Disruption of Cell-Cell Junctions and Induction of Pathological Cytokines in the Retinal Pigment Epithelium of Light-Exposed Mice

Abstract

Purpose.: Toelucidate the influences of light exposure on the retinal pigment epithelium (RPE) in vivo that may be involved in the pathogenesis of AMD.

Methods.: Six- to 7-week-old BALB/c mice were exposed to light at 2000 lux for 3 hours. Flat-mount RPE samples were immunostained with anti-ZO-1 antibody for evaluating tight junction, anti-N-cadherin, and anti-β-catenin antibodies for adherens junction, and stained with phalloidin for actin cytoskeleton. The reactive oxygen species (ROS) level was measured using DCFH-DA; Rho-associated coiled-coil forming kinase (ROCK) activity was by ELISA. Cytokine expression was analyzed by real-time RT-PCR and/or ELISA in the RPE-choroid, and macrophage recruitment was by real-time RT-PCR and immunohistochemistry. Either an antioxidant, N-Acetyl-L-cysteine (NAC), or a ROCK inhibitor, Y-27632, were administered to analyze the roles of ROS and ROCK activation, respectively.

Results.: Light exposure disrupted staining patterns of tight junctions, adherens junctions, and actin cytoskeleton in the RPE, where ROS was elevated. However, NAC treatment avoided the RPE changes, reducing ROS. ROCK activity increased after light exposure was suppressed by NAC, and the structural disruptions were suppressed by Y-27632. The levels of MCP-1, CCL11, and IL-6 increased after light exposure were suppressed by NAC. Light-induced MCP-1 and IL-6 were suppressed by Y-27632. Macrophage recruitment after light exposure was also suppressed either by NAC or Y-27632.

Conclusions.: Light exposure induced ROS and Rho/ROCK activation, which caused disruption of cell-cell junctions (tight junctions and adherens junctions) and actin cytoskeleton, the RPE's barrier structure, and induced AMD-associated pathological changes in the RPE-choroid.

Introduction

Oxidative stress is induced in response to endogenous or exogenous stimuli and contributes to the pathogenesis of diseases. In addition to its role in cancer ¹ and diabetes, ² oxidative stress is involved in AMD, a leading cause of blindness worldwide, and one that is rapidly increasing in prevalence as the population ages. ^3–5 It is well accepted that the effects of oxidative stress on the retinal pigment epithelium (RPE) contribute to AMD's pathogenesis, ^3–6 and light exposure can be involved as a risk of AMD. ^3,5,7 Light exposure is known to cause oxidative stress in the neural retina ^7–9 ; however, the influences of light stimuli on the RPE are not fully understood. 

Eyes receive light stimuli and create vision by transforming the stimuli into electric signals, which are relayed from the retina to the visual centers of the brain. The process of light-to-neural signal transformation is closely associated with the visual cycle, in which interactions between photoreceptor cells and RPE cells cause the sequential isomerization of retinoid molecules to regenerate rhodopsin. When the light stimulus is very high, both the toxicity of the products from visual cycle and excessive metabolic action in the cells can cause oxidative stress–induced apoptosis in the photoreceptors, ^7–9 and this apoptosis can be prevented by administering antioxidants, such as lutein. ^8,10 Given that the RPE cells perform metabolic functions related to the visual cycle, oxidative stress accumulating in the RPE after light exposure could induce the pathological changes in the RPE that are associated with AMD. 

In addition to its role in metabolizing retinoid molecules in the visual cycle, the RPE functions as the blood-retinal barrier (BRB), a monolayered cuboidal epithelium held together by cell-cell junctional structures, such as tight junctions and adherens junctions. ¹¹ These junctional structures, as well as actin cytoskeleton, can be influenced by Rho family GTPases that ultimately control cell motility, cell proliferation, and morphogenesis. ^12,13 In the downstream of Rho proteins, Rho-associated coiled-coil forming kinase (ROCK) is activated and involved in regulating the shape and movement of cells by acting on cytoskeleton. ROCK activation can be inhibited by a ROCK inhibitor, Y-27632, a low-molecular compound that is cell-permeable. The RPE also secretes cytokines that maintain the microenvironment or, under certain conditions, causes pathological changes including macrophage recruitment. In AMD, breakdown of the BRB may allow choroidal neovascularization (CNV) and related exudative fluids to invade the subretinal space, resulting in visual loss; abnormal cytokine secretion could also promote disease activity. 

