Lipofuscin-mediated photic stress inhibits phagocytic activity of ARPE-19 cells; effect of donors’ age and antioxidants

The risk of chronic oxidative stress in the retinal pigment epithelium (RPE) increases with age due to accumulation of the photoreactive age pigment lipofuscin (LFG). Here, we asked whether sublethal and weakly lethal photic stress, induced by irradiation of ARPE-19 cells containing phagocytised LFG, affected the cell specific phagocytic activity, which is critically important for proper functioning and survival of the retina, and if natural antioxidants could modify the observed outcomes. ARPE-19 cells preloaded with LFG isolated from human donors of different age or containing LFG enriched with zeaxanthin and α-tocopherol (LFG-A), were irradiated with blue light. Phagocytosis of fluorescein-5-isothiocyanate (FITC)-labelled photoreceptor outer segments was determined by flow cytometry. Photoreactivity of LFG and LFG-A was analysed by measuring photoconsumption of oxygen and photogeneration of singlet oxygen mediated by the granules. LFG-mediated photic stress in ARPE-19 cells induced significant inhibition of their specific phagocytosis. The inhibitory effect increased with age of LFG donors and was reduced by enrichment of the granules with antioxidants. Oxygen consumption and generation of singlet oxygen induced by the photoexcited LFG increased with donor’s age and was partially quenched by antioxidants. Although the phototoxic potential of lipofuscin increased with age, natural antioxidants reduced photoreactivity of LFG and their efficiency to induce oxidative stress. This study has demonstrated, for the first time, that mild oxidative stress, mediated by the age pigment lipofuscin, impairs specific phagocytic activity of RPE, and that natural antioxidants can protect this important cellular function by reducing lipofuscin photoreactivity.     

Inhibition of phagocytic activity of ARPE-19 cells by free radical mediated oxidative stress

Oxidative stress is a main factor responsible for key changes leading to the onset of age-related macular degeneration (ARMD) that occur in the retinal pigment epithelium (RPE), which is involved in phagocytosis of photoreceptor outer segments (POS). In this study, hydrogen peroxide (H₂O₂), H₂O₂ and iron ions (Fe) or rose Bengal (RB) in the presence of NADH and Fe were used to model free radical mediated oxidative stress to test if free radicals and singlet oxygen have different efficiency to inhibit phagocytosis of ARPE-19 cells. Free radical mediated oxidative stress was confirmed by HPLC-EC(Hg) measurements of cholesterol hydroperoxides in treated cells. Electron paramagnetic resonance (EPR) spin trapping was employed to detect superoxide anion. Cell survival was analyzed by the MTT assay. Specific phagocytosis of fluorescein-5-isothiocyanate-labeled POS and non-specific phagocytosis of fluorescent beads were measured by flow cytometry. HPLC analysis of cells photosensitized with RB in the presence of NADH and Fe indicated substantial increase in formation of free radical-dependent 7α/7β-hydroperoxides. EPR spin trapping confirmed the photogeneration of superoxide anion in samples enriched with RB, NADH and Fe. For all three protocols sub-lethal oxidative stress induced significant inhibition of the specific phagocytosis of POS. In contrast, non-specific phagocytosis was inhibited only by H₂O₂ or H₂O₂ and Fe treatment. Inhibition of phagocytosis was transient and recoverable by 24?h. These results suggest that free radicals may exert similar to singlet oxygen efficiency in inhibiting phagocytosis of RPE cells, and that the effect depends on the location where initial reactive species are formed.     

Oxysterol-induced toxicity in R28 and ARPE-19 cells

Studies have shown an intimate relationship between cholesterol and retinal diseases; we examined the effects of cholesterol oxides on cultured cells. Using the rat retinal precursor cell line R28 and the human RPE cell line ARPE-19, we investigated the potential cytotoxicity of cholesterol oxides. Cultured R28 and ARPE-19 cells were treated with either 25-hydroxycholesterol and 7-ketocholesterol (0–50 µg/ml). Cell viability was determined by the WST-1 colorimetric assay. Production of reactive oxygen intermediate (ROI) was assessed by a fluorescent probe–based assay (2,7-dichlorodihydrofluorescein diacetate [H₂DCFDA]). To detect the presence of apoptosis, DNA fragmentation gel analysis and Hoescht nuclear staining were performed. Both cholesterol oxides tested were toxic in a time- and dose-dependent fashion to the two cell lines used in this study. Treatment of R28 cells with either 25-hydroxycholesterol or 7-ketocholesterol at a concentration of 25 µg/ml resulted in greater than 50% loss of cell viability after 24 h. ARPE-19 cells were slightly less affected, with a loss of cell viability of approximately 20% and 40% after 24 h-exposure of 25-hydroxycholesterol and 7-ketocholesterol, respectively. DNA fragmentation and chromatin condensation demonstrated apoptotic events occurring in 7-ketocholesterol–treated cells. The fluorescent assay for ROI production showed that after an hour of exposure to 7-ketocholesterol, R28 cells responded with increased levels of ROIs, whereas no immediate production of ROIs were detected with treated ARPE-19 cells. These in vitro findings provide evidence that cholesterol oxides can directly damage cultured retinal and RPE cells. The oxysterol-induced oxidative stress in these cells may be a factor in the pathology of retinal degenerative diseases.     

Knockdown of FOXO6 inhibits high glucose–induced oxidative stress and apoptosis in retinal pigment epithelial cells

Oxidative stress and apoptosis in retinal pigment epithelium cells are involved in the pathogenesis of diabetic retinopathy (DR). Forkhead box class O 6 (FOXO6) is a member of the FOXO family that can regulate diabetes-induced oxidative stress. However, the role of FOXO6 in DR has not been clarified. The aim of the present study was to investigate the effects of FOXO6 on high glucose (HG)-induced oxidative stress and apoptosis in ARPE-19 cells. The results showed that FOXO6 was overexpressed in clinical vitreous samples from DR patients and in HG-induced ARPE-19 cells. Knockdown of FOXO6 by small interfeing RNA targeting FOXO6 (si-FOXO6) mitigated the HG-induced the production of reactive oxygen species and malondialdehyde, as well as the inhibition of superoxide dismutase activity. Knockdown of FOXO6 reduced the rate of cell apoptosis in HG-induced ARPE-19 cells. The increase in bax expression and decrease in bcl-2 expression caused by HG stimulation were reversed by si-FOXO6 transfection. Furthermore, knockdown of FOXO6 enhanced the activation of Akt/Nrf2 pathway in HG-stimulated ARPE-19 cells. Taken together, suppression of FOXO6 protects ARPE-19 cells from HG-induced oxidative stress and apoptosis, which is in part mediated by the activation of Akt/Nrf2 pathway.     

Retinal pigment epithelial cell multinucleation in the aging eye – a mechanism to repair damage and maintain homoeostasis

Retinal pigment epithelial (RPE) cells are central to retinal health and homoeostasis. Dysfunction or death of RPE cells underlies many age‐related retinal degenerative disorders particularly age‐related macular degeneration. During aging RPE cells decline in number, suggesting an age‐dependent cell loss. RPE cells are considered to be postmitotic, and how they repair damage during aging remains poorly defined. We show that RPE cells increase in size and become multinucleate during aging in C57BL/6J mice. Multinucleation appeared not to be due to cell fusion, but to incomplete cell division, that is failure of cytokinesis. Interestingly, the phagocytic activity of multinucleate RPE cells was not different from that of mononuclear RPE cells. Furthermore, exposure of RPE cells in vitro to photoreceptor outer segment (POS), particularly oxidized POS, dose‐dependently promoted multinucleation and suppressed cell proliferation. Both failure of cytokinesis and suppression of proliferation required contact with POS. Exposure to POS also induced reactive oxygen species and DNA oxidation in RPE cells. We propose that RPE cells have the potential to proliferate in vivo and to repair defects in the monolayer. We further propose that the conventionally accepted ‘postmitotic’ status of RPE cells is due to a modified form of contact inhibition mediated by POS and that RPE cells are released from this state when contact with POS is lost. This is seen in long‐standing rhegmatogenous retinal detachment as overtly proliferating RPE cells (proliferative vitreoretinopathy) and more subtly as multinucleation during normal aging. Age‐related oxidative stress may promote failure of cytokinesis and multinucleation in RPE cells.     

