October 2013
Volume 54, Issue 10
Free
Retina  |   October 2013
Effects of Semaphorin 3A on Retinal Pigment Epithelial Cell Activity
Author Affiliations & Notes
  • Yujing Bai
    Key Laboratory of Vision Loss and Restoration, Ministry of Education, Department of Ophthalmology, Peking University People's Hospital, Beijing, China
  • Wenzhen Yu
    Key Laboratory of Vision Loss and Restoration, Ministry of Education, Department of Ophthalmology, Peking University People's Hospital, Beijing, China
  • Na Han
    Department of Orthopedics and Trauma, Peking University People's Hospital, Beijing, China
  • Fei Yang
    Key Laboratory of Vision Loss and Restoration, Ministry of Education, Department of Ophthalmology, Peking University People's Hospital, Beijing, China
  • Yaoyao Sun
    Key Laboratory of Vision Loss and Restoration, Ministry of Education, Department of Ophthalmology, Peking University People's Hospital, Beijing, China
  • Lijuan Zhang
    Key Laboratory of Vision Loss and Restoration, Ministry of Education, Department of Ophthalmology, Peking University People's Hospital, Beijing, China
  • Min Zhao
    Key Laboratory of Vision Loss and Restoration, Ministry of Education, Department of Ophthalmology, Peking University People's Hospital, Beijing, China
  • Lvzhen Huang
    Key Laboratory of Vision Loss and Restoration, Ministry of Education, Department of Ophthalmology, Peking University People's Hospital, Beijing, China
  • Aiyi Zhou
    Key Laboratory of Vision Loss and Restoration, Ministry of Education, Department of Ophthalmology, Peking University People's Hospital, Beijing, China
  • Fei Wang
    Key Laboratory of Vision Loss and Restoration, Ministry of Education, Department of Ophthalmology, Peking University People's Hospital, Beijing, China
  • Xiaoxin Li
    Key Laboratory of Vision Loss and Restoration, Ministry of Education, Department of Ophthalmology, Peking University People's Hospital, Beijing, China
  • Correspondence: Xiaoxin Li, Key Laboratory of Vision Loss and Restoration, Ministry of Education, Department of Ophthalmology, Peking University People's Hospital, Xizhimen South Street 11, Xi Cheng District, 100044 Beijing, China; drlixiaoxin@163.com
Investigative Ophthalmology & Visual Science October 2013, Vol.54, 6628-6637. doi:10.1167/iovs.13-12625
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      Yujing Bai, Wenzhen Yu, Na Han, Fei Yang, Yaoyao Sun, Lijuan Zhang, Min Zhao, Lvzhen Huang, Aiyi Zhou, Fei Wang, Xiaoxin Li; Effects of Semaphorin 3A on Retinal Pigment Epithelial Cell Activity. Invest. Ophthalmol. Vis. Sci. 2013;54(10):6628-6637. doi: 10.1167/iovs.13-12625.

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

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Abstract

Purpose.: Semaphorin 3A (Sema3A), a chemorepellant guidance protein, has been shown to be crucial for neural and vascular remodeling. This study is designed to examine the effects of Sema3A on RPE cell activity both in vitro and in vivo.

Methods.: Retinal pigment epithelial were incubated with Sema3A, or VEGF- and Sema3A-containing medium. Cell proliferation, migration, cell cycle, apoptosis, cocultured human umbilical vein endothelial cells tube formation, VEGF receptor 2 (VEGFR2) and neuropilin 1 (Nrp1) receptor expression, VEGF- and pigment epithelium–derived factor (PEDF) concentration, and c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases (p38MAPK) signaling pathway studies were measured. A rabbit proliferative vitreoretinopathy (PVR) model was used for in vivo study. Subconfluent ARPE19 cells were injected intravitreously with or without Sema3A. Data were analyzed with Graphpad Prism 5.0 software.