In this study, we first confirmed that excessive light exposure induced oxidative stress in the RPE of mice, and analyzed the influence of light, focusing on the cell-cell junctions, actin cytoskeleton, cytokine expression, and macrophage recruitment. The causative signaling mechanisms leading to light-induced tissue damage were also analyzed. 

Methods

Animals

Six-week-old male BALB/c mice were purchased (CLEA Japan, Tokyo, Japan) and housed in an air-conditioned room (22 ± 2°C) under a 12-hour dark/light cycle (light on from 8 AM to 8 PM), with free access to a standard diet (CLEA Japan) and tap water. All animal experiments were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

Light Exposure

The light-exposure experiments were performed as described previously. ^8,14 Briefly, the mice were rested for several days and used at the age of 6 to 7 weeks. Before light exposure, the mice were dark-adapted by keeping them in complete darkness for 12 hours. The pupils of the mice in the light exposure group were dilated with a mixed solution of 0.5% tropicamide and 0.5% phenylephrine (Mydrin-P; Santen Pharmaceutical, Osaka, Japan) just before light exposure. They were then exposed to light from a white fluorescent lamp (FHD100ECW; Panasonic, Osaka, Japan) at 2000 lux in actual measurement for 3 hours, starting at 9 AM, in a dedicated exposure box with stainless-steel mirrors on each wall and on the floor (Tinker N, Kyoto, Japan). The temperature of the box was set at 22 ± 2°C, maintained with an air conditioner and fans, and monitored with a thermometer. After the light exposure, the mice were returned to their cages and maintained under dim cyclic light (5 lux, 12 hours on/off) until they were euthanized at the time of sampling in different time points according to each experiment. Control mice were also kept under dim cyclic light and euthanized at the time of sampling. Tropicamide and phenylephrine were confirmed to have no effects on morphological changes and cytokine expressions (data not shown). 

Measurement of Reactive Oxygen Species

The protocol for measuring reactive oxygen species (ROS) was described previously. ¹⁵ Briefly, the eyes were enucleated and the cornea, lens, vitreous, and retina were carefully removed, and the RPE, together with the choroid, was carefully scraped from the eyecups and placed into 100 μL PBS. The RPE and choroid could not be separated for technical reasons, so we called the sample the “RPE-choroid complex.” The sample from both eyes of an individual mouse were mixed and analyzed as one sample. The samples were incubated with 1 μL diluted cell-permeant fluorescent ROS detection reagent, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) (Invitrogen, Carlsbad, CA; the stock was made by diluting 2 mg DCFH-DA with DMSO [Sigma-Aldrich, St. Louis, MO]) at 37°C, and the fluorescence intensity was measured according to the manufacturer's protocol with an absorption spectrometer (Wallac ARVO SX 1420 Multilabel Counter; PerkinElmer, Waltham, MA) every 30 minutes from the beginning of the incubation. 

Administration of N-Acetyl-L-cysteine or a ROCK Inhibitor, Y-27632

For experiments with an antioxidant, N-Acetyl-L-cysteine (NAC) (Nakalai Tesque, Kyoto, Japan), mice were grouped and received two intraperitoneal injections of either NAC diluted with PBS (500 mg/kg body weight) or vehicle before dark adaptation and before light exposure. 

For experiments of ROCK inhibition, a ROCK inhibitor, Y-27632 (Wako Pure Chemical Industries, Osaka, Japan) was first dissolved in distilled water (10 mM), and the solution was diluted with PBS. Mice received two intraperitoneal injections of either Y-27632 (20 mg/kg body weight) or vehicle just before and after light exposure. This dose concentration was determined after preliminary experiments, using 200 and 500 mg/kg body weight for NAC, and 5, 15, and 20 mg/kg body weight for Y-27632, to find the minimum effective concentration for inhibiting the morphological changes. 