Cynaroside protects the blue light-induced retinal degeneration through alleviating apoptosis and inducing autophagy in vitro and in vivo

BackgroundBlue light can directly penetrate the lens and reach the retina to induce retinal damage, causing dry age-related macular degeneration (dAMD). Cynaroside (Cyn), a flavonoid glycoside, was proved to alleviate the oxidative damage of retinal cells in vitro. However, whether or not Cyn also exerts protective effect on blue light-induced retinal degeneration and its mechanisms of action are unclear.PurposeThis study aims to evaluate the protective effects of Cyn against blue-light induced retinal degeneration and its underlying mechanisms in vitro and in vivo.Study design/methodsBlue light-induced N-retinylidene-N-retinylethanolamine (A2E)-laden adult retinal pigment epithelial-19 (ARPE-19) cell damage and retinal damage in SD rats were respectively used to evaluate the protective effects of Cyn on retinal degeneration in vitro and in vivo. MTT assay and AnnexinV-PI double staining assay were used to evaluate the in vitro efficacy. Histological analysis, TUNEL assay, and fundus imaging were conducted to evaluate the in vivo efficacy. ELISA assay, western blot, and immunostaining were performed to investigate the mechanisms of action of Cyn.ResultsCyn decreased the blue light-induced A2E-laden ARPE-19 cell damage and oxidative stress. Intravitreal injection of Cyn (2, 4 μg/eye) reversed the retinal degeneration induced by blue light in SD rats. Furthermore, Cyn inhibited the nuclear translocation of NF-κB and induced autophagy, which led to the clearance of overactivated pyrin domain containing 3 (NLRP3) inflammasome in vitro and in vivo.ConclusionCyn protects against blue light-induced retinal degeneration by modulating autophagy and decreasing the NLRP3 inflammasome.     

Cleavage of Mer Tyrosine Kinase (MerTK) from the Cell Surface Contributes to the Regulation of Retinal Phagocytosis

Phagocytosis of apoptotic cells by macrophages and spent photoreceptor outer segments (POS) by retinal pigment epithelial (RPE) cells requires several proteins, including MerTK receptors and associated Gas6 and protein S ligands. In the retina, POS phagocytosis is rhythmic, and MerTK is activated promptly after light onset via the αvβ5 integrin receptor and its ligand MFG-E8, thus generating a phagocytic peak. The phagocytic burst is limited in time, suggesting a down-regulation mechanism that limits its duration. Our previous data showed that MerTK helps control POS binding of integrin receptors at the RPE cell surface as a negative feedback loop. Our present results show that a soluble form of MerTK (sMerTK) is released in the conditioned media of RPE-J cells during phagocytosis and in the interphotoreceptor matrix of the mouse retina during the morning phagocytic peak. In contrast to macrophages, the two cognate MerTK ligands have an opposite effect on phagocytosis and sMerTK release, whereas the integrin ligand MFG-E8 markedly increases both phagocytosis and sMerTK levels. sMerTK acts as a decoy receptor blocking the effect of both MerTK ligands. Interestingly, stimulation of sMerTK release decreases POS binding. Conversely, blocking MerTK cleavage increased mostly POS binding by RPE cells. Therefore, our data suggest that MerTK cleavage contributes to the acute regulation of RPE phagocytosis by limiting POS binding to the cell surface.     

Photobleaching of pheomelanin increases its phototoxic potential: Physicochemical studies of synthetic pheomelanin subjected to aerobic photolysis

Although melanin is a photoprotective pigment, its elevated photochemical reactivity could lead to various phototoxic processes. Photoreactivity of synthetic pheomelanin, derived from 5‐S‐cysteinyldopa (5SCD‐M) and its photodegradation products obtained by subjecting the melanin to aerobic irradiation with UV‐visible light, was examined employing an array of advanced physicochemical methods. Extensive photolysis of 5SCD‐M was accompanied by partial bleaching of the melanin, modification of its paramagnetic properties, and significant increase in the ability to photogenerate singlet oxygen. The changes correlated with a substantial decrease in the melanin content of benzothiazine (BT) units and increase of modified benzothiazole (BZ) units. Synthetically prepared BZ exhibited higher efficiency to photogenerate singlet oxygen than the synthetic BT, and the free radical form of BZ, unlike that of BT, did not show measurable spin density on nitrogen atom, which was confirmed by quantum chemical calculations. Formation of modified BZ units in the photobleached 5SCD‐M is responsible for the paramagnetic and photochemical changes of the melanin and its elevated phototoxic potential. Given a relatively constant pheomelanin–eumelanin ratio, such undesirable changes could occur in individual of all skin types.     