Results.: In vitro, Sema3A not only induced RPE cell cycle arrest and inhibited RPE migration under normal culture conditions, but also inhibited exogenous and endogenous VEGF165-induced cell activities. These activities included proliferation, migration, cell cycle arrest, JNK and p38MAPK signaling pathway phosphorylation, and cocultured endothelial cell tube formation. It is shown that both VEGF165 and Sema3A induced the upregulation of VEGFR2 and Nrp1 receptors. Activity inhibition was mediated by impeding VEGF165 utilization and possibly mediated by competitive inhibition of VEGF165 binding to its receptor VEGFR2, but not by the suppression of VEGF165 secretion. In vivo, Sema3A inhibited PVR, which is induced by RPE proliferation.

Conclusions.: These results suggested that Sema3A could be a useful therapeutic strategy for preventing RPE malfunction.

Introduction
In the vertebrate eye, the RPE is composed of a single layer of hexagonally packed cells, which contain pigment granules and organelles, connected by tight-junctions. 1,2 The functions of the RPE layer include the absorption of light, maintenance of the visual cycle, phagocytosis of photoreceptor outer membranes, secretion, epithelial transport, and immune privilege of the eye. 3 Among the essential functions, the most important role is working as the metabolic gatekeeper between the light-sensitive outer segments, photoreceptors, and the blood supply of the retinal outer layer, the choriocapillaris. 4 As a highly metabolic eye tissue, the RPE suffers cumulative endogenous and exogenous oxidative injury over its lifetime, thus, leading to abnormalities in proliferation, secretion, phagocytic capacity, and accumulation of extracellular matrix, which can cause blindness due to proliferative vitreoretinopathy (PVR), 3 diabetic retinopathy (DR), 5 and AMD. 3,6 Although modern vitreoretinal microsurgery has improved treatment in proliferative retinal diseases 7 and injectable intravitreal medicines impede the development of neovascularization diseases, 8 no therapy is currently available that perfectly inhibits the genesis and development of proliferative eye diseases. Therefore, investigation of new treatment methods and adjunctive treatment strategies is greatly needed. 
The semaphorins constitute a large family of endogenous secreted and transmembrane associated proteins that have been characterized as repulsive axonal and vessel network guidance signals. 9,10 They primarily bind and signal through neuropilins (Nrps) and plexins (Plxns) receptor complexes, and can also signal through other multimeric receptors, such as integrins, and so on. 11 Semaphorins are grouped into eight major classes based on structure and phylogenetic tree analyses. 10 Among these classes, Semaphorin 3A (Sema3A), which belongs to the class 3 semaphorin family (Sema3), has been proven to play an important role in neural system and tumor therapy. 12,13 Sema3A, as a special member of Sema3s, exclusively binds to neuropilins 1 receptor (Nrp1) first and then combines with plexins A 1-4 receptor (PlexA1-4) to form a complex (Nrp1/PlexA1-4). 14 In this receptor complex, Nrp1 acts as a binding element, while PlexA1-4 acts as a signal transducing element. Since the discovery of Sema3A, a variety of studies have reported the effects of Sema3A on neuronal cell migration, tumor metastasis, and vascular genesis. 15 17 However, the effects of Sema3A on RPE have not been documented. Therefore, in the present study, we investigated the effects and possible mechanisms of Sema3A on RPE behavior for the first time. The encouraging results of our study provided a new strategy for the therapeutic treatment of retinal proliferative eye diseases. 
Methods
Cell Proliferation Assay
Human RPE cells (ARPE19 cell line, CRL-2302; American Type Culture Collection [ATCC], Manassas, VA) were used in this study as previously described, 18 and cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 medium (HyClone; Hyclone, Grand Island, NY) containing 10% fetal bovine serum (HyClone FBS; Hyclone) as recommended by ATCC. Semaphorin 3A (50631-M01H; Sino Biological, Inc., Beijing, China) was incubated with RPE in 96-well plates for 24, 48, and 72 hours at concentrations of 250 and 500 ng/mL 19,20 in a general culture medium or in VEGF165 (25 ng/mL, Cat# 293-VE; BD Biosciences, San Diego, CA)-containing medium. 21,22 Cell Counting Kit-8 (CCK-8; Dojindo, Shanghai, China) assays were performed according to the manufacturer's instructions and read by an ELISA microplate reader (Finstruments Multiskan Models 347; MTX Lab Systems, Inc., Vienna, VA). Each experiment was performed in five wells and repeated at least three times. 
Migration Assay
The RPE migration study was performed using Transwell (Cat#3422; Corning, Tewksbury, MA) as described previously. 23 Briefly, 2 × 104 cells in 200-μL serum-free medium were placed in the top part of a Transwell. Dulbecco's modified Eagle's medium (containing 10% FBS) with 250 or 500 ng/mL Sema3A or Sema3A with VEGF165 (25 ng/mL) was placed in the bottom chamber at a final volume of 600 μL. All migration assays were conducted at 37°C for 6 hours, and then the cells were fixed and stained with 4,6-diamidino-2-phenylindole (DAPI; Roche Diagnostics, Indianapolis, IN). The cells that had not migrated through the membrane were removed with a cotton swab, and the membrane was imaged with fluorescence microscopy (Zeiss Axiophot, Thornwood, NY). Retinal pigment epithelial cells from five random fields of view were counted. Each experiment was repeated three times. 
Flow Cytometry Analysis of RPE Apoptosis and Cell Cycle
The RPE apoptosis study (FITC Annexin V Apoptosis Detection Kit; BD Biosciences) and cell cycle analysis (Cycletest Plus DNA Reagent Kit; BD Biosciences) were performed according to the manufacturer's instructions and as previously reported. 23 Briefly, RPE cells (1 × 106) were seeded in 6-well plates and incubated for 24, 48, and 72 hours, with Sema3A, VEGF165 plus Sema3A, or controls. The samples were analyzed by flow cytometry (FACSCalibur; BD Biosciences, Franklin Lakes, NJ). The apoptotic rate was calculated as the percentage of early apoptotic cells (LR) plus late apoptotic cells (UR). 
Western Blot Analysis
Retinal pigment epithelium cells were treated by different stimulators for 48 hours, and were prepared with protein extraction and protease inhibitor kits (Pierce, Rockford, IL). After centrifugation, the supernatant was collected, and the protein lysate was measured with a BCA protein assay kit (Pierce) according to the manufacturer's instructions. Equal amounts of protein were separated by 12% SDS-PAGE and transferred electrophoretically to nitrocellulose membranes (Amersham, Little Chalfont, UK). The proteins were visualized with enhanced chemiluminescence Western blot detection reagents (Pierce). Band densities of c-Jun N-terminal kinase (JNK, 1:1000), phosphorylation-c-Jun N-terminal kinase (p-JNK, 1:1000), p38 mitogen-activated protein kinases (p38MAPK, 1:1000) and phosphorylation-p38 mitogen-activated protein kinases (p-p38MAPK, 1:1000) (Cell Signaling Technology, Danvers, MA), followed by incubation with an HRP-conjugated goat antibody against rabbit IgG (1:1000; Cell Signaling Technology, Danvers, MA) were tested. To identify the expression pattern of VEGFR2 and Nrp1, VEGFR2 (1:1000, Cell Signaling Technology) and Nrp1 (1:1000, Abcam, Cambridge, MA) were tested in each treatment group. For sequential blotting with additional antibodies, the membranes were stripped with a restorative Western blot stripping buffer and reprobed with the indicated antibodies. Western blot analyses were repeated three times, and qualitatively similar results were obtained. 
RNA Extraction and Real-Time PCR of VEGFR2 and Nrp1
ARPE19 cells treated with VEGF165 and Sema3A were lysed in Trizol, and RNA was extracted according to the manufacturer's protocol. Reverse transcriptase reactions were performed using the RevertAid First Strand cDNA Synthesis Kit with oligo-dT primer (Fermentas, Pittsburgh, PA). Real-time PCR reactions were performed with the SYBR Green PCR mix (Thermo, Pittsburgh, PA) using the ABI7300 real-time PCR system (Applied Biosystems, Life Technologies, Foster City, CA). The primers used in real-time PCR were VEGFR2: forward 5′-GACGGCTTCACCATCGAAT-3′; reverse 5′-CAGCGCAGATGCTCGTACTT-3′; Nrp1: forward 5′-CGCTACGACCGGCTAGAAAT-3′; reverse 5′-AGAGAATGCCCGATGAGGAT-3′; and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH): forward 5′-GAGTCCACTGGCGTCTTCAC-3′; reverse 5′-GTTCACACCCATGACGAACA-3.′ Data were normalized to the housekeeping gene GAPDH, and the results were expressed as fold amplifications. Each experiment was repeated five times. 
The Concentration of VEGF165 and Pigment Epithelium–Derived Factor (PEDF) Measurement by ELISA Assay
Vascular endothelial growth factor and PEDF are the most important growth factors in the pathologic of retinal diseases. 3 Enzyme-linked immunosorbent assay was used to detect the concentration (pg/mL) of these two factors in the cell culture supernatant. The 0% and 10% FBS containing culture medium group was treated as control, where the measured concentration of VEGF and PEDF reflects the secreted amount by RPE cell. In drug treatment group, Sema3A (250 ng/mL and 500 ng/mL) was incubated with RPE in 96-well plates for 24, 48, and 72 hours in a general culture medium (to test VEGF and PEDF secreted by RPE during Sema3A treatment) or in VEGF165 (25 ng/mL)-containing medium (to test the effects of Sema3A on VEGF and PEDF utilization). At indicated time-points, the cell culture supernatant were harvested and centrifuged. Free VEGF165 protein in the culture medium was measured by a VEGF165 (human) ELISA Kit (EK0575; Bostar, Wuhan, China), and the concentration of PEDF protein was tested using a PEDF (human) ELISA Kit (EK0896; Bostar) according to the manufacturer's instructions. 
To test the effects of Sema3A on the function of endogenous VEGF165 and PEDF, the chemical hypoxic model (application of 200 μM/L cobalt chloride (CoCl2) to RPE cell culture medium for 12 hours, which can induce the expression of endogenous VEGF165) was used. 24,25 Sema3A (250 ng/mL and 500 ng/mL) was incubated with RPE in 96-well plates for 24, 48, and 72 hours with or without CoCl2. At indicated time-points, the cell culture supernatant were collected to measure VEGF165 and PEDF concentration. In this experiment, the VEGF165 or PEDF secreted by RPE in 10% FBS–containing culture medium was treated as control group. All of other treatment groups were compared with the control group, and the ratio was presented in the results. 
HUVECs Tube Formation Study
A human umbilical vein endothelial cell line (HUVECs, CRL-1730; ATCC) was used in this study for in vitro evaluation of angiogenesis. The tube formation study is a quantifiable assay to test the angiogenic/antiangiogenic properties of compounds on vascular endothelial cells. 23 In this study, RPE cells were cultured in 200 μM/L CoCl2-containing medium with or without Sema3A (250 and 500 ng/mL) for 12 hours. Then, the supernatants were collected and used immediately. In total, 150 μL Matrigel (Cat#354234; BD Biosciences) solution was poured into 48-well plates and then incubated at 37°C for 30 minutes. Human umbilical vein endothelial cells line (5 × 104 per well) incubated in the collected supernatants were seeded on the Matrigel and cultured for 8 to 10 hours. The networks in Matrigel from five randomly chosen fields were counted and photographed. Tube formation is typically quantified by measuring the number, length, or area of these capillary-like structures in two-dimensional microscope images. In our study, the length of the tube was measured by ImageJ software (National Institutes of Health, Bethesda, MD). The experiments were performed in triplicate. 
Rabbit PVR Models
Twenty-seven adult pigmented rabbits, 2.0 to 2.5 kg each, were used in this in vivo study. All experiments adhered to the ARVO statements for the Use of Animals in Ophthalmology and Vision Research. All procedures were approved by Animal Care Use Committee of Peking University. The animals were housed with free access to laboratory food and water and kept in a 12:12 hour light-dark cycle. 
Subconfluent ARPE19 cells were used for the intravitreous injection. Before RPE cell injection, 0.1 mL of aqueous humor was removed from each rabbit eye using a 29-gauge needle. For RPE cell injection, a 29-gauge insulin injection syringe was loaded with 100 μL RPE cells (2.5 × 105) 26 containing PBS, 100 ng of Sema3A or 200 ng of Sema3A. 27 The needle was inserted through the sclera, 1.5-mm posterior to the limbus. On day 14, transpupillary optical coherence tomography (Humphrey Instruments, San Leandro, CA) was performed in each rabbit. Classification of PVR was performed as in a previous paper 27 as follows: stage 0: healthy retina; stage 1: