Search

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 β5 integrin subunits and Mer tyrosine kinase (MerTK) were quantified by western blotting. The intact functional heterodimer αvβ5 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.

The epidermis of Eisenia is covered by a cuticle and rests on a basement lamella. The cuticle, which is resistant to a variety of enzymes, is composed of non-striated, bundles of probable collagen fibers that are orthogonally oriented and are embedded in a proteoglycan matrix. The basement lamella consists of striated collagen fibers with a 560 A major periodicity. Proximity and morphology suggest that the epidermis may contribute to both the cuticle and the basement lamella — that is, the single tissue may synthesize at least two types of collagen. The epidermis is a pseudostratified epithelium containing three major cell types (columnar, basal and gland) and a rare fourth type with apical cilia. The esophagus is lined by a simple cuticulated epithelium composed predominantly of a single cell type, which resembles the epidermal columnar cell. Rare gland cells occur in the esophageal epithelium, but basal cells are lacking.

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.

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.

Macular xanthophylls (MXs) lutein and zeaxanthin are dietary carotenoids that are selectively concentrated in the human eye retina, where they are thought to protect against age-related macular degeneration (AMD) by multiple mechanisms, including filtration of phototoxic blue light and quenching of singlet oxygen and triplet states of photosensitizers. These physical protective mechanisms require that MXs be in their intact structure. Here, we investigated the protection of the intact structure of zeaxanthin incorporated into model membranes subjected to oxidative modification by water- and/or membrane-soluble small nitroxide free radicals. Model membranes were formed from saturated, monounsaturated, and polyunsaturated phosphatidylcholines (PCs). Oxidative modification involved autoxidation, iron-mediated, and singlet oxygen-mediated lipid peroxidation. The extent of chemical destruction (bleaching) of zeaxanthin was evaluated from its absorption spectra and compared with the extent of lipid peroxidation evaluated using the thiobarbituric acid assay. Nitroxide free radicals with different polarity (membrane/water partition coefficients) were used. The extent of zeaxanthin bleaching increased with membrane unsaturation and correlated with the rate of PC oxidation. Protection of the intact structure of zeaxanthin by membrane-soluble nitroxides was much stronger than that by water-soluble nitroxides. The combination of zeaxanthin and lipid-soluble nitroxides exerted strong synergistic protection against singlet oxygen-induced lipid peroxidation. The synergistic effect may be explained in terms of protection of the intact zeaxanthin structure by effective scavenging of free radicals by nitroxides, therefore allowing zeaxanthin to quench the primary oxidant, singlet oxygen, effectively by the physical protective mechanism. The redox state of nitroxides was monitored using electron paramagnetic resonance spectroscopy. Both nitroxide free radicals and their reduced form, hydroxylamines, were equally effective. Obtained data were compared with the protective effects of α-tocopherol, which is the natural antioxidant and protector of MXs within the retina. The new strategies employed here to maintain the intact structure of MXs may enhance their protective potential against AMD.

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)-, and60 (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 from60 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.

Euphotic water samples were collected monthly from 16 sites in Lake of the Woods, Ontario, Canada from July to September, 2006 and 2007. Sites represented a range of inorganic nitrogen and phosphorus concentrations and mixing regimes, which was reflected in the spatial variability in phytoplankton biomass and community composition, and cyanobacterial toxin [microcystin-LR (MC-LR)] concentrations. Nutrient concentrations decreased along a south to north transect, concomitant with distance from the main river inlet at the south end of the lake. Filamentous, nitrogen-fixing Aphanizomenon spp. and Anabaena spp. were dominant at most sites: Aphanizomenon was more abundant at southern sites and Anabaena was more abundant at northern sites. MC-LR was detected at all sites at least once (maximum concentration 0. 69 μg L -1 ). However, there was no correlation between MC-LR concentration and the biomass of Anabaena, a documented MC-LR producer. For the pooled data set, total phosphorus and ammonium concentrations were the two parameters most strongly related with MC-LR concentration. Principal components analysis was used to analyze the data set; it indicated that two factors accounted for 81 % of the variability among the 12 environmental parameters tested. Factor 1 represented physicochemical characteristics and phytoplankton biomass and Factor 2 represented Anabaena and Woronichinia biomass.

Photoexcited melanin from retinal pigment epithelium (RPE) has been shown to induce photo-oxidation of ascorbate with concomitant generation of hydrogen peroxide. The aim of this study was to test whether the age-related changes in melanin content and distribution in the RPE affect the susceptibility of RPE cells to ascorbate-mediated photo-oxidation. Our results demonstrate that there is an age-dependent shift in the pathways with which ascorbate interacts in human RPE. In young RPE, melanin-ascorbate interactions may lead to pro-oxidant effects, but in the aged there is no net increase in photo-oxidation in the presence of ascorbate in comparison with samples without ascorbate. However, as ascorbate undergoes light-induced depletion and photogenerates ascorbyl free radical in the old RPE cells with initial yields similar to that observed for young RPE, an influence of ascorbate on oxidation pathways is revealed in the old RPE as well. Interestingly, the pro-oxidant effects of photoexcited melanolipofuscin-ascorbate interactions are greater than for photoexcited melanosomes when normalized to the same melanin content. The pro-oxidant effects of photoexcited melanin-ascorbate interactions are strongly dependent on the irradiation wavelength, this being the greatest for the shortest wavelength studied (340 nm) and steeply decreasing with increasing wavelength but still detectable even at 600 nm.