Photobleaching kinetics, photoproduct formation, and dose estimation during ALA induced PpIX PDT of MLL cells under well oxygenated and hypoxic conditions.

Fluorescence photobleaching and photoproduct formation were investigated during delta-aminolevulinic acid (ALA) induced protoporphyrin IX (PpIX) PDT of MLL cells in vitro. Cells were incubated in either 0.1 or 1.0 mM ALA for 4 h and were treated with 532 nm or 635 nm light under well oxygenated or hypoxic conditions. Fluorescence spectra were acquired during treatment. Photobleaching and photoproduct formation were quantified using singular value decomposition fitting of fluorescence spectra to experimentally determined basis spectra for PpIX, photoprotoporphyrin (Ppp), product II (peak at 655 nm), and product III (peak at 618 nm). PpIX photobleaching occurred under both normal and hypoxic conditions. The photobleaching kinetics could not be explained by purely first- or second-order photobleaching kinetics, and were attributed to differences in PpIX binding at the two ALA incubation concentrations. Ppp was the main photoproduct and accumulated in higher levels in the absence of oxygen, likely a result of reduced Ppp photobleaching under hypoxia. Increases in product II fluorescence occurred mainly in the presence of oxygen. To assess potential fluorescence based PDT dose metrics, cell viability was measured at select times during treatment using a colony formation assay. Cell survival correlated well to changes in product II fluorescence, independent of oxygenation, sensitizer concentration, and treatment wavelength, suggesting that this product is primarily a result of singlet oxygen mediated reactions and may potentially be useful to quantify singlet oxygen dose during PDT.     

Mechanisms of the damaging effect of fluorescent dyes on the retina

Mechanisms of the photo-damage of a fluorescent dye (methylene blue) and of the protective action of antioxidants and quenchers of singlet oxygen on the outer retinal rod segments (ORRS) and retinal function in situ and in vivo were studied. The methylene blue-induced formation of singlet oxygen in the ORRS resulted in accumulation of lipid peroxidation products, oligomerization of rhodopsin, and in a decrease in rhodopsin thermal resistance. Modification of the lipid and protein components of the visual cells by singlet oxygen inhibited the electrical activity of both isolated frog retina in situ and rabbit retina in vivo (waves a and b on the electroretinogram). The antioxidants (alpha-naphthol, alpha-tocopherol, 4-methyl-2,6-di-tert-butylphenol) and singlet oxygen quenchers, [1,4-diazabicyclo (2,2,2)octane] and alpha-tocopherol prevented the damaging effects of the fluorescent dye induced by singlet oxygen formation.     

Toxicity of Selected Cationic Drugs in Retinoblastomal Cultures and in Cocultures of Retinoblastomal and Retinal Pigment Epithelial Cell Lines

Tamoxifen and toremifene are antiestrogenic drugs successfully used in the therapy of breast cancer. Rheumatoid arthritis and malaria have been treated with chloroquine for decades. Unfortunately, tamoxifen and chloroquine are reported to induce retinal changes as a side effect. We now studied the effects of tamoxifen, toremifene, and chloroquine on the viability of the human retinoblastomal cell line Y79, using the WST-1 test or measurement of the cellular ATP content. The studies were made on Y79 cell cultures and on cocultures of Y79 cells and retinal pigment epithelial cell line ARPE-19. The cocultures were used to clarify the effect of retinal pigment epithelium on toxicity to Y79 cells. In the coculture, the drugs were applied to ARPE-19 cells growing in the culture inserts on top of Y79 cells and the viability of ARPE-19 and Y79 cells was assessed separately. Tamoxifen, toremifene, and chloroquine reduced dose-dependently the viability of Y79 cells after 24-h exposure. The ARPE-19 cells proved to be protective after chloroquine exposure in the coculture. The results shed light on the toxicity of tamoxifen and chloroquine in Y79 cells in vitro. With the coculture we were able to simulate the in vivo route of chloroquine to the retina via the retinal pigment epithelium.     

