October 2015
Volume 56, Issue 11
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Retina  |   October 2015
Protective Effects of Antiplacental Growth Factor Antibody Against Light-Induced Retinal Damage in Mice
Author Affiliations & Notes
  • Hiroshi Izawa
    Molecular Pharmacology Department of Biofunctional Evaluation, Gifu Pharmaceutical University, Gifu, Japan
  • Yuki Inoue
    Molecular Pharmacology Department of Biofunctional Evaluation, Gifu Pharmaceutical University, Gifu, Japan
  • Yuta Ohno
    Molecular Pharmacology Department of Biofunctional Evaluation, Gifu Pharmaceutical University, Gifu, Japan
  • Kazuki Ojino
    Molecular Pharmacology Department of Biofunctional Evaluation, Gifu Pharmaceutical University, Gifu, Japan
  • Kazuhiro Tsuruma
    Molecular Pharmacology Department of Biofunctional Evaluation, Gifu Pharmaceutical University, Gifu, Japan
  • Masamitsu Shimazawa
    Molecular Pharmacology Department of Biofunctional Evaluation, Gifu Pharmaceutical University, Gifu, Japan
  • Hideaki Hara
    Molecular Pharmacology Department of Biofunctional Evaluation, Gifu Pharmaceutical University, Gifu, Japan
  • Correspondence: Hideaki Hara, Molecular Pharmacology, Department of Biofunctional Evaluation, Gifu Pharmaceutical University, 1-25-4 Daigakunishi, Gifu 501-1196, Japan; hidehara@gifu-pu.ac.jp
Investigative Ophthalmology & Visual Science October 2015, Vol.56, 6914-6924. doi:10.1167/iovs.15-16748
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      Hiroshi Izawa, Yuki Inoue, Yuta Ohno, Kazuki Ojino, Kazuhiro Tsuruma, Masamitsu Shimazawa, Hideaki Hara; Protective Effects of Antiplacental Growth Factor Antibody Against Light-Induced Retinal Damage in Mice. Invest. Ophthalmol. Vis. Sci. 2015;56(11):6914-6924. doi: 10.1167/iovs.15-16748.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Placental growth factor (PlGF) is part of the VEGF family and is known to be involved in angiogenesis, vasopermeability, and neuroprotection. Recently, PlGF has been reported as a novel therapeutic target for wet AMD. However, there are few reports about the effect of PlGF against dry AMD. Previously, we reported that PlGF has protective effects against retinal neuronal cell damage in vitro. Therefore, we investigated the effects of PlGF against photoreceptor degeneration.

Methods: In this study, mice were exposed to white light at 8000 lx for 3 hours to induce retinal damage, which was evaluated by recording the electroretinogram amplitude and measuring the outer nuclear layer (ONL) thickness. The mice were injected intravitreally with PlGF before light exposure, PlGF after light exposure, or anti-PlGF antibody before light exposure. RPE–choroid–sclera flat mounts were immunostained with anti–ZO-1 antibody to evaluate the disruption of retinal pigmented epithelium (RPE) cell–cell junctional integrity after light exposure. Furthermore, the expression of VEGF receptor in the retina and RPE–choroid complex after light exposure was measured using Western blot analysis.

Results: Contrary to the expected outcome, PlGF treatment exacerbated the light-induced retinal functional damage and ONL thinning. In contrast, anti-PlGF treatment significantly improved the light-induced retinal degeneration. The disruption of RPE cell–cell junctional integrity after light exposure was suppressed by anti-PlGF treatment. Moreover, the VEGF receptor, which is involved in blood–retinal barrier breakdown, was up-regulated after light exposure.

Conclusions: These findings suggest that anti-PlGF antibody has protective effects against light-induced retinal degeneration in the murine retina through inhibition of RPE breakdown after light exposure. Thus, anti-PlGF antibody may be useful therapeutic agents in dry AMD.