Immunohistochemistry

The eyes were enucleated, and the cornea, lens, vitreous, and retina were carefully removed to prepare the flat-mount eyecups. The eyecups were prefixed with 4% paraformaldehyde (PFA) for 30 minutes and flattened by making 4 radial cuts. The samples were further fixed with 4% PFA, or for β-catenin staining, with methanol. The samples were activated with Liberate Antibody Binding Solution (Polysciences, Warrington, PA) for 20 minutes at room temperature, blocked with TNB blocking buffer (0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl, and 0.5% TSA Blocking Reagent [PerkinElmer]) for 30 minutes at room temperature, and incubated overnight with a rabbit anti-ZO-1 (Mid) antibody (1:100; Invitrogen), a rabbit anti-N-cadherin antibody (1:100; Abcam, Cambridgeshire, UK), or a rabbit anti-β-catenin (6B3) antibody (1:25; Cell Signaling Technology, Beverly, MA) at 4°C. After washing with PBS, the samples were incubated with Alexa Fluor 488 goat anti-rabbit IgG antibody (H+L) (1:250; Invitrogen) and 10 μg/mL Hoechst bisbenzimide 33258 (for nuclei; Sigma-Aldrich) with shaking for 1 hour at room temperature. 

For F-actin staining, the prefixed samples were incubated with Alexa Fluor 546 Phalloidin (1:40; Invitrogen) for 30 minutes at room temperature, and then with 10 μg/mL Hoechst bisbenzimide 33258 (Sigma-Aldrich) for 1 hour at room temperature. All the samples were mounted with VECTASHIELD mounting medium H-1000 (Vector Laboratories, Burlingame, CA). Fluorescent images of the flat mounts were obtained using a confocal fluorescence microscope FV 1000 (Olympus, Tokyo, Japan). 

For the immunostaining in the retinal sections, eyes were fixed with 4% PFA and prepared for cryosections (8 μm) through the vertical meridian including optic nerve head. After blocking with the normal goat serum (Vector Laboratories), the sections were incubated with anti-F4/80 antibody (1:100; Abcam) followed by Alexa Fluor 488 goat anti-rat IgG antibody (H+L) (1:300; Invitrogen) and 10 μg/mL Hoechst bisbenzimide 33258 (for nuclei; Sigma-Aldrich). All the sections were examined using a confocal fluorescence microscope FV 1000 (Olympus). 

ELISA

The RPE-choroid complex samples were placed in a lysis buffer containing protease inhibitor cocktail (cOmplete, EDTA-free; Roche, Basel-Stadt, Switzerland), according to the manufacturer's protocol. The tissues from both eyes of an individual mouse were mixed and analyzed as one sample. The sample was exposed to 5 periods of sonication for 10 seconds each on ice, using a Handy Sonic UR-20 (Tomy Seiko, Tokyo, Japan) at a power control dial setting of 4. The sonicated suspension was left on ice for 30 minutes and then centrifuged at 20,400g for 15 minutes at 4°C. The supernatant was transferred to a new 1.5-mL tube, and subjected to ELISA assays using the 96-Well ROCK Activity Assay Kit (Cell Biolabs, San Diego, CA), Quantikine Mouse CCL2/JE/MCP-1 Immunoassay kit (for MCP-1; R&D Systems, Minneapolis, MN), or Quantikine Mouse Eotaxin Immunoassay kit (for CCL11; R&D Systems), according to the respective manufacturer's instructions. The absorbance was measured with an absorption spectrometer (Wallac ARVO SX 1420 Multilabel Counter; PerkinElmer). 

