a reporter for local protein oxidation. Isolated porcine RPE melanosomes were photobleached with visible light to simulate aging, then photobleached melanosomes, untreated melanosomes and control particles (black latex beads) were actin coated and illuminated in a photosensitized cell free system. Protein was re-stripped from particles and analyzed for carbonylation by Western blotting. Quantitative densitometry showed no reproducible differences for protein associated with untreated melanosomes when compared with control particles. Melanin has both anti- and pro-oxidant functions when light irradiated, but neither of these functions predominated in the protein oxidation assay when untreated melanosomes were used. However, protein extracted from photobleached melanosomes showed markedly increased carbonylation, both of associated actin and of endogenous melanosomal protein(s), and the effect increased with extent of granule photobleaching. Photobleaching of RPE melanosomes therefore changes the oxidation state of protein endogenous to the organelle and reduces the ability of the granule to modify the oxidation of exogenous protein near the particle surface. The results support the growing body of evidence that photobleaching of RPE melanosomes, which is believed to occur with aging, changes the physicochemical properties of the organelle and reduces the likelihood that the granules perform an antioxidant function.

Photobleaching of retinal pigment epithelium melanosomes reduces their ability to inhibit iron-induced peroxidation of lipids.

Melanin in the human retinal pigment epithelium (RPE) is believed to play an important photoprotective role. However, unlike in skin, melanosomes in the RPE are rather long-lived organelles, which increases their risk of modifications resulting from significant fluxes of light and high oxygen tension. In this work, we subjected purified bovine RPE melanosomes to prolonged aerobic exposure with intense visible and near ultraviolet radiation and studied the effects of irradiation on the melanosome's capacity to inhibit peroxidation of lipids induced by iron/ascorbate. We found that control, untreated melanosomes show a concentration-dependent inhibition of the accumulation of lipid hydroperoxides and the accompanying consumption of oxygen, but photolysed melanosomes lose their antioxidant efficiency and even became prooxidant. The prooxidant action of partially photobleached melanosomes was observed for pigment granules with a melanin content reduced by about 50% compared with untreated melanosomes, as determined by electron spin resonance spectroscopy. We have previously shown that a similar loss in the content of the RPE melanin occurs during human lifetime, which may suggest that the normal antioxidant properties of human RPE melanin become compromised with aging.