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.

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.

Summary 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.

The retinal pigment epithelium (RPE) is a monolayer of cells that appear phenotypically regular, but which exhibit striking cell-cell variability in content of melanin and lipofuscin granules, and in expression of many proteins. This naturally occurring cell heterogeneity likely arises by normal mechanisms regulating gene expression during development and postnatal aging. The consequence is a tissue in which individual cells may differ in their ability to support adjacent photoreceptors, and which may respond differentially to oxidative stress and other environmental influences that contribute to cell dysfunction during aging. The inherent variability of RPE cells is probably one factor contributing to the characteristically patchy pattern of retinal diseases like age-related macular degeneration.

Retinal photoreceptors and retinal pigment epithelial (RPE) cells are among the cell types that are sensitive to poisoning with methanol and its toxic metabolite formic acid. When exposed to formic acid in vitro, cultured cell lines from photoreceptors (661W) and the RPE (ARPE-19) were previously shown to accumulate similar levels of formate, but cytotoxic effects are greater in 661W cells. Here catalase and glutathione were analyzed in the two retinal cell lines to determine whether differences in these antioxidant systems contributed to cell-type-specific differences in cytotoxicity. Cells were exposed to formic acid (pH 6.8) in the culture medium in the presence or absence of a catalase activity inhibitor, 3-amino-1,2,4-triazole (AT), or a glutathione synthesis inhibitor, buthionine L-sulfoximine (BSO). Catalase protein, catalase enzyme activity, glutathione, glutathione peroxidase activity, cellular ATP, and cytotoxicity were analyzed. Compared to ARPE-19, 661W cells show lower antioxidant levels: 50% less glutathione, glutathione peroxidase and catalase protein, and 90% less catalase enzyme activity. In both cell types, formic acid treatment produced decreases in glutathione and glutathione peroxidase, and glutathione synthesis inhibition with BSO produced greater ATP depletion and cytotoxicity than formic acid treatment alone. In contrast, formate exposure produced decreases in catalase protein and activity in 661W cells, but increases in activity in ARPE-19. Treatment with the catalase inhibitor AT increased the formate sensitivity only of the ARPE-19 cells. ARPE-19 cells, therefore, may be less susceptible to formate toxicity due to higher levels of antioxidants, especially catalase, which increases on formate treatment and which has a significant cytoprotective effect for the RPE cell line.

purpose. Lipofuscin accumulates in human retinal pigment epithelium (RPE) cells with age and may be the main factor responsible for the increasing susceptibility of RPE to photo-oxidation with age. As the composition, absorption, and fluorescence of lipofuscin undergo age-related changes, the purpose of this study was to determine whether photoreactivity of lipofuscin granules also changes with the donor age.\ud \ud methods. To determine whether the photoreactivity of lipofuscin itself is age related, lipofuscin granules were isolated from human RPE and pooled into age groups. Photoreactivity was assessed by measuring action spectra of photo-induced oxygen uptake and photogeneration of reactive oxygen species. Separation of chloroform-soluble (ChS) and -insoluble (ChNS) components by Folch’s extraction was used to determine the factors responsible for the age-related increase in lipofuscin photoreactivity.\ud \ud results. The observed rates of photo-induced oxygen uptake and photo-induced accumulation of superoxide-derived spin adducts indicated that when normalized to equal numbers of lipofuscin granules, aerobic photoreactivity of lipofuscin increased with age. Both ChS and ChNS mediated photogeneration of singlet oxygen, superoxide radical anion, and photo-oxidation of added lipids and proteins. Although both ChS and ChNS exhibited substantial photoreactivities, neither exhibited significant age-related changes when normalized to equal dry mass. In contrast, ChNS contents in lipofuscin granules significantly increased with aging.\ud \ud conclusions. Aerobic photoreactivity of RPE lipofuscin substantially increases with aging. This effect may be ascribed to the increased content of insoluble components.

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.