Quantitative Real-Time RT-PCR

The RPE-choroid complex samples from both eyes of an individual mouse were mixed and analyzed as one sample. The sample was placed in 100 μL TRIzol reagent (Invitrogen) to extract the total RNA. The cDNA was made by adding 1 μg of the total RNA to SuperScript VILO Master Mix (Invitrogen), and performing reverse-transcription according to the manufacturer's instructions. PCR was performed using the StepOnePlus Real Time PCR system (Applied Biosystems, Foster City, CA), and the ΔΔCT method was used to quantify gene expression. For mcp-1, ccl11, and f4/80, the following gene-specific primers designed by TaqMan Gene Expression Assays (Applied Biosystems) were used: Mm00656886_g1 (mcp-1), Mm00441238_m1 (ccl11), Mm00802529_m1 (f4/80), and Catalog Number 4352339E (gapdh). The il-6 analysis was done using SYBR system; the primers were, il-6 forward: 5′-GAGGATACCACTCCCAACAGACC-3′ and reverse: 5′-AAGTGCATCATCGTTGTTCATACA-3′; and gapdh forward: 5′-TGTCTTCACCACCATGGAGA-3′ and reverse: 5′-AGTCTTCTGGGTGGCAGTGA-3′. Each mRNA level was normalized to that of the gapdh mRNA as an internal control. 

Statistical Analysis

All results were expressed as means ± SD. The values were processed for statistical analyses (one-way ANOVA with Tukey's post hoc test) using SPSS Statistics 20 (IBM, Armonk, NY) software, and differences were considered statistically significant at P < 0.05. 

Results

Cell-Cell Junctions and Actin Cytoskeleton Were Disrupted in the RPE of the Light-Exposed Mice

We first analyzed how light exposure to the mice influenced the RPE in vivo. To examine the morphological changes of the RPE directly, we prepared flat-mount samples of the eyecup, removing the cornea, lens, vitreous, and neural retina. Because the RPE is a monolayered cuboidal epithelium, the cell-cell boundary was clearly observable by this method. 

We first checked the localization of ZO-1, which is a component of tight junctions, at the apical side of the RPE layer, by immunohistochemistry (Figs. 1A, 1B). The membrane pattern of ZO-1 immunostaining was clearly observed in the control mice; however, the pattern was largely disrupted in the RPE 24 hours after light exposure. We then analyzed the other junctional structure, the adherens junction, by immunostaining for N-cadherin (Figs. 1C, 1D) and β-catenin (Figs. 1E, 1F), and found that their membrane pattern was also disrupted by the light exposure at 24 hours. In parallel, F-actin (Figs. 1G, 1H), which forms microfilaments at the cytoplasmic face of these junctional structures, showed a scattered pattern in the cytoplasm 24 hours after light exposure, in contrast to the membrane-associated pattern observed under the control condition. Hoechst staining showed no obvious cell death with or without light exposure (Figs. 1I, 1J). These changes were observed in all the mice examined at maximum levels 24 hours after light exposure, and the disruptions were most obvious in the central part of the retina, where the light had hit straight on, compared with the peripheral part. Therefore, we used the morphology of the RPE in the central part, 200 μm away from the optic disc, 24 hours after light exposure, for comparisons throughout this study. 

Figure 1

 

Disruption of cell-cell adhesion and the actin cytoskeleton in the RPE of light-exposed mice. Images of the flat-mount RPE of mice 24 hours after light exposure (A–H). Representative images from an individual mouse in each group with or without light exposure are shown. Flat-mount samples of control, nonirradiated mice immunostained for ZO-1 (A), N-cadherin (C), and β-catenin (E), and labeled for F-actin (G) all showed a membrane pattern in the RPE. In contrast, each staining showed a scattered pattern in the cytoplasm, and the cell-cell boundary was undetectable 24 hours after light exposure (B, D, F, H). Hoechst staining showed no obvious cell death with or without light exposure (I, J); n = 6. Scale bar: 50 μm.

Oxidative Stress Was Increased in the RPE-Choroid Complex of the Light-Exposed Mice

Next, we analyzed the level of oxidative stress using DCFH-DA. This probe detects hydroxyl radicals, peroxyl radicals, and other ROS, after its deacetylation by endogenous esterases. In its deacetylated form, it will fluoresce on reacting with the ROS. We found that the ROS level in the RPE-choroid complex was elevated 6 hours after light exposure (Fig. 2). 