Age-related macular degeneration (AMD) is the most common cause of severe and irreversible vision loss in the world. The prevalence of AMD rapidly increases, and it is estimated that globally about 200 million people will have AMD in 2020, increasing to 300 million in 2040.1 There are two forms of AMD: dry and wet. Dry AMD is characterized by soft drusen, retinal pigmented epithelium (RPE) breakdown, and geographic atrophy.2 Patients with dry AMD progress to visual loss in approximately 10 years and eventual have complete loss of central vision.3 Moreover, about 10% to 15% of dry AMD turns into wet AMD.4 In contrast, wet AMD is characterized by choroidal neovascularization with leakage of blood and serous fluid and rapidly leads to loss of central vision over a short period of time.5,6 Currently, anti-VEGF treatment and photo-dynamic therapy are widely used for wet AMD. However, there is no approved treatment for dry AMD. 
Excessive light exposure is considered a risk factor for AMD.7,8 The light-induced photoreceptor degeneration model is widely used because it represents characteristic of dry AMD.9 Previous reports have suggested that exposure to light generates reactive oxygen species (ROS), and ROS induces photoreceptor cell damage and disrupts RPE structure.1013 Thus, it has been reported that antioxidants have protective effects on light-induced retinal damage.14,15 Moreover, many researchers have reported the efficacy of neurotrophic factors such as brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor against light-induced retinal degeneration.16 Nevertheless, antioxidants and neurotrophic factors have not been clinically approved for the treatment of dry AMD. Therefore, alternative therapeutic targets for dry AMD are strongly desired. 
Placental growth factor (PlGF) is a member of the VEGF family of growth factors and plays an important role in angiogenesis, inflammation, and vasopermeability.1719 More specifically, PlGF induces pathologic angiogenesis and increases vascular permeability in specific eye diseases such as wet AMD and diabetic retinopathy (DR). Thus, PlGF has been reported as a novel therapeutic target for wet AMD and DR lately.20,21 On the other hand, previous reports have shown that the VEGF family has protective effects on neurologic diseases, such as cerebral ischemia, amyotrophic lateral sclerosis, and glaucoma.2225 Blocking VEGF specifically in RPE cells prevented RPE disruption and retinal degeneration after light exposure.26 Moreover, previous reports indicated that VEGF up-regulates PlGF expression via VEGF receptor (VEGFR)-2.27 Therefore, it is suggested that PlGF participates in photoreceptor cell death after light exposure and dry AMD. 
Therefore, in the present study, we aimed to clarify the effects of PlGF against photoreceptor cell death. 
Materials and Methods
Animals
All experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved and monitored by the Institutional Animal Care and Use Committee of Gifu Pharmaceutical University (Ethic nos. 2011-399, 2012-028, 2013-254, 2013-213, 2014-210, and 2015-015). All experiments were conducted using male adult ddY albino mice (8 weeks old; body weight, 30–40 g; Japan SLC Ltd., Hamamatsu, Japan). Animals were housed in an air-conditioned room maintained at 22 ± 2°C under controlled lightning conditions (12-hour light/dark cycle, with free access to a standard diet [CLEA Japan, Inc., Tokyo, Japan] and tap water). The operators (H.I., Y.I., Y.O., and K.O.) were blinded to the treatment status of the animals in all experiments. 
Exposure to Light
Light exposure was performed as previously described.14,2830 Mice were adapted to dark conditions 24 hours prior to light exposure by covering with a completely black box, and they had free access to food and water. Mice pupils were dilated via administration of 1% cyclopentolate hydrochloride eye drops (Santen Pharmaceuticals Co., Ltd., Osaka, Japan) 30 minutes prior to exposure to light. Nonanesthetized mice were exposed to 8000 lx of visible light emitted by white fluorescent lamps (rated lamp wattage, 30 W; color temperature, 4200 K) (FL30SW; Toshiba, Tokyo, Japan) for 3 hours in cages with reflective interiors. These experiments were started at 9 AM and equally applied to all mice. The temperature during the exposure to light was maintained at 25 ± 1.5°C. This temperature was checked every 10 minutes. 
Electroretinogram Analysis
An electroretinogram (ERG) was recorded 5 days after light exposure (Mayo, Aichi, Japan) as previously described.14 Mice were maintained in a completely dark room for 24 hours. They were then anesthetized intraperitoneally with a mixture of ketamine (120 mg/kg; Daiichi-Sankyo, Tokyo, Japan) and xylazine (6 mg/kg; Bayer Health Care, Tokyo, Japan). The pupils were dilated with 1% tropicamide and 2.5% phenylephrine (Santen Pharmaceuticals Co., Ltd.). A flash ERG was recorded (PowerLab/8SP and LabChart software; AD Instruments, New South Wales, Australia) from the left eye of each dark-adapted mouse by placing a golden-ring electrode (Mayo, Aichi, Japan) in contact with the cornea, and a reference electrode (Nihon Kohden, Tokyo, Japan) was placed to touch the tongue. A neutral electrode (Nihon Kohden) was inserted subcutaneously near the tail. Light pulses of 3.98 cds/m2 and 0.3-ms duration were delivered through a commercial stimulator. The ERGs were measured in response to flash at an intensity ranging from −2.92 to 0.98 log cds/m2. The different flash intensities were generated by a white light source. The digital band-pass filter ranging from 0.3 to 500 Hz was used to isolate signals after the waves were recorded. All procedures were performed under dim red light, and the mice were kept on a heating pad to maintain a constant body temperature during the ERG recording. The a-wave amplitude was measured from baseline to the trough of the a-wave, whereas the b-wave was measured from the trough of the a-wave to the peak of the b-wave. 
Histologic Analysis
Histologic analysis was carried out by staining retinal cross sections with hematoxylin and eosin as previously described.14 The mice were euthanized by cervical dislocation, and each eye was enucleated and kept immersed for at least 24 hours at 4°C in a fixative solution containing 4% paraformaldehyde (PFA). Six paraffin-embedded sections (thickness, 5 μm) were cut through the optic disc of each eye, prepared in the standard manner, and stained with hematoxylin and eosin. The damage induced by light exposure was then evaluated, with six sections from each eye being used for the morphometric analysis described below. Images were photographed using a fluorescence microscope (BZ-9000; Keyence, Osaka, Japan), and the thickness of the outer nuclear layer (ONL) was measured on sections parallel to the vertical meridian at 240-μm intervals from the optic disc toward the superior and inferior ora serrata using ImageJ (National Institutes of Health, Bethesda, MD, USA). Data from three sections (selected randomly from the six sections) were averaged for each eye. 