Maspin, an ov-serpin, inhibits tumor invasion and induces cell adhesion to extracellular matrix molecules. Here, we use maspin/ovalbumin chimeric proteins and the maspin reactive site loop (RSL) peptide to characterize the role of the RSL in maspin-mediated functions. Replacement of the RSL plus the C-terminal region or the RSL alone of maspin with that of ovalbumin resulted in the loss of the stimulatory effect on adhesion of corneal stromal cells to type I collagen, fibronectin, and laminin and of mammary carcinoma MDA-MB-231 cells to fibronectin. Maspin with ovalbumin as the C-terminal region retained activity, suggesting the maspin C-terminal polypeptide is not required. An R340Q mutant retained full maspin activity; however, an R340A mutant lost activity. This indicates the arginine side chain at the putative P1 site forms a hydrogen bond and not an ionic bond. The RSL peptide (P10-P5', amino acids 330-345) alone induced cell-matrix adhesion of mammary carcinoma cells and corneal stromal cells and inhibited invasion of the carcinoma cells. Substitution of the RSL of ovalbumin with that of maspin converted inactive ovalbumin into a fully active molecule. Maspin bound specifically to the surface of the mammary carcinoma cells with a kd of 367 +/- 67 nM and 32.0 +/- 2.2 x 10(6) binding sites/cell. The maspin RSL peptide inhibited binding, suggesting the RSL is involved in maspin binding to cells. Sufficiency of the maspin RSL for activity suggests the mechanism by which maspin regulates cell-matrix adhesion and tumor cell invasion does not involve the serpin mechanism of protease inhibition.

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.

In MDCK epithelial cells, cell contact at confluency initiates a protracted process of morphogenesis during which several proteins known to bind the cytoskeleton become progressively associated with the detergent-resistant cell fraction and distributed to their characteristic polarized domains. Using extraction protocols that identify this tight cytoskeletal linkage, here we show a similar but slower, time-dependent enrichment in the detergent resistant fraction of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a highly abundant glycolytic enzyme that is traditionally considered soluble. Similar enrichment did not occur for two other glycolytic enzymes, phosphoglycerate mutase or lactate dehydrogenase. Insoluble GAPDH was not homogeneously distributed in the cytoplasm but rather displayed several discrete patterns that varied within and among MDCK cells. It also localized prominently to a few nuclei in the phenotypically heterogeneous cells of late confluency cultures. Disruptors of cytoskeletal filaments were relatively ineffective in the postconfluent epithelial monolayers, although use of disrupting agents implicated actin as the cytoplasmic filament that tethers insoluble GAPDH. Catalytic activity could be demonstrated in the insoluble fraction of GAPDH from postconfluent cultures, but only after release by mechanical disruption of insoluble extracts. Treatment of postconfluent cells with agents that deplete ATP diminished the fraction of cytoskeletally associated GAPDH, and levels of insoluble GAPDH were restored with ATP repletion, suggesting that ATP levels may regulate cytoskeletal linkage and thereby local enzyme activity. We conclude that the highly abundant and ubiquitous enzyme GAPDH becomes progressively enriched in detergent stable subcellular compartments during the process of epithelial morphogenesis. The process that produces GAPDH compartments is slow, suggesting that epithelial cells just at confluency, when they are typically analyzed, have not yet maximized the organizational state that can be attained in monolayer culture.

To determine the role of major chromophores of the human retinal pigment epithelium (RPE) in photooxidation of ascorbate, we monitored spectrophotometrically rates of ascorbate depletion, induced by blue light, in suspensions of human RPE melanin, melanolipofuscin and lipofuscin and in preparation of pigmented and nonpigmented bovine RPE cells. The results clearly show that melanin is the key retinal pigment responsible for the photosensitized oxidation of exogenous ascorbate. To elucidate the mechanism of the photooxidation process, we used purified RPE melanin granules and synthetic dopa (dihydroxyphenylalanine) melanin and employed electron spin resonance (ESR) spectroscopy, ESR oximetry and oxidase electrode. Our data indicate that photoinduced melanin radicals oxidize ascorbate via one-electron transfer reaction. The reduced melanin is reoxidized by molecular oxygen with the formation of superoxide anion and hydrogen peroxide, while the ascorbate radicals decay by disproportionation. Because in the absence of oxygen, no measurable oxidation of ascorbate is observed, it can be concluded that melanin acts as an electron transfer agent. Biological implications of this study remain unclear; however, the formation of oxygen-reactive species that accompany melanin-mediated photooxidation of ascorbate may represent a potential risk to the RPE that should be minimized by yet unknown cellular mechanisms.