Figure 2

 

Light-induced increase in oxidative stress and its suppression by NAC in the RPE-choroid complex of light-exposed mice. ROS in the RPE-choroid complex of mice 6 hours after light exposure were measured by the fluorescence intensity of DCFH-DA, with an absorption spectrometer. The fluorescence intensity of DCFH-DA was measured every 30 minutes up to 180 minutes, after incubation with RPE-choroid samples. The intensity of DCFH-DA fluorescence was upregulated after light exposure. However, treatment with NAC decreased the intensity. ▪, control mice with no light exposure; •, light-exposed mice treated with vehicle; ○, light-exposed mice treated with NAC. n = 6. *P < 0.05; **P < 0.01. Both * and ** showed significant changes of the intensity between the light-exposed mice treated with vehicle and with NAC.

Suppression of Oxidative Stress by NAC Avoided the Disruption of Cell-Cell Junctions and the Actin Cytoskeleton in the RPE of Light-Exposed Mice

Based on our hypothesis that oxidative stress after light exposure can cause the pathological changes in the RPE, we administered NAC to reduce the ROS level after light exposure. The ability of NAC to significantly decrease the ROS level compared with vehicle in the RPE-choroid complex of light-exposed mice was first observed 6 hours after light exposure that was confirmed by DCFH-DA (Fig. 2). 

We then analyzed the effects of NAC on the immunostaining of ZO-1 (Fig. 3A), N-cadherin (Fig. 3B), and β-catenin (Fig. 3C) in the RPE of the light-exposed mice at 24 hours. All these components showed a membrane-associated localization pattern in the light-exposed mice treated with NAC that was similar to their pattern in control mice. The disruption of the microfilament organization detected by F-actin staining in mice that were not treated with NAC was prevented by NAC treatment at the same time point (Fig. 3D). Hoechst staining showed no obvious changes (Fig. 3E). These data together indicated that the disruption of cell-cell junctions and actin cytoskeleton of RPE was caused by light-induced oxidative stress. 

Figure 3

 

Suppression of the light-induced disruption of cell-cell adhesion by NAC in the RPE of light-exposed mice. Images of the flat-mount RPE of NAC-treated mice 24 hours after light exposure. Representative images of ZO-1 (A), N-cadherin (B), β-catenin (C), and F-actin (D) from an individual mouse are shown. All stainings showed the membrane-associated pattern in the RPE of the light-exposed mice treated with NAC. Hoechst staining showed no obvious changes (E). n = 6. Scale bar: 50 μm.

ROCK Activity Was Upregulated by Oxidative Stress in the RPE-Choroid Complex of Light-Exposed Mice

Because the regulation of the junctional complexes and actin cytoskeleton generally involves Rho/ROCK signaling, ^12,13 we next asked whether this signal played a role in the structural photo-damage of the RPE, and whether it was induced by ROS. Importantly, the level of ROCK activity in the RPE-choroid complex became clearly increased 9 hours after light exposure (Supplementary Fig. S1), and this increase was prevented by NAC treatment and ROS reduction (Fig. 4). 

Figure 4

 

Light-induced increase in ROCK activity and its suppression by NAC in the RPE-choroid complex of light-exposed mice. ELISA assay. The level of ROCK activation in the RPE-choroid complex was significantly increased in mice 9 hours after light exposure, and the level was attenuated by NAC treatment. n = 6. *P < 0.05; **P < 0.01.

ROCK Inhibition by Y-27632 Suppressed the Disruption of Cell-Cell Junctions and Actin Cytoskeleton in the RPE of Light-Exposed Mice

We next analyzed whether ROCK activity was involved in the disruption of cell-cell adhesion, using a ROCK inhibitor, Y-27632. Notably, the flat-mount RPE samples showed that ROCK inhibition preserved the membrane-bound expression of ZO-1 (Fig. 5A), N-cadherin (Fig. 5B), β-catenin (Fig. 5C), and F-actin (Fig. 5D) 24 hours after light exposure. Hoechst staining showed no obvious changes (Fig. 5E). Therefore, ROCK activity was responsible for the alterations in cell-cell junctions and the actin cytoskeleton induced by light exposure. We confirmed that Y-27632 suppressed ROCK activity 9 hours after light exposure (Fig. 5F) when ROCK activity peaked. 