PlGF and Anti-PlGF Antibody Injection
In humans, PlGF has four isoforms (PlGF-1, -2, -3, -4), whereas mice only express a single isoform, PlGF-2, which is comparable to human PlGF-2.3135 Thus, this study focused on PlGF-2. Mouse recombinant PlGF-2 (2.5 and 25 ng/mL; final concentrations at 5 and 50 pg/eye, respectively; R&D Systems, Minneapolis, MN, USA) was injected (2 μL) into the vitreous body of the left eyes 2 hours before light exposure or immediately after light exposure under isoflurane (Merck Hoei Ltd., Osaka, Japan) anesthesia according to previously reported methods.36 On the other hand, anti–PlGF-2 antibody (50 and 500 μg/mL; final concentrations at 0.1 and 1 μg/eye; GeneTex, Irvine, CA, USA) was injected (2 μL) into the vitreous body of the left eyes 2 hours before light exposure. For buffer control experiments, mice were injected intravitreally with 2 μL of 0.01 M PBS into the left eyes. All solutions were suspended in 0.01 M PBS and injected into the vitreous space by pricking the eye at corneal–scleral junction using a 10-μL Hamilton glass syringe (701N; Hamilton Co., Reno, NV, USA) fitted with a sterile 33-gauge needle (Terumo, Tokyo, Japan). One drop of 0.01% levofloxacin ophthalmic solution (Santen Pharmaceuticals Co., Ltd.) was applied topically to the treated eye immediately after intravitreal injection. 
Western Blot
Western blot analysis was conducted as previously reported.37 Mice were euthanized by cervical dislocation, and each eye was rapidly removed. Next, the retinas and RPE–choroid complex were carefully separated from the eyeballs and quickly frozen in dry ice. For protein extraction, the tissue was homogenized in cell lysis buffer using a Physcotron homogenizer (Microtec Co., Ltd., Chiba, Japan). The lysate was centrifuged at 12,000g for 20 minutes, and the supernatant was used for this study. The protein concentrations were measured by comparison with a known concentration of BSA using a bicinchoninic acid protein assay kit (Pierce Chemical, Rockford, IL, USA). A mixture of equal parts of an aliquot of protein and sample buffer with 10% 2-mercaptoethanol was subjected to 15% SDS-PAGE. The separated protein was then transferred onto a polyvinylidene difluoride membrane (Immobilon-P; Millipore Corporation, Billerica, MA, USA). Transfers were blocked for 30 minutes at room temperature with 5% Block One-P (Nacalai Tesque, Inc., Kyoto, Japan) in 10 mM Tris-buffered saline with 0.05% Tween 20, and then incubated overnight at 4°C with the primary antibody. For immunoblotting, the following primary antibodies were used: goat anti–VEGFR-1 antibody (R&D Systems, Minneapolis, MN, USA; 1:2000), rabbit anti–VEGFR-2 antibody (Cell Signaling, Danvers, MA, USA; 1:1000), and mouse anti–β-actin antibody (Sigma-Aldrich Corp., St. Louis, MO, USA; 1:5000). The secondary antibodies used were horseradish peroxidase (HRP)-conjugated rabbit anti-goat IgG (1:100,000; Thermo Fisher Scientific, Waltham, MA, USA), HRP-conjugated goat anti-rabbit IgG (1:2000; Pierce Biotechnology, Rockford, IL, USA), or HRP-conjugated goat anti-mouse IgG (1:200; Pierce Biotechnology) for 1 hour at room temperature. The immuno-reactive bands were visualized using Immunostar LD (Wako Pure Chemical, Osaka, Japan) and then measured using LAS-4000 Mini (Fuji Film Co., Ltd., Tokyo, Japan). 
Immunohistochemistry
Immunohistochemistry was performed as described previously.13,26 Eyes were enucleated and immediately fixed in 4% PFA for 30 minutes and then transferred to PBS. The cornea, lens, and retina were carefully removed from each eye, and the eyes were flat mounted by making eight radial cuts. Whole mounts of RPE–choroid–sclera were briefly washed in PBS, before blocking with 10% normal goat serum in 0.3% Triton X-100 in PBS for 1 hour at room temperature. Whole mounts of RPE–choroid–sclera were washed three times in PBS and subsequently incubated overnight at 4°C with a rabbit anti–ZO-1 (Mid) antibody (Invitrogen, Carlsbad, CA, USA; 1:100). After washing with PBS, whole mounts of RPE–choroid–sclera were incubated with Alexa Fluor 488 goat anti-rabbit IgG antibody (H+L) (1:250; Invitrogen) for 1 hour at room temperature. After washing with PBS, the samples were mounted with FluoromountTM (Diagnostic BioSystems, Pleasanton, CA, USA) and stored at 4°C. Images were photographed (60×, 4.5 × 104 μm2 and 120×, 1.12 × 104 μm2) for four quadrants of each eye at a distance of 200 μm from the optic disc using a confocal laser scanning microscope (FluoView FV10; Olympus, Tokyo, Japan). Data from the four parts of each eye were quantified (total area, 4.48 × 104 μm2). 
Quantification of Junctional Linearity
Junctional linearity was quantified as previously described.38,39 The junctional “linearity index” was defined as the ratio of actual junction length to the straight line length between vertices. Cell junctions were manually traced and measured along the shape of each cell using ImageJ (National Institutes of Health). Values closest to 1 represent a high degree of linearity. 
Statistical Analyses
The data are presented as means ± SEM. Statistical comparisons were made by using the Student's t-test, Aspin-Welch's t-test, or Dunnett's test with SPSS Statistics (IBM, Armonk, NY, USA) software. P < 0.05 was considered statistically significant. 
Results
Effects of PlGF Pretreatment on Light-Induced Retinal Function Damage and Histologic Changes in the Mouse Retina
The treatment protocol for this experiment is shown in Figure 1A. To investigate the effects of PlGF pretreatment against light-induced damage of retinal function in the mouse retina, we conducted ERG analysis. Retinal function was evaluated by ERG recordings 5 days after light exposure. The a-wave represents photoreceptor function, whereas the b-wave reflects subsequent neuron function, such as bipolar cells and Müller cells. In the light-exposed group, both a- and b- wave amplitudes were significantly decreased 5 days after exposure to light compared with the control group. Intravitreal injection of PlGF at 25 ng/mL before light exposure exacerbated the light-induced retinal dysfunction, whereas intravitreal injection of PlGF at 2.5 ng/mL had no effect (Figs. 1B–D). In particular, at 0.98 log cds/m2, the amplitudes of the a- and b-waves in administration of PlGF (25 ng/mL) were reduced by 48% and 43%, respectively, versus the vehicle-treated group. In addition, to explore the effects of PlGF pretreatment on light-induced photoreceptor degeneration, histologic analysis was performed. Representative retinal images were obtained 5 days after exposure to light (Fig. 1E). The ONL thickness decreased in the light-exposed group compared with the control group. The PlGF treatment (25 ng/mL) before exposure to light significantly aggravated the light-induced ONL thinning compared with the vehicle-treated group, whereas intravitreal injection of PlGF at 2.5 ng/mL had no effect (Figs. 1E, 1F). The PlGF treatment (25 ng/mL) exacerbated the reduction of ONL thickness by a maximum of 53% compared with the vehicle-treated group. 
Figure 1
 