A2E-epoxides damage DNA in retinal pigment epithelial cells. Vitamin E and other antioxidants inhibit A2E-epoxide formation

The autofluorescent pigments that accumulate in retinal pigment epithelial cells with aging and in some retinal disorders have been implicated in the etiology of macular degeneration. The major constituent is the fluorophore A2E, a pyridinium bisretinoid. Light-exposed A2E-laden retinal pigment epithelium exhibits a propensity for apoptosis with light in the blue region of the spectrum being most damaging. Efforts to understand the events precipitating the death of the cells have revealed that during irradiation (430 nm), A2E self-generates singlet oxygen with the singlet oxygen in turn reacting with A2E to generate epoxides at carbon-carbon double bonds. Here we demonstrate that A2E-epoxides, independent of singlet oxygen, exhibit reactivity toward DNA with oxidative base changes being at least one of these lesions. Mass spectrometry revealed that the antioxidants vitamins E and C, butylated hydroxytoluene, resveratrol, a trolox analogue (PNU-83836-E), and bilberry extract reduce A2E-epoxidation, whereas single cell gel electrophoresis and cell viability studies revealed a corresponding reduction in the incidence of DNA damage and cell death. Vitamin E, a lipophilic antioxidant, produced a more pronounced decrease in A2E-epoxidation than vitamin C, and treatment with both vitamins simultaneously did not confer additional benefit. Studies in which singlet oxygen was generated by endoperoxide in the presence of A2E revealed that vitamin E, butylated hydroxytoluene, resveratrol, the trolox analogue, and bilberry reduced A2E-epoxidation by quenching singlet oxygen. Conversely, vitamin C and ginkgolide B were not efficient quenchers of singlet oxygen under these conditions.     

Effects of white light on beta-catenin signaling pathway in retinal pigment epithelium

This study investigated the effect of visible light exposure on retinal pigment epithelium (RPE). The activation of Wnt/β-catenin pathway was investigated by immunofluorescence and Western blot analysis using human retinal pigment epithelial (ARPE-19) cells, which demonstrated that the exposure of white light induced the activation of the Wnt/β-catenin pathway. Real time RT-PCR demonstrated that the mRNA of α-smooth muscle actin (α-SMA), and vimentin increased 2.5-4-fold and that of zona occludens 1 (ZO-1) decreased approximately 0.8-fold after white light exposure. The up-regulation of vimentin expression and the down-regulation of ZO-1 were evident by Western blot analysis and immunohistochemistry. Moreover, the ability of phagocytosis of ARPE-19 cells decreased 0.6-fold after light exposure. Together, white light exposure was supposed to induce the activation of Wnt/β-catenin pathway, the changes in the expression markers of epithelial and mesenchymal cells in RPE cells, and the concomitant impairment of the ability of phagocytosis.     

Zeaxanthin in combination with ascorbic acid or alpha-tocopherol protects ARPE-19 cells against photosensitized peroxidation of lipids

The antioxidant action of carotenoids is believed to involve quenching of singlet oxygen and scavenging of reactive oxygen radicals. However, the exact mechanism by which carotenoids protect cells against oxidative damage, particularly in the presence of other antioxidants, remains to be elucidated. This study was carried out to examine the ability of exogenous zeaxanthin alone and in combination with vitamin E or C, to protect cultured human retinal pigment epithelium cells against oxidative stress. The survival of ARPE-19 cells, subjected to merocyanine 540-mediated photodynamic action, was determined by the MTT test and the content of lipid hydroperoxides in photosensitized cells was analyzed by HPLC with electrochemical detection. We found that zeaxanthin-supplemented cells, in the presence of either alpha-tocopherol or ascorbic acid, were significantly more resistant to photoinduced oxidative stress. Cells with added antioxidants exhibited increased viability and accumulated less lipid hydroperoxides than cells without the antioxidant supplementation. Such a synergistic action of zeaxanthin and vitamin E or C indicates the importance of the antioxidant interaction in efficient protection of cell membranes against oxidative damage induced by photosensitized reactions.     