Figure 5

 

Suppression of light-induced disruption of cell-cell adhesion and the actin cytoskeleton by a ROCK inhibitor, Y-27632, in the RPE of light-exposed mice. Images of the flat-mount RPE from Y-27632-treated mice 24 hours after light exposure. Representative images of ZO-1 (A), N-cadherin (B), β-catenin (C), and F-actin (D) from an individual mouse are shown. The stainings show a membrane-associated pattern in the RPE of light-exposed mice treated with Y-27632. Hoechst staining showed no obvious changes (E). ROCK activity was indeed attenuated by Y-27632, 9 hours after light exposure (F). n = 6. *P < 0.05. Scale bar: 50 μm.

Cytokine Expression Was Affected in the RPE-Choroid Complex of Light-Exposed Mice Through Oxidative Stress

We further analyzed the light-induced changes in the mouse RPE-choroid complex. Because the expression of MCP-1, which can attract macrophages, is one of the main events in the pathogenesis of AMD, ^17,18 we measured the MCP-1 level at both the mRNA and protein levels (Figs. 6A, 6B). Six hours after light exposure when ROS level was significantly increased, the level of mcp-1 mRNA expression was elevated, and subsequently, the MCP-1 protein was elevated 24 hours after light exposure. Both of these increases were effectively suppressed by NAC treatment, which reduced ROS. 

Figure 6

 

Induction of cytokines in the RPE-choroid complex of light-exposed mice and effects of NAC and Y-27632. Real time RT-PCR (A, C, E) and ELISA assays (B, D, F). The mRNA levels were measured 6 hours after light exposure and are shown relative to the control, and the protein levels were measured 24 hours after light exposure, both in RPE-choroid complex samples. The mRNA and protein levels of MCP-1 (A, B) and CCL11 (C, D) were all upregulated by light exposure, and these increases were suppressed by NAC treatment. The mRNA and protein levels of MCP-1 were also suppressed by a ROCK inhibitor, Y-27632 (E, F). n = 6. *P < 0.05; **P < 0.01.

Another cytokine associated with AMD in animal experiments is CCL11 (eotaxin 1), which can recruit vascular endothelial cells as well as eosinophils. We found that the mRNA and protein levels of this cytokine also increased following light exposure, and that the increases were suppressed by NAC treatment (Figs. 6C, 6D). Taken together, these findings indicated that AMD-associated cytokine expressions are induced in the RPE-choroid complex after light exposure in vivo, through oxidative stress. 

We also analyzed the involvement of ROCK in the alteration of cytokine expressions, and found that the light-exposure–associated increase in MCP-1 expression was mediated by ROCK activation (Figs. 6E, 6F), but the increase of CCL11 was not (data not shown). 

Macrophage Recruitment Was Induced in the Light-Exposed Mice Through Oxidative Stress

Then, we analyzed whether macrophages were recruited after light exposure. Interestingly, the macrophage marker, F4/80 mRNA was upregulated in the RPE-choroid 24 hours after light exposure, and this pathological event was suppressed either by NAC or Y-27632 (Figs. 7A, 7B). Moreover, immunohistochemistry showed that the macrophage recruited to the choroid 24 hours after light exposure was also suppressed by NAC or Y-27632 (Figs. 7C–J). Some of the macrophages were infiltrated above the RPE (Figs. 7D, 7H) that was also suppressed by either of the treatments. These data suggested that macrophage recruitment was also induced by ROS increase and ROCK activation after light exposure. 

Figure 7. 

 

Recruitment of macrophages in the light-exposed mice and effects of NAC and Y-27632. Real-time RT-PCR (A, B) and immunohistochemistry (C–J). The mRNA was measured 24 hours after light exposure and is shown relative to the control in RPE-choroid complex samples. The mRNA level of F4/80 was upregulated by light exposure, and this increase was suppressed either by NAC treatment (A) or Y-27632 treatment (B). The F4/80 immunostaining showed that the macrophage recruitment to the choroid and above the RPE was observed 24 hours after light exposure ([C], control; [D], after light exposure), and these changes were suppressed either by NAC treatment (E) or Y-27632 treatment (F). Hoechst staining showed nuclei (G–J). Arrowheads show the F4/80-positive macrophages in the choroid and above the RPE (D). n = 6. *P < 0.05; **P < 0.01. Scale bar: 100 μm.