Aggravation effects of PlGF pretreatment on light-induced visual function impairment and histologic changes after exposure to light in the mouse retina. (A) Experimental protocol. (B) Representative dark-adapted ERG responses of both a- and b-wave amplitudes 5 days after light exposure. (C, D) Amplitudes of a- and b-waves of individual mice (control: n = 8, vehicle treated: n = 8, PlGF 2.5 ng/mL treated: n = 8, and PlGF 25 ng/mL treated: n = 8). (E) Representative photographs of hematoxylin and eosin staining of retinal sections (control: n = 8, vehicle treated: n = 8, PlGF 2.5 ng/mL treated: n = 7, PlGF 25 ng/mL treated: n = 7). Scale bar: 30 μm. (F) Thickness measurements in the ONL 5 days after light exposure. Data are shown as means ± SEM. #P < 0.05 and ##P < 0.01 versus control group (t-test); *P < 0.05 and **P < 0.01 versus vehicle-treated group (Dunnett's test).
Figure 1
 
Aggravation effects of PlGF pretreatment on light-induced visual function impairment and histologic changes after exposure to light in the mouse retina. (A) Experimental protocol. (B) Representative dark-adapted ERG responses of both a- and b-wave amplitudes 5 days after light exposure. (C, D) Amplitudes of a- and b-waves of individual mice (control: n = 8, vehicle treated: n = 8, PlGF 2.5 ng/mL treated: n = 8, and PlGF 25 ng/mL treated: n = 8). (E) Representative photographs of hematoxylin and eosin staining of retinal sections (control: n = 8, vehicle treated: n = 8, PlGF 2.5 ng/mL treated: n = 7, PlGF 25 ng/mL treated: n = 7). Scale bar: 30 μm. (F) Thickness measurements in the ONL 5 days after light exposure. Data are shown as means ± SEM. #P < 0.05 and ##P < 0.01 versus control group (t-test); *P < 0.05 and **P < 0.01 versus vehicle-treated group (Dunnett's test).
Effects of PlGF Posttreatment on Light-Induced Retinal Function Damage and Histologic Changes in the Mouse Retina
We further investigated the effects of PlGF posttreatment against light-induced retinal function damage in the mouse retina (the experimental protocol is shown in Fig. 2A). Retinal function was evaluated by ERG recordings 5 days after light exposure. Both a- and b- wave amplitudes were significantly decreased 5 days after exposure to light compared with the control group. Intravitreal injections of PlGF at 2.5 and 25 ng/mL just after exposure to light exacerbated the light-induced retinal functional damage (Figs. 2B–D). In particular, at 0.98 log cds/m2, the amplitudes of the a-wave in administration of PlGF (2.5 and 25 ng/mL) were reduced by 42% and 36%, respectively, and the b-wave amplitudes were reduced by 55% and 50% versus the vehicle-treated group. In addition, to evaluate the effects of PlGF posttreatment on light-induced photoreceptor degeneration, histologic analysis was performed. Representative retinal images were taken 5 days after light exposure (Fig. 2E). The ONL thickness decreased in the light-exposed group compared with the control group. The PlGF treatment (25 ng/mL) just after light exposure significantly aggravated the light-induced ONL thinning compared with the vehicle-treated group, whereas intravitreal injection of PlGF at 2.5 ng/mL had no effect (Figs. 2E, 2F). The PlGF treatment (25 ng/mL) exacerbated the reduction of ONL thickness by a maximum of 45% compared with the vehicle-treated group. 
Figure 2
 
Aggravation effects of PlGF posttreatment on light-induced visual function impairment and histologic changes after exposure to light in the mouse retina. (A) Experimental protocol. (B) Representative dark-adapted ERG responses of both a- and b-wave amplitudes 5 days after light exposure. (C, D) Amplitudes of a- and b-waves in individual mice (control: n = 8, vehicle treated: n = 8, PlGF 2.5 ng/mL treated: n = 8, and PlGF 25 ng/mL treated: n = 8). (E) Representative photographs of hematoxylin and eosin staining of retinal sections (control: n = 8, vehicle treated: n = 8, PlGF 2.5 ng/mL treated: n = 8, PlGF 25 ng/mL treated: n = 8). Scale bar: 30 μm. (F) Thickness measurements in the ONL 5 days after light exposure. Data are shown as means ± SEM. #P < 0.05 and ##P < 0.01 versus control group (t-test); *P < 0.05 and **P < 0.01 versus vehicle-treated group (Dunnett's test).
Figure 2
 
Aggravation effects of PlGF posttreatment on light-induced visual function impairment and histologic changes after exposure to light in the mouse retina. (A) Experimental protocol. (B) Representative dark-adapted ERG responses of both a- and b-wave amplitudes 5 days after light exposure. (C, D) Amplitudes of a- and b-waves in individual mice (control: n = 8, vehicle treated: n = 8, PlGF 2.5 ng/mL treated: n = 8, and PlGF 25 ng/mL treated: n = 8). (E) Representative photographs of hematoxylin and eosin staining of retinal sections (control: n = 8, vehicle treated: n = 8, PlGF 2.5 ng/mL treated: n = 8, PlGF 25 ng/mL treated: n = 8). Scale bar: 30 μm. (F) Thickness measurements in the ONL 5 days after light exposure. Data are shown as means ± SEM. #P < 0.05 and ##P < 0.01 versus control group (t-test); *P < 0.05 and **P < 0.01 versus vehicle-treated group (Dunnett's test).
Effects of Anti-PlGF Antibody on Light-Induced Retinal Function Damage and Histologic Changes in the Mouse Retina
We next evaluated the effects of the anti-PlGF antibody against light-induced retinal function damage and histologic changes in the mouse retina (the experimental protocol is presented in Fig. 3A). Retinal function was evaluated by ERG recordings 5 days after light exposure. Both a- and b-wave amplitudes were significantly decreased 5 days after exposure to light compared with the control group. Intravitreal injection of the anti-PlGF antibody before exposure to light partial protected the reduction in amplitudes induced by light exposure (Figs. 3B–D). In particular, at −0.02 log cds/m2, the amplitudes of the a-wave in administration of anti-PlGF antibody (50 and 500 μg/mL) were improved by 60% and 80%, respectively, and b-wave amplitudes were improved by 54% and 78% versus the vehicle-treated group. In addition, to explore the effects of the anti-PlGF antibody on light-induced photoreceptor degeneration, histologic analysis was performed. Representative retinal images show the retina 5 days after exposure to light (Fig. 3E). The ONL thickness decreased in the light-exposed group compared with the control group. Anti-PlGF antibody treatment significantly suppressed the light-induced ONL thinning compared with the vehicle-treated group (Figs. 3E, 3F). Anti-PlGF antibody treatment (50 and 500 μg/mL) improved the reduction of ONL thickness by a maximum of 57% and 66%, respectively, compared with the vehicle-treated group. 
Figure 3
 