Most studies of cell growth of the RPE employ cultures which have been previously passaged, but here we investigated freshly-explanted RPE cells, which may have been growth-quiescent for years, within the eye to determine whether co-culture affects initial outgrowth in primary culture.Bovine or human RPE were co-cultured in primary culture with several cell types to test the effects of homologous or heterologous cell association. For bovine RPE, cell number was measured over 14 days in cultures of RPE alone, or RPE in co-culture with irradiated living cells, with fixed-killed cells, with cells separated from the RPE by a semipermeable membrane, or in medium conditioned by the cell types used for co-culture. For human RPE, isolates from all donors were randomized, over a 13-month period, to co-culture or to culture alone. The number of cultures attaining a cell number at one month that was sufficient for further propagation was compared.Co-culture with irradiated living cells increased the growth of primary cultures of both bovine and human RPE. Living cells were required; fixed-killed cells were ineffective. The outgrowth-promoting activity was not tissue or species specific, and it appeared to require close cell association between the RPE and the co-culture cell population. Conditioned media were ineffective and rather were slightly growth inhibitory. Primary RPE cells showed an earlier expression of vimentin (a marker of Gzero-G1 transition), more rapid cell spreading, and a greater increase in cell number between 7 and 14 days after explant when grown in co-culture than when cultured alone.The results indicate that cell association between non-mitotic RPE cells and previously cultured cells of many types increases the outgrowth of the RPE by accelerating the early stages of growth activation in vitro. Co-culture methods offer a practical means for increasing the likelihood of producing cultures from small RPE isolates. Further, should cells involved in proliferative pathologies in situ associate with non-mitotic RPE within the monolayer, the latter cells may also be activated, leading to an augmentation of the pathology.

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.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.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.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.

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.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.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.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.

Exposure of the eye to intense light, particularly blue light, can cause irreversible, oxygen-dependent damage to the retina. However, no key chromophores that trigger such photooxidative processes have been identified yet. We have found that illumination of human retinal pigment epithelium (RPE) cells with light induces significant uptake of oxygen that is both wavelength- and age-dependent. Analysis of photoreactivity of RPE cells and their age pigment lipofuscin indicates that the observed photoreactivity in RPE cells is primarily due to the presence of lipofuscin, which, under aerobic conditions, generates several oxygen-reactive species including singlet oxygen, superoxide anion, and hydrogen peroxide. We have also found that lipofuscin-photosensitized aerobic reactions lead to enhanced lipid peroxidation as measured by accumulation of lipid hydroperoxides and malondialdehyde in illuminated pigment granules. Hydrogen peroxide is only a minor product of aerobic photoexcitation of lipofuscin. We postulate that lipofuscin is a potential photosensitizer that may increase the risk of retinal photodamage and contribute to the development of age-related maculopathy.

Summary 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 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.

Like most epithelial cells that are isolated from tissues and placed in culture, the phenotype of human retinal pigment epithelial cells (RPE) in vitro is more variable than for the same cells in situ. The phenotypic heterogeneity of the cultures has been attributed to varying stages of differentiation of the cells induced by the culture environment. In this study we show that within the same cultures RPE cells exist as phenotypic variants which are distinct and stable. Two phenotypically distinct subpopulations were identified, one epithelioid and one fusiform, that were present from the first spreading event in primary culture through multiple serial passages and when maintained for extended periods at confluency. Cell aggregation studies indicated differences in cellular adhesions, known determinants of cell shape, between the subpopulations. Methods to separate the subpopulations were developed which are based on the differential trypsin sensitivity of adhesions. The separated subpopulations had the same RPE cytokeratins by immunoblotting, but cytokeratin filaments (and actin filaments) had different organizations. The study indicates that RPE cell cultures contain at least two subpopulations of phenotypically distinct cells under identical culture conditions that can be separated and propagated independently. The phenotypic variants offer a model system for investigating determinants of epithelial cell shape in RPE. Further, the separation methods might be applied to test for phenotypic variants in other types of cultured epithelia.

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.

We have previously observed that the density of Na/K ATPase pumps is lower in RPE cells in the posterior pole of both bovine and human eyes. The posterior pole in human eyes includes the macula, a region which is predisposed to aging pathology. In this study we examined the effect of age on the sodium pump using cultures of bovine (bRPE) and human (hRPE) RPE cells that were aged in vitro by repeated passage. The cultures were assayed for cell number, protein, pump density (specific binding of [ 3 H]ouabain) and pump activity (specific uptake of 86 Rb) at confluency at each passage. In culture, bRPE had more pumps per cell (3·2 × 10 6 ) than hRPE (1·2 × 10 6 ), but the bRPE pumps were less active so the pumping capacity per cell was nearly equal. Bovine RPE aged more rapidly in vitro (survived fewer passages) than hRPE. With aging, RPE cells from both species showed declines in cell number at confluency. Pump number and pump activity per cell remained constant. Because cell number declined, the pumping capacity per unit area of confluent epithelium was diminished with culture aging. RPE cell number is known to decline with age in situ, especially in the macula. If Na/K ATPase pump number and activity per RPE cell remain constant with aging in vivo as shown here in vitro, the effective pumping capacity of the RPE per unit area of 'monolayer' would decline in aged eyes. Diminished pumping capacity with aging could affect the ability of the RPE to transport ions and other metabolites especially in the aging macula where pump density is lower even in normal young eyes.