Green fluorescent protein photobleaching: a model for protein damage by endogenous and exogenous singlet oxygen

Characterization of protein damage during photosensitization of chlorin e6-treated cells was performed using the green fluorescent protein (GFP). The GFP-chromophore damage caused by singlet oxygen was studied in COS 7 kidney cells and E. coli bacteria following light irradiation. Electron spin resonance (ESR) revealed the generation of endogenous singlet oxygen (1O2) by photoactivated GFP, an effect similar to that produced by the exogenous photosensitizer chlorin e6. A light dose-dependent photobleaching effect of GFP was pronounced at low pH or upon photosensitization with chlorin e6. However, the 1O2 quenchers beta-carotene and sodium azide minimized GFP photo-bleaching. Gel electrophoresis of photosensitized GFP followed by fluorescence multi-pixel spectral imaging revealed the binding of chlorin e6 to GFP, affecting the photobleaching efficacy. Fluorescence multi-pixel spectral imaging of GFP-transfected COS 7 cells demonstrated the presence of GFP in the cytoplasm and nucleus, while chlorin e6 was found to be concentrated in the perinuclear vesicles. Exposure of the cells to light induced GFP photobleaching in the close vicinity of chlorin e6 vesicles. We conclude that photoactivated GFP generates endogenous 1O2, inducing chromophore damage, which can be enhanced by the cooperation of exogenous chlorin e6.     

The role of molecular oxygen in the photodynamic effect of phthalocyanines

Phthalocyanines are a class of mammalian cell photosensitizers which may be useful in photodynamic therapy for cancer. Chloroaluminum phthalocyanine tetrasulfonate was incubated with Chinese hamster cells in culture and exposed to white light at different concentrations of oxygen. The ability of the cells to form colonies served as an end point for the photobiological effect of the dye. The efficiency of photoinactivation of the sensitized cells decreased with decreasing oxygen concentration. Very little photoinactivation was observed when the atmosphere equilibrated with the cells was oxygen-free nitrogen. At an oxygen partial pressure of 2.5 mm Hg, photoinactivation was reduced by 50% compared to ambient atmosphere. In an attempt to understand the nature of the interaction between excited dyes and oxygen, the ability of several phthalocyanines to photogenerate singlet oxygen was measured. Thus phthalocyanines containing paramagnetic ions (copper, iron, vanadyl) do not generate 1O2 in contradistinction to diamagnetic metals (zinc and aluminum). The latter are efficient photosensitizers, while the former have little if any photobiological activity. In spite of this correlation, singlet oxygen may not be the intermediate involved in cytotoxicity. The reasons are discussed.     

贴壁培养细胞台盼蓝拒染试验的方法学探讨

本工作旨在探讨贴壁培养细胞台盼蓝拒染试验的方法,为其合理应用提出建议.用正常培养及NaN3作致死处理的人视网膜色素上皮细胞株-19,进行原位贴壁细胞和培养体系上悬液中细胞的台盼蓝拒染实验,并比较其原位法与计数板法所测活细胞率.与计数板法相似,随染液浓度增高或染色时间延长,原位台盼蓝拒染实验的活细胞率检测值逐渐增加.培养...     

Exosomes derived from RPE cells under oxidative stress mediate inflammation and apoptosis of normal RPE cells through Apaf1/caspase-9 axis

This study aims to explore the effects of exosomes, secreted by retinal pigment epithelial (RPE) cells under oxidative stress (OS), on apoptosis and inflammation of normal RPE cells. Exosomes secreted by normal RPE cells (named as exo) and rotenone (2.5 µmol/L) stimulated RPE cells (named as rot-exo) were isolated and extracted by multi-step differential centrifugation for morphology observation under a transmission electron microscopy. pcDNA3.1a, pcDNA3.1a-Apaf1, and p3xFlag-CMV-caspase-9 plasmids were constructed and transfected into ARPE-19 cells. Exosomes secreted by ARPE-19 cells were injected into the vitreous body of rats to verify the effect of Apaf1 and caspase-9 on cell apoptosis and inflammation. Co-immunoprecipitation was applied to clarify the interaction of Apaf1 with caspase-9. Exosomes secreted by rotenone stimulated ARPE-19 cells could induce cell apoptosis, oxidative injury, and inflammation in ARPE-19 cells. Exosomes secreted under OS can damage retinal functions of rats and have upregulated expression of Apaf1. Overexpression of Apaf1 in exosomes secreted under OS can cause the inhibition of cell proliferation, the increase of cell apoptosis and elicitation of inflammatory response in ARPE-19 cells. Exosomes derived from ARPE-19 cells under OS regulate Apaf1 expression to increase cell apoptosis and to induce oxidative injury and inflammatory response through a caspase-9 apoptotic pathway.