Neuroprotective effects of anti-PlGF antibody against light-induced visual function impairment and histologic changes after light exposure in the mouse retina. (A) Experimental protocol. (B) Representative dark-adapted ERG responses of both a- and b-wave amplitudes 5 days after light exposure. (C, D) Amplitudes of a- and b-waves of individual mice (control: n = 10, vehicle treated: n = 10, anti-PlGF 50 μg/mL treated: n = 10, and anti-PlGF 500 μg/mL treated: n = 8). (E) Representative photographs of hematoxylin and eosin staining of retinal sections (control: n = 10, vehicle treated: n = 10, anti-PlGF 50 μg/mL treated: n = 10, and anti-PlGF 500 μg/mL treated: n = 9). Scale bar: 30 μm. (F) Thickness measurements in the ONL 5 days after light exposure. Data are shown as means ± SEM. #P < 0.05 and ##P < 0.01 versus control group (t-test); *P < 0.05 and **P < 0.01 versus vehicle-treated group (Dunnett's test).
Figure 3
 
Neuroprotective effects of anti-PlGF antibody against light-induced visual function impairment and histologic changes after light exposure in the mouse retina. (A) Experimental protocol. (B) Representative dark-adapted ERG responses of both a- and b-wave amplitudes 5 days after light exposure. (C, D) Amplitudes of a- and b-waves of individual mice (control: n = 10, vehicle treated: n = 10, anti-PlGF 50 μg/mL treated: n = 10, and anti-PlGF 500 μg/mL treated: n = 8). (E) Representative photographs of hematoxylin and eosin staining of retinal sections (control: n = 10, vehicle treated: n = 10, anti-PlGF 50 μg/mL treated: n = 10, and anti-PlGF 500 μg/mL treated: n = 9). Scale bar: 30 μm. (F) Thickness measurements in the ONL 5 days after light exposure. Data are shown as means ± SEM. #P < 0.05 and ##P < 0.01 versus control group (t-test); *P < 0.05 and **P < 0.01 versus vehicle-treated group (Dunnett's test).
Effects of PlGF and Anti-PlGF Antibody on Normal Non–Light-Damaged Mouse Retina
We investigated the effects of PlGF and anti-PlGF antibody treatment on retinal function in the normal murine retina without excess light exposure, and we conducted ERG analysis. Retinal function was evaluated by ERG recordings 5 days after intravitreal injection of PlGF (25 ng/mL) and anti-PlGF antibody (500 μg/mL). There were no effects of PlGF and anti-PlGF antibody on normal non–light-damaged mice in a-waves or b-waves on ERG recording (Figs. 4A–D). In addition, to explore the effects of PlGF and anti-PlGF antibody treatment on photoreceptors in the normal mouse, histologic analysis was performed: PlGF and anti-PlGF antibody by itself did not affect retinal thickness on normal non–light-damaged mice (Figs. 4E, 4F). 
Figure 4
 
No effects of PlGF and anti-PlGF antibody on normal mouse. (A, B) Amplitudes of a- and b-waves of individual mice 5 days after intravitreal injection of PlGF (25 ng/mL) (control: n = 6, vehicle treated: n = 5, PlGF 25 ng/mL treated: n = 6) and (C, D) anti-PlGF antibody (500 μg/mL) (control: n = 6, vehicle treated: n = 5, anti-PlGF 500 μg/mL treated: n = 6). (E) Thickness measurements in the ONL 5 days intravitreal injection of PlGF (25 ng/mL) (control: n = 6, vehicle treated: n = 5, PlGF 25 ng/mL treated: n = 5) and (F) anti-PlGF antibody (500 μg/mL) (control: n = 6, vehicle treated: n = 5, anti-PlGF 500 μg/mL treated: n = 6).
Figure 4
 
No effects of PlGF and anti-PlGF antibody on normal mouse. (A, B) Amplitudes of a- and b-waves of individual mice 5 days after intravitreal injection of PlGF (25 ng/mL) (control: n = 6, vehicle treated: n = 5, PlGF 25 ng/mL treated: n = 6) and (C, D) anti-PlGF antibody (500 μg/mL) (control: n = 6, vehicle treated: n = 5, anti-PlGF 500 μg/mL treated: n = 6). (E) Thickness measurements in the ONL 5 days intravitreal injection of PlGF (25 ng/mL) (control: n = 6, vehicle treated: n = 5, PlGF 25 ng/mL treated: n = 5) and (F) anti-PlGF antibody (500 μg/mL) (control: n = 6, vehicle treated: n = 5, anti-PlGF 500 μg/mL treated: n = 6).
Effects of the Anti-PlGF Antibody on Light-Induced Disruption of Cell–Cell Junctions in the Mouse RPE
Previous reports have shown that light exposure causes loss of RPE cell–cell junction integrity.13,26 In addition, it has been reported that PlGF contributes to RPE breakdown.40,41 Therefore, we analyzed whether PlGF is involved in the disruption of cell–cell junction integrity after light exposure using an anti-PlGF antibody. Cell–cell junctional integrity was measured using a linearity index as described in the Materials and Methods. Immunostaining for ZO-1, a component protein of tight junctions, was clearly hexagonal shaped in the RPE of control mice. However, the shape of ZO-1 staining was largely disrupted in the RPE of the light-exposed mice at 24 hours. Anti-PlGF antibody treatment significantly improved the disruption of cell–cell junctional integrity (Figs. 5A, B). 
Figure 5
 