Cytochrome c oxidase (CO) is a mitochondrial enzyme complex that was used in this study as a marker enzyme of aerobic metabolism. The activity of the enzyme was measured in the RPE of posterior and peripheral regions of bovine and human eyes. The posterior regions included the bovine area centralis or human macula. To determine if aging affected CO activity, activity was compared in RPE cells from human donors of varying age. An in vitro aging model was also used in which CO activity was measured in bovine and human RPE cells that were aged by repeated culture passage. CO activity was found to be significantly lower in posterior RPE cells from both bovine and human eyes suggesting that aerobic metabolism in RPE cells differs regionally. For human RPE samples, CO activity was lower in posterior cells at all donor ages and no significant age-related changes in CO activity were observed except that the highest activity samples were from older donors. Similarly, RPE cells that were aged in culture showed no significant changes in CO activity per cell although there were some high activity samples among the aged cultures. With aging, RPE cell density at confluency declined resulting in lower total CO activity in the aged epithelial monolayer. The data suggest that the ability of individual RPE cells to generate energy via respiration is maintained in the aged eye, but because RPE cell number declines with age and the activity of remaining RPE cells does not increase commensurately, there may declines in the ability of the aged pigment epithelium to perform tissue-level functions (e.g., transepithelial transport).(ABSTRACT TRUNCATED AT 250 WORDS)

A multimodal retinal imaging system that combines the merits of photoacoustic ophthalmoscopy, optical coherence tomography, confocal laser scanning ophthalmoscopy, and autofluorescence imaging has been developed.

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.

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.

Raman microspectroscopy is applied for the first time to study the effect of high-power light irradiation on isolated melanosomes and melanosomes in retinal pigmented epithelium cells.

The number and activity of ouabain-sensitive Na/K ATPase pumps expressed by many cell types in vitro, including human retinal pigment epithelial cells (RPE), have been shown to decline with increasing culture density. Cell proliferation also declined as cultures became dense so it was unclear if pump number was modulated by cell proliferation or culture confluency. By exposing RPE cultures to various feeding regimens, using culture medium containing or lacking serum, it was possible to produce RPE cultures with a range of culture densities and growth rates. These were analyzed for proliferative activity by quantifying [3H]thymidine incorporation and for Na/K ATPase pump number by measuring specific [3H]ouabain binding. The results suggest that pump number is modulated by culture density and, further, that the density-dependent regulation of pump number requires serum. Although density-dependent modulation of culture growth is also serum requiring, cell proliferation and pump number did not appear to be related; cultures of similar density which differed significantly in growth rate had similar numbers of pumps. The view that elevated numbers of pumps were not necessarily found in proliferating cells was further supported by qualitative examination of radioautographs of cells dually labeled with [3H]thymidine and [3H]ouabain. Cycling cells which had [3H]thymidine-labeled nuclei did not have notably higher labeling with [3H]ouabain. However, [3H]ouabain labeling, as an indicator of pump site number and distribution, did vary among cells in an RPE population and also within individual cells. This latter observation suggests that unpolarized RPE cells in sparse cultures may have regionally different requirements for ionic regulation.

Pigment granules (melanosomes), phagosomes, and other membrane-bound organelles translocate within cells along cytoskeletal scaffolds of microfilaments or microtubules.1-4 Both proteins constituting these scaffolds, actin and tubulin, are sensitive to modification under conditions of oxidative stress, which can produce cytoskeletal disruption.5-11 This observation motivated us to analyze whether oxidative stress impairs organelle movement. Stress-induced impairments of organelle motility, should they occur, could affect the ability of cells to maintain normal subcellular distribution of organelles and thus their overall cytoplasmic organization. A focus of this investigation was the motility of melanosomes within retinal pigment epithelial (RPE) cells subjected to oxidative stress induced by irradiation with visible light. This issue is of particular interest for several reasons. The retinal pigment epithelium is a monolayer of long-lived, postmitotic pigmented cells subjected to a lifetime of visible light irradiation. Photic damage to the retinal pigment epithelium caused by visible light, especially blue light, is believed to cause tissue dysfunction over time, contributing to age-related degenerations of the adjacent photoreceptors that the retinal pigment epithelium supports.12,13 Among the age-related structural changes that take place within RPE cells is redistribution of melanosomes from predominantly apical to more basal regions of the cell cytoplasm,14 suggesting that aging may affect mechanisms used to localize organelles. The functions that melanosomes perform within pigmented cells are not fully understood, but optical screening by melanin pigments is a well-established property of the organelle. Given that light strikes the retinal pigment epithelium from the apical side, the function of screening light-sensitive molecules in the RPE cytoplasm is best performed when the pigment granules are located apically. Melanin is also believed to protect cells from oxidative stress by virtue of its antioxidant properties.15 Little is known about the subcellular location of pro-oxidant and antioxidant species within cells, but the positioning of melanosomes within pigmented cells may be one determinant of whether the granules are located near sites where reactive oxygen species are generated so that the pigment can exert its putative antioxidant effect. To determine whether organelle motility in RPE cells is susceptible to oxidative stress induced by light, isolated porcine melanosomes were introduced by phagocytosis into cultures of the human RPE cell line ARPE-19. Parameters were then established for subjecting cultures to sublethal stress using visible light irradiation. We focused on inducing low levels of stress that might produce small functional decrements rather than overt cell death. Mild stress is likely more relevant during aging, when stress is not acute and fatal but rather subacute and chronic or recurrent and when it produces mild damage that could have cumulative consequences over time. Accordingly, methods were devised to quantify organelle motility in stressed cells that remained viable. Although RPE melanosome movement was one focus of this investigation, in most experiments the pigment granule was phagocytized and, therefore, encapsulated within a phagosome. This model of phagocytized granules has several benefits for analyses of organelle movement and photic stress. Granule uptake can be adjusted to control particle number within cells, and, because the granule is dark, phagosomes containing them can be tracked by bright-field microscopy, thereby avoiding the use of fluorescent tags that could confound studies involving light treatment. Further, as explained later, properties of the pigment can be exploited to alter the magnitude and location of photic stress. In addition to phagocytized melanosomes, a few experiments were conducted to determine the effects of photic stress on other organelles: phagosomes containing black latex beads in ARPE-19 cells and endogenous melanosomes in primary cultures of porcine retinal pigment epithelium.

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.

Raman microspectroscopy is used for the first time to study chemical and structural transformations in RPE cells exposed to cw and short-pulsed high-intensity light irradiation.

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.\ud 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.\ud 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.\ud 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.

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.

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.

Best vitelliform macular dystrophy (BMD) is an autosomal dominant inherited macular degenerative disease caused by mutations in the gene BEST1 (formerly VMD2). Prior reports indicate that BMD is characterized histopathologically by accumulation of lipofuscin in the retinal pigment epithelium (RPE). However, this accumulation has not been quantified and the chemical composition of lipofuscin in BMD has not been examined. In this study we characterize the histopathology of a donor eye from a rare individual homozygous for a mutation (W93C) in BEST1. We find that this individual's disease was not any more severe than has been described for heterozygotes. We then used this tissue to quantify lipofuscin accumulation by enriching intracellular granules from RPE cells on sucrose gradients and counting the granules in each density fraction. Granules from the homozygous donor eye as well as a donor eye from an individual heterozygous for the mutation T6R were compared with age-matched control eyes. Interestingly, the least dense fraction, representing classical lipofuscin granules was either not present or significantly diminished in the BMD donor eyes and the autoflourescence associated with lipofuscin had shifted to denser fractions. However, a substantial enrichment for granules in fractions of higher density was also noted in the BMD samples. Inspection of granules from the homozygous donor eye by electron microscopy revealed a complex abnormal multilobular structure. Analysis of granules by HPLC indicated a approximately 1.6- and approximately fourfold overall increase in A2E in the BMD eyes versus age-matched control eyes, with a shift of A2E to more dense granules in the BMD donor eyes. Despite the increase in A2E and total intracellular granules, the RPE in the homozygous donor eyes was relatively well preserved. Based on these data we conclude that the clinical and histopathologic consequences to the homozygous donor were not any more severe than has been reported previously for individuals who are established or presumptive heterozygotes. We find that A2E is a component of the lipofuscin accumulated in BMD and that it is more abundant than in control eyes suggesting that the etiology of BMD is similar to Stargardt's disease and Stargardt-like macular dystrophy. Finally, the changes we observe in the granules suggest that the histopathology and eventual vision loss associated with BMD may be due to defects in the ability of the RPE to fully degrade phagocytosed photoreceptor outer segments.

Cadherins are a family of glycoproteins that mediate calcium-dependent cell–cell adhesion at adherens junctions and participate in complex signaling pathways affecting cell phenotype.1 Dominant cadherin type differs among tissues, with E-cadherin the major cadherin of most monolayer epithelial cells. RPE cells differ in that they preferentially express N-cadherin, which localizes to a zonular junction2 resembling the E-cadherin adhesion in other epithelial cells. E-cadherin is not completely absent from the RPE, however. Junctional E-cadherin is found in scattered groups of both human and bovine RPE cells within the monolayer in situ.3,4 It is also found in patches of cells cultured from human RPE, but only after the cells have been held for long intervals at confluence.3 E-cadherin expression by the RPE is of interest because it is competent to induce basolateral polarity of Na,K-ATPase,5,6 which is the opposite of the normal polarity found in RPE cells in the eye.7–9 How apical polarity of the sodium pump develops in the RPE remains unresolved, but it may be essential for the cells to at least limit the basal inductive potential of E-cadherin. One mechanism to limit E-cadherin function would be to limit protein abundance, which led us to question why E-cadherin accumulates so slowly at RPE junctions in vitro. To address this question, we first examined the timing of E-cadherin gene expression after plating RPE cell cultures, expecting to find upregulation in the late postconfluence period when the protein is detected.3 Unexpectedly, mRNA for E-cadherin was found in RPE cultures even shortly after plating (shown herein), which prompted us to look more closely at E-cadherin protein at this early culture interval. As we now show, E-cadherin does not accumulate substantially in early RPE cultures. Instead small amounts of newly synthesized E-cadherin are detectable only transiently, peaking within a few hours after plating, at which time the protein is in the form of peptides of variable size rather the predicted 120-kDa molecular mass. Immunoreactive E-cadherin peptides codistribute with several organelles, notably those involved in protein degradation, rather than trafficking to the cell surface and localizing to junctions. The results suggest that posttranscriptional mechanisms involving altered processing and rapid turnover exist in the RPE to limit E-cadherin protein accumulation in early cultures when junctions comprised of N-cadherin are first forming.

Unlike most monolayer epithelial cells, cultured RPE are competent to form a zonular adhesion of N- rather than E-cadherin. To determine whether other normal epithelial cells do likewise, cells with high endogenous N-cadherin were cloned from the typically E-cadherin dominant epithelial line Madin-Darby canine kidney cells (MDCK) to analyze cell and junction phenotype in the presence of N-cadherin.A MDCK subclonal line, clone-YH, was selected for high endogenous N-cadherin and was compared with the RPE line hTERT-RPE1 with regard to cell phenotype, cadherin gene expression and cadherin protein distribution, glycosylation state, and catenin complex composition.In early cultures, hTERT-RPE1 cells are moderately epithelioid with junctional N-cadherin, but clone-YH cells are initially highly fusiform with N-cadherin in multiple sites. With time, N-cadherin in clone-YH becomes deglycosylated, resistant to detergent extraction, and zonular, and cells become epithelioid. Treatment with the N-glycosylation inhibitor tunicamycin induces an epithelioid phenotype in clone-YH, like time in culture but disrupts the hTERT-RPE1 phenotype. N-cadherin traffics to surface membranes and complexes with catenins regardless of cell type or glycosylation state, although catenin complex composition varied, showing enriched alpha-catenin under the cell-type-specific conditions in which N-cadherin was junctional. Clone-YH continued to express E-cadherin as a very minor cadherin, which trafficked to membranes but did not accumulate at junctions.RPE cells are not unique in localizing N-cadherin to a zonular adhesion typical of a monolayer epithelium, because even epithelial cells derived from a typically E-cadherin dominant line (clone-YH) form a zonular N-cadherin junction if the protein is abundant. However, there are cell and cadherin differences in mechanisms of cadherin accumulation in a zonular pattern, and a previously unrecognized cell-type-specific role for protein glycosylation in epithelial phenotype development.

Melanosomes of the retinal pigment epithelium (RPE) are relatively long-lived organelles that are theoretically susceptible to changes induced by exposure to visible light. Here melanosomes were isolated from porcine RPE cells and subjected to high intensity visible light to determine the effects of illumination on melanosome structure and on the content and antioxidant properties of melanin. As compared to untreated melanosomes, illuminated granules showed morphologic changes consistent with photodegradation, which included variable reductions in electron density demonstrated by transmission electron microscopy (TEM), and particle fragmentation and surface disruption revealed by scanning electron microscopy (SEM) and atomic force microscopy. Illuminated melanosomes had lower melanin content, indicated by measures of absorbance and electron spin resonance (ESR) signal intensity, and reduced ability to bind iron, shown by chemical and ESR analyses. Compared to untreated melanosomes, ESR-spin trapping analyses further indicated that illuminated melanosomes show increased photogeneration of superoxide anion and reduced ability to inhibit the iron ion-catalyzed free radical decomposition of hydrogen peroxide. It appears therefore that visible light irradiation can disrupt the structure of RPE melanosomes and reduce the amount and antioxidant properties of melanin. Some of these changes occur in human RPE melanosomes with aging and the results obtained here suggest that visible light irradiation is at least partly responsible. The consequence of light-induced changes in RPE melanosomes may be a diminished capacity of melanin to help protect aged cells from oxidative damage, perhaps increasing the risk of diseases with an oxidative stress component such as age-related macular degeneration.

The pigment melanin has antioxidant properties that could theoretically reduce oxidative damage to the retinal pigment epithelium (RPE), perhaps protecting against retinal diseases with an oxidative stress component like age-related macular degeneration. To determine whether melanin confers cytoprotection on RPE cells, melanosomes or control particles were introduced by phagocytosis into the human cell line ARPE-19 and oxidative stress was induced chemically (H2O2 or tert-butyl hydroperoxide) or with visible light. Since the iron-binding capacity of melanin is important for its antioxidant function, experiments were performed to confirm that the melanosomes were not iron saturated. Cytotoxicity was assessed by measures of plasma or lysosomal membrane integrity, mitochondrial function, and cell-substrate reattachment. Oxidative stress protocols were critically evaluated to produce modest cytotoxicity, which might allow detection of a small cytoprotective effect as expected for melanosomes. Particle internalization alone had no effect on baseline metabolic activity or on major RPE antioxidants. Particles were tested in multiple oxidative stress experiments in which culture conditions known to affect stress-induced cytotoxicity, notably culture density, were varied. No testing condition or outcome measure revealed a consistent protective (or cytotoxic) effect of melanosomes, indicating that measures of lysosome stability or whole cell viability do not demonstrate an antioxidant role for RPE melanosomes. If the melanosome, an insoluble particle, performs a cytoprotective function within cells, its effects may be limited to the local environment of the organelle and undetectable by conventional methods.

Cyanobacteria possess many adaptations to develop population maxima or "blooms" in lakes and reservoirs. A potential consequence of freshwater blooms of many cyanobacterial species is the production of potent toxins, including the cyclic hepatotoxins, microcystins (MCs). Approximately 70 MC variants have been isolated. Their toxicity to humans and other animals is well studied, because of public health concerns. This review focuses instead on the production and degradation of MCs in freshwater environments and their effects on aquatic organisms. Genetic research has revealed the existence of MC-related genes, yet the expression of these genes seems to be regulated by complex mechanisms and is influenced by environmental factors. In natural water bodies, the species composition of cyanobacterial communities and the ratio of toxic to nontoxic species and strains are largely responsible for total toxin production. Cyanobacteria play vital roles in aquatic food webs, yet production, accumulation, and toxicity patterns of MCs within aquatic food webs remain obscure.

Objective To determine whether human immunoglobulin attenuates the toxic effectsof Staphylococcus aureus culture supernatant in arabbit model of endophthalmitis. Methods Immunoglobulin binding to products of S aureus strainRN4220 was tested by Western blot analysis using known toxins (β-hemolysinand toxic shock syndrome toxin-1) and a concentrated culture supernatant containing S aureus exotoxins (pooled toxin). To induce endophthalmitis,pooled toxin was injected into the rabbit vitreous. For immunoglobulin treatment,immunoglobulin and pooled toxin were either mixed and injected simultaneouslyor immunoglobulin was injected immediately after or 6 hours after pooled toxininjection. Severity of endophthalmitis was graded according to a 9-day coursewith clinical examination (slitlamp biomicroscopy or indirect ophthalmoscopy)and evaluation of histologic sections. Results The toxic effects of pooled toxin were markedly reduced when immunoglobulinwas mixed with pooled toxin and injected simultaneously. Delayed injectionof immunoglobulin diminished its ability to reduce toxicity. Clinical andhistologic signs of toxicity were partially attenuated when immunoglobulinwas injected immediately after pooled toxin, but only minimal clinically detectablereductions in toxicity were observed when immunoglobulin injection was delayedfor 6 hours. Conclusion Pooled human immunoglobulin can attenuate the toxic intravitreal effectsof a concentrated culture supernatant containing S aureus exotoxins. Clinical Relevance Immunoglobulin may represent a novel adjuvant in the treatment of bacterialendophthalmitis. To optimize the potential therapeutic benefit, maximizingthe mixture of immunoglobulin with bacterial products and early interventionare likely to be important.

Epithelial (E)-cadherin plays a critical role in developing a normal epithelial phenotype but neural (N)-cadherin can disrupt epithelial shape, at least in carcinoma-derived cells. Here the normal epithelial cell line MDCK was used to select for a trypsin-sensitive (TS-MDCK) subpopulation that expresses low levels of endogenous N-cadherin. Similar amounts of E-cadherin and all catenins are found in both TS-MDCK and trypsin-resistant cells (TR-MDCK), but TS-MDCK are less phenotypically epithelioid and more motile, and junctional proteins are more detergent soluble. In TS-MDCK, N-cadherin is largely nonjunctional; a similar N-cadherin distribution and mesenchymal phenotype are found in TR-MDCK transfected to express low levels of exogenous N-cadherin. Little N-cadherin was attracted to junctions between TS-MDCK and hTERT-RPE1 cells, a retinal pigment epithelium-derived line that expresses dominantly N-cadherin. No differences were seen in E-cadherin-catenin complexes in TS- and TR-MDCK, but N-cadherin-catenin complexes in TS-MDCK have more abundant p120 catenin. Overall, the results indicate that E- and N-cadherin assemble stoichiometrically different complexes with p120 in the same cells. Further, N-cadherin does not participate with E-cadherin in a zonular epithelial junction in normal MDCK epithelial cells. Rather, even low levels of endogenous N-cadherin contribute to a disrupted epithelial phenotype, resembling the effect of N-cadherin on carcinoma-derived epithelial cells.