Discussion

We found that light exposure caused the disruption of tight junctions, adherens junctions, and the actin cytoskeleton (Fig. 1), via oxidative stress (Figs. 2, 3), in the mouse RPE in vivo. Downstream of the oxidative stress, Rho/ROCK signaling was upregulated (Fig. 4), and the Rho/ROCK signaling was responsible for the photo-damage to the RPE-choroid complex (Fig. 5). The light-induced oxidative stress also promoted the expression of cytokines MCP-1 and CCL11 at both the mRNA and protein levels, and of these, the increase in MCP-1 was also mediated by ROCK (Fig. 6). Macrophages were recruited to the choroid and above the RPE after light exposure, and this pathological event also turned out to be mediated by ROS increase and ROCK activation after light exposure (Fig. 7). 

Previous studies have shown that light exposure induces excessive activation of the visual cycle, resulting in photoreceptor cell death, ^7,18,19 through the accumulation of oxidative stress. ^7–9,20 In this study, we showed that light exposure also affected the RPE in vivo, by promoting ROS induction. 

Some of the reactions in the visual cycle occur in the RPE, in addition to those in photoreceptor cells, suggesting that excessive activation of the visual cycle might also cause ROS accumulation in the RPE. Moreover, a large amount of retinosomes, which are formed by the accumulation of retinyl ester (i.e., retinol esterified with fatty acid) in lipid droplets that are found in visual cycle, ²¹ might also have caused decompensation of the cellular activity, resulting in ROS. 

Alternatively, a previous report indicates that light exposure of the human RPE cell line, ARPE19, produces ROS independent of the rhodopsin in photoreceptor cells, ²² suggesting that light exposure may also have induced ROS independent of rhodopsin and the visual cycle, also in the light-exposed mice. 

The antioxidative machinery of the RPE has been established. Knocking down superoxide dismutase (SOD) 2 in the RPE by infection with an adeno-associated virus in mice leads to ROS accumulation and apoptosis, ²³ and SOD2-deficient mice show mitochondrial abnormalities in the RPE without exogenous stimulation. ²⁴ Thus, superoxide anion is physiologically generated in the RPE but deleted by SOD2 and subsequent reactions. In this study, we observed an increase in ROS using DCFH-DA, which mainly detects the hydroxyl radical, an intermediate form of ROS downstream of the superoxide anion, although the involvement of other ROS cannot be excluded, suggesting that even if SOD2 functioned to some extent, the overall antioxidative reactions were eventually overwhelmed, resulting in ROS accumulation after light exposure. Alternatively, ROS induced in the choroid might also have affected RPE. ²⁵  

Light-induced disruption of the cell-cell junctions (consisting of tight junctions and adherens junctions) and the actin cytoskeleton was prevented by NAC, which reduced the level of ROS, indicating that the changes occurred through oxidative stress. Given that a substantial level of hydroxyl radicals would lead to lipid peroxidation in the RPE, changes in the status of the cytoplasmic membrane could easily have altered the calcium kinetics and perturbed the cytoskeletal proteins. ^11,26 In brain endothelial cells, oxidization of the membrane causes the formation of Michael adducts, following activity changes in the focal adhesion kinase, Src family of nonreceptor kinases, paxillin, and Rho. ^27,28 Moreover, in the ARPE19 cell line, stimulation with H₂O₂ promotes Src-kinase activation, the translocation of p120-catenin, a stabilizer of cadherins, and subsequent Rho/ROCK activation, which causes the dissociation of cellular junctions. ²⁹  

Importantly, we observed that light-induced ROS activated ROCK, a downstream effector of Rho protein, and that the inhibition of ROCK prevented the light-induced RPE degeneration, indicating that light-induced ROCK activation was responsible for some of the RPE changes observed in mouse eyes in vivo. It is biologically important that the in vitro observation described above was actually observed in the living animal. The morphological change was eventually reversible in the acute model used in the current study (data not shown), which would be because the ROCK activity level returned in the later phase in this model (Supplementary Fig. S1). 

Light exposure also influenced cytokine expressions. Light-induced ROS increased the level of MCP-1, at both the transcriptional and protein levels. This was consistent with a previous article showing that oxidized lipids added to ARPE19 culture increase the MCP-1 expression. ¹⁷ MCP-1 is known to induce macrophage migration, and it is also reported to induce the remodeling of F-actin and the disruption of tight junctions through the caveolae-mediated internalization of tight junction proteins, such as ZO-1, occludin, and claudin-5. ³⁰ MCP-1 also causes the redistribution of tight junction proteins through ROCK activation, ³¹ which explains how the blood-brain barrier (BBB) is disrupted by MCP-1 administration to the brain. ³² MCP-1 may have elicited a similar mechanism in the light-induced disruption of cell-cell junctions and the actin cytoskeleton in this study. Moreover, MCP-1′s induction was also regulated by ROCK activity, suggesting that the events involved in the induction of photo-damage in the RPE are closely associated. 

We also showed that CCL11 (eotaxin 1) increased as a result of light exposure–induced oxidative stress. This cytokine, as well as MCP-1, is increased in an AMD mouse model with laser-induced CNV. ^16,17,33 MCP-1′s role in CNV formation may be determined by whether the recruited macrophages are proinflammatory ¹⁷ or anti-inflammatory. ³⁴ Further analyses would be necessary, but at least mRNA level of a proinflammatory cytokine, interleukin 6 (IL-6), was upregulated in the light-exposed RPE-choroid complex and repressed either by NAC or Y-27632 (Supplementary Fig. S2), suggesting that proinflammatory status was induced in the RPE-choroid after light exposure. Because CCL11 promotes the recruitment and proliferation of vascular endothelial cells together with the remodeling of F-actin, ³³ this cytokine could also play a role in the RPE structural changes observed in the current study. CCL11 was regulated by ROS but not by ROCK, suggesting that ROS may have activated other pathways not only ROCK, which may have induced CCL11. 

In this study, light exposure to mice induced macrophage recruitment in parallel with MCP-1 induction, through ROS increase and ROCK activation. Therefore, light-induced MCP-1 may have been, at least in part, responsible for the macrophage recruitment in this model. The importance of macrophages in CNV development is well accepted. ^20,35 Thus, this light exposure–induced event related to macrophage recruitment may have a role in AMD development. 

In the pathogenesis of AMD, CNV invading above the RPE and/or CNV-related exudative fluid penetrating the RPE can worsen the visual outcome. These pathological changes may be suppressed if the RPE barrier is intact; otherwise, the retinal and visual condition can easily deteriorate. Therefore, the light-induced disruption of cell-cell junctions and the actin cytoskeleton, and the induction of AMD-associated cytokines help to explain how light could increase the risk for AMD progression. ^3,5,7,20,34  

In summary, we demonstrated that light exposure caused ROS accumulation in the mouse RPE-choroid complex, which activated ROCK signaling, and led to impairment of RPE integrity. The pathological changes involved the disruption of cell-cell junctions and the actin cytoskeleton in the RPE, and the induction of cytokines (MCP-1 and CCL11) and macrophage recruitment in the RPE and/or choroid, all of which are candidates for promoting AMD progression and worsening the retinal condition, which leads to poor visual outcome. 

Supplementary Materials

pdf S1 

Acknowledgments

The authors thank Haruna Koizumi-Mabuchi, Eriko Toda, Hiroe Sato, and Mari Muto for their technical assistance. 

Supported in part by Grant-in-aid 05-045-2071 from the Ministry of Education, Culture, Sports, Science and Technology (YO). 

Disclosure: T. Narimatsu, None; Y. Ozawa, None; S. Miyake, None; S. Kubota, None; M. Hirasawa, None; N. Nagai, None; S. Shimmura, None; K. Tsubota, None