Suppression of the light-induced breakdown of RPE integrity by anti-PlGF treatment. (A) Top row, representative images of ZO-1 staining of the flat-mounted RPE in each of the mice 24 hours after light exposure (control: n = 8, vehicle-treated: n = 8, and anti-PlGF 500 μg/mL treated: n = 9). Boxed areas are higher magnification (bottom row). Scale bar: 20 μm. (B) Quantification of junction linearity index. “Linearity index'' was quantified surrounding a lesion as the ratio of actual junctional length to the linear junction length (straight line between vertices). Data are shown as means ± SEM. ##P < 0.01 versus control group; **P < 0.01 versus vehicle-treated group (t-test).
Figure 5
 
Suppression of the light-induced breakdown of RPE integrity by anti-PlGF treatment. (A) Top row, representative images of ZO-1 staining of the flat-mounted RPE in each of the mice 24 hours after light exposure (control: n = 8, vehicle-treated: n = 8, and anti-PlGF 500 μg/mL treated: n = 9). Boxed areas are higher magnification (bottom row). Scale bar: 20 μm. (B) Quantification of junction linearity index. “Linearity index'' was quantified surrounding a lesion as the ratio of actual junctional length to the linear junction length (straight line between vertices). Data are shown as means ± SEM. ##P < 0.01 versus control group; **P < 0.01 versus vehicle-treated group (t-test).
Activation of VEGFR-1 and -2 After Light Exposure in the Retina and RPE–Choroid Complex
It has been reported that PlGF only binds to VEGFR-1.42 In addition, activation of VEGFR-1 by PlGF amplifies VEGFR-2 signaling.18 To determine whether VEGFR-1 and -2 are involved in the light-induced retinal damage model, the quantity of these proteins was quantified using Western blot. VEGFR-1 level in the retina showed a tendency to be up-regulated after light exposure (Fig. 6A). Moreover, 48 hours after light exposure, VEGFR-2 in the retina was significantly increased (Fig. 6B), and both VEGFR-1 and -2 levels in the RPE–choroid complex increased significantly 6 hours after exposure to light (Figs. 6C, 6D). 
Figure 6
 
VEGFR-1 and -2 expression levels after light exposure in the retina and RPE–choroid complex. (A) VEGFR-1 expression level in the retina at 0, 6, 12, 24, and 48 hours after exposure to light. (B) VEGFR-2 in the retina. (C) VEGFR-1 in the RPE–choroid complex. (D) VEGFR-2 in the RPE–choroid complex. Data are shown as means ± SEM (n = 5 in each group). #P < 0.05 and ##P < 0.01 versus control (Dunnett's test).
Figure 6
 
VEGFR-1 and -2 expression levels after light exposure in the retina and RPE–choroid complex. (A) VEGFR-1 expression level in the retina at 0, 6, 12, 24, and 48 hours after exposure to light. (B) VEGFR-2 in the retina. (C) VEGFR-1 in the RPE–choroid complex. (D) VEGFR-2 in the RPE–choroid complex. Data are shown as means ± SEM (n = 5 in each group). #P < 0.05 and ##P < 0.01 versus control (Dunnett's test).
Discussion
In the present study, we investigated the effects of PlGF against dry AMD using light-induced retinal damage in vivo mouse model. We found that PlGF treatment aggravated the light-induced retinal function damage and ONL thinning (Figs. 1, 2). In contrast, anti-PlGF antibody treatment significantly suppressed the light-induced retinal functional damage and ONL thinning (Fig. 3). There were no effects of PlGF and anti-PlGF on normal non–light-damaged tissues (Fig. 4). In addition, anti-PlGF antibody treatment suppressed the light-induced disruption of cell–cell junctional integrity (Fig. 5). Also, VEGFR-1 and -2 levels in the RPE–choroid complex significantly increased 6 hours after light exposure (Fig. 6). 
It has been reported that many neurotrophic factors and growth factors such as BDNF and fibroblast growth factor protect against light-induced retinal damage.16 We previously described that PlGF has protective effects on an in vitro retinal neuronal cell damage model.43 However, the present study showed that PlGF aggravated the light-induced retinal damage in an in vivo mouse model. Moreover, anti-PlGF antibody treatment protected against light-induced retinal degeneration. Conversely, a previous report showed that blocking VEGF specifically in RPE cells prevented RPE disruption and retinal degeneration after light exposure,26 suggesting that its effects on the permeability of the RPE barrier plays a pivotal role in the light-induced retinal damage. Light exposure increased expression of VEGF and PlGF in human primary RPE cells.44 Moreover, increased VEGF levels in the RPE of VEGFhyper mice caused RPE disfunction and not the choriocapillaris.45,46 These reports suggested that VEGF can disrupt RPE barrier directly, in either a paracrine or an autocrine manner. PIGF induces RPE hyperpermeability and leads to blood–retinal barrier (BRB) breakdown.40 Indeed, disruption of RPE cell–cell junctional integrity was observed after light exposure. In the present study, anti-PlGF antibody treatment significantly decreased the disruption of cell–cell junctional integrity. These findings indicate that PlGF, as well as VEGF, is involved in the disruption of cell–cell junction integrity after light exposure. 
A previous report has shown that RPE breakdown after light exposure was involved in VEGF signaling and photoreceptor degeneration through induction of pathologic cytokines and macrophages by the collapse of the RPE.26 In the present study, expression levels of VEGFR-1 and -2 increased in the RPE–choroid complex 6 hours after light exposure. Previous reports have shown that PlGF induces tight junction hyperpermeability by activating Rho/Rho-associated coiled-coil forming kinase (ROCK) signaling.47 Moreover, activated Rho/ROCK signaling is implicated in disruption of the RPE barrier structure 24 hours after light exposure.13 Therefore, PlGF may contribute to RPE barrier breakdown via both VEGF signaling and Rho/ROCK signaling. 
Conversely, the VEGFR-2 expression level was up-regulated in the retina 48 hours after light exposure, although this was consistent with peak expression of TUNEL-positive cells after light exposure in our previous study.14 A previous report has shown that the oxidative stress-induced peroxynitrite inactivates the VEGF/PI3-kinase/Akt-1 prosurvival pathway and stimulates cell death via activation of p38 MAP kinase pathway.48 Nitration of PI3-kinase via oxidative stress switches off the VEGF prosurvival pathway and triggers the proapoptotic pathway. Therefore, VEGF signaling indirectly may participate in photoreceptor apoptosis pathway after light exposure. 
We recently reported that PlGF had protective effects on in vitro models of retinal neuronal cell damage.43 However, the present study indicated that PlGF treatment aggravates in vivo light-induced retinal degeneration. The cause of this difference may be due to the influence PlGF has on the RPE: PlGF affects RPE junctional integrity and contributes to BRB breakdown. The BRB breakdown promotes induction of pathologic cytokine production and macrophage recruitment in the RPE/choroid and retina, resulting in photoreceptor cell death. In this study, intravitreal injection of PlGF by itself without light exposure did not affect retinal tissue. It has been reported that the PlGF participates in RPE disruption.40 However, it is thought that RPE disruption does not directly cause photoreceptor cell death. The induction of pathologic cytokines and macrophage recruitment are caused in RPE/choroid after light exposure. The RPE disruption promotes them and exacerbated light-induced retinal damage.13 Therefore, PlGF might aggravate the retinal damage only after light exposure. Taken together, these findings indicate that photoreceptor cell damage induced by the BRB breakdown induced by PlGF outweighs the neuroprotective action of PlGF and, consequently, anti-PlGF and PlGF treatments can prevent and aggravate, respectively, in the in vivo light-induced retinal damage. However, further experiments are needed to clarify the detailed mechanism underlying these actions. Thus, anti-PlGF treatment may represent a potential therapeutic target in dry AMD. 
Acknowledgments
Disclosure: H. Izawa, None; Y. Inoue, None; Y. Ohno, None; K. Ojino, None; K. Tsuruma, None; M. Shimazawa, None; H. Hara, None 
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Figure 1
 
Aggravation effects of PlGF pretreatment on light-induced visual function impairment and histologic changes after exposure to light in the mouse retina. (A) Experimental protocol. (B) Representative dark-adapted ERG responses of both a- and b-wave amplitudes 5 days after light exposure. (C, D) Amplitudes of a- and b-waves of individual mice (control: n = 8, vehicle treated: n = 8, PlGF 2.5 ng/mL treated: n = 8, and PlGF 25 ng/mL treated: n = 8). (E) Representative photographs of hematoxylin and eosin staining of retinal sections (control: n = 8, vehicle treated: n = 8, PlGF 2.5 ng/mL treated: n = 7, PlGF 25 ng/mL treated: n = 7). Scale bar: 30 μm. (F) Thickness measurements in the ONL 5 days after light exposure. Data are shown as means ± SEM. #P < 0.05 and ##P < 0.01 versus control group (t-test); *P < 0.05 and **P < 0.01 versus vehicle-treated group (Dunnett's test).
Figure 1
 
Aggravation effects of PlGF pretreatment on light-induced visual function impairment and histologic changes after exposure to light in the mouse retina. (A) Experimental protocol. (B) Representative dark-adapted ERG responses of both a- and b-wave amplitudes 5 days after light exposure. (C, D) Amplitudes of a- and b-waves of individual mice (control: n = 8, vehicle treated: n = 8, PlGF 2.5 ng/mL treated: n = 8, and PlGF 25 ng/mL treated: n = 8). (E) Representative photographs of hematoxylin and eosin staining of retinal sections (control: n = 8, vehicle treated: n = 8, PlGF 2.5 ng/mL treated: n = 7, PlGF 25 ng/mL treated: n = 7). Scale bar: 30 μm. (F) Thickness measurements in the ONL 5 days after light exposure. Data are shown as means ± SEM. #P < 0.05 and ##P < 0.01 versus control group (t-test); *P < 0.05 and **P < 0.01 versus vehicle-treated group (Dunnett's test).
Figure 2
 
Aggravation effects of PlGF posttreatment on light-induced visual function impairment and histologic changes after exposure to light in the mouse retina. (A) Experimental protocol. (B) Representative dark-adapted ERG responses of both a- and b-wave amplitudes 5 days after light exposure. (C, D) Amplitudes of a- and b-waves in individual mice (control: n = 8, vehicle treated: n = 8, PlGF 2.5 ng/mL treated: n = 8, and PlGF 25 ng/mL treated: n = 8). (E) Representative photographs of hematoxylin and eosin staining of retinal sections (control: n = 8, vehicle treated: n = 8, PlGF 2.5 ng/mL treated: n = 8, PlGF 25 ng/mL treated: n = 8). Scale bar: 30 μm. (F) Thickness measurements in the ONL 5 days after light exposure. Data are shown as means ± SEM. #P < 0.05 and ##P < 0.01 versus control group (t-test); *P < 0.05 and **P < 0.01 versus vehicle-treated group (Dunnett's test).
Figure 2
 
Aggravation effects of PlGF posttreatment on light-induced visual function impairment and histologic changes after exposure to light in the mouse retina. (A) Experimental protocol. (B) Representative dark-adapted ERG responses of both a- and b-wave amplitudes 5 days after light exposure. (C, D) Amplitudes of a- and b-waves in individual mice (control: n = 8, vehicle treated: n = 8, PlGF 2.5 ng/mL treated: n = 8, and PlGF 25 ng/mL treated: n = 8). (E) Representative photographs of hematoxylin and eosin staining of retinal sections (control: n = 8, vehicle treated: n = 8, PlGF 2.5 ng/mL treated: n = 8, PlGF 25 ng/mL treated: n = 8). Scale bar: 30 μm. (F) Thickness measurements in the ONL 5 days after light exposure. Data are shown as means ± SEM. #P < 0.05 and ##P < 0.01 versus control group (t-test); *P < 0.05 and **P < 0.01 versus vehicle-treated group (Dunnett's test).
Figure 3
 
Neuroprotective effects of anti-PlGF antibody against light-induced visual function impairment and histologic changes after light exposure in the mouse retina. (A) Experimental protocol. (B) Representative dark-adapted ERG responses of both a- and b-wave amplitudes 5 days after light exposure. (C, D) Amplitudes of a- and b-waves of individual mice (control: n = 10, vehicle treated: n = 10, anti-PlGF 50 μg/mL treated: n = 10, and anti-PlGF 500 μg/mL treated: n = 8). (E) Representative photographs of hematoxylin and eosin staining of retinal sections (control: n = 10, vehicle treated: n = 10, anti-PlGF 50 μg/mL treated: n = 10, and anti-PlGF 500 μg/mL treated: n = 9). Scale bar: 30 μm. (F) Thickness measurements in the ONL 5 days after light exposure. Data are shown as means ± SEM. #P < 0.05 and ##P < 0.01 versus control group (t-test); *P < 0.05 and **P < 0.01 versus vehicle-treated group (Dunnett's test).
Figure 3
 
Neuroprotective effects of anti-PlGF antibody against light-induced visual function impairment and histologic changes after light exposure in the mouse retina. (A) Experimental protocol. (B) Representative dark-adapted ERG responses of both a- and b-wave amplitudes 5 days after light exposure. (C, D) Amplitudes of a- and b-waves of individual mice (control: n = 10, vehicle treated: n = 10, anti-PlGF 50 μg/mL treated: n = 10, and anti-PlGF 500 μg/mL treated: n = 8). (E) Representative photographs of hematoxylin and eosin staining of retinal sections (control: n = 10, vehicle treated: n = 10, anti-PlGF 50 μg/mL treated: n = 10, and anti-PlGF 500 μg/mL treated: n = 9). Scale bar: 30 μm. (F) Thickness measurements in the ONL 5 days after light exposure. Data are shown as means ± SEM. #P < 0.05 and ##P < 0.01 versus control group (t-test); *P < 0.05 and **P < 0.01 versus vehicle-treated group (Dunnett's test).
Figure 4
 
No effects of PlGF and anti-PlGF antibody on normal mouse. (A, B) Amplitudes of a- and b-waves of individual mice 5 days after intravitreal injection of PlGF (25 ng/mL) (control: n = 6, vehicle treated: n = 5, PlGF 25 ng/mL treated: n = 6) and (C, D) anti-PlGF antibody (500 μg/mL) (control: n = 6, vehicle treated: n = 5, anti-PlGF 500 μg/mL treated: n = 6). (E) Thickness measurements in the ONL 5 days intravitreal injection of PlGF (25 ng/mL) (control: n = 6, vehicle treated: n = 5, PlGF 25 ng/mL treated: n = 5) and (F) anti-PlGF antibody (500 μg/mL) (control: n = 6, vehicle treated: n = 5, anti-PlGF 500 μg/mL treated: n = 6).
Figure 4
 
No effects of PlGF and anti-PlGF antibody on normal mouse. (A, B) Amplitudes of a- and b-waves of individual mice 5 days after intravitreal injection of PlGF (25 ng/mL) (control: n = 6, vehicle treated: n = 5, PlGF 25 ng/mL treated: n = 6) and (C, D) anti-PlGF antibody (500 μg/mL) (control: n = 6, vehicle treated: n = 5, anti-PlGF 500 μg/mL treated: n = 6). (E) Thickness measurements in the ONL 5 days intravitreal injection of PlGF (25 ng/mL) (control: n = 6, vehicle treated: n = 5, PlGF 25 ng/mL treated: n = 5) and (F) anti-PlGF antibody (500 μg/mL) (control: n = 6, vehicle treated: n = 5, anti-PlGF 500 μg/mL treated: n = 6).
Figure 5
 
Suppression of the light-induced breakdown of RPE integrity by anti-PlGF treatment. (A) Top row, representative images of ZO-1 staining of the flat-mounted RPE in each of the mice 24 hours after light exposure (control: n = 8, vehicle-treated: n = 8, and anti-PlGF 500 μg/mL treated: n = 9). Boxed areas are higher magnification (bottom row). Scale bar: 20 μm. (B) Quantification of junction linearity index. “Linearity index'' was quantified surrounding a lesion as the ratio of actual junctional length to the linear junction length (straight line between vertices). Data are shown as means ± SEM. ##P < 0.01 versus control group; **P < 0.01 versus vehicle-treated group (t-test).
Figure 5
 
Suppression of the light-induced breakdown of RPE integrity by anti-PlGF treatment. (A) Top row, representative images of ZO-1 staining of the flat-mounted RPE in each of the mice 24 hours after light exposure (control: n = 8, vehicle-treated: n = 8, and anti-PlGF 500 μg/mL treated: n = 9). Boxed areas are higher magnification (bottom row). Scale bar: 20 μm. (B) Quantification of junction linearity index. “Linearity index'' was quantified surrounding a lesion as the ratio of actual junctional length to the linear junction length (straight line between vertices). Data are shown as means ± SEM. ##P < 0.01 versus control group; **P < 0.01 versus vehicle-treated group (t-test).
Figure 6
 
VEGFR-1 and -2 expression levels after light exposure in the retina and RPE–choroid complex. (A) VEGFR-1 expression level in the retina at 0, 6, 12, 24, and 48 hours after exposure to light. (B) VEGFR-2 in the retina. (C) VEGFR-1 in the RPE–choroid complex. (D) VEGFR-2 in the RPE–choroid complex. Data are shown as means ± SEM (n = 5 in each group). #P < 0.05 and ##P < 0.01 versus control (Dunnett's test).
Figure 6
 
VEGFR-1 and -2 expression levels after light exposure in the retina and RPE–choroid complex. (A) VEGFR-1 expression level in the retina at 0, 6, 12, 24, and 48 hours after exposure to light. (B) VEGFR-2 in the retina. (C) VEGFR-1 in the RPE–choroid complex. (D) VEGFR-2 in the RPE–choroid complex. Data are shown as means ± SEM (n = 5 in each group). #P < 0.05 and ##P < 0.01 versus control (Dunnett's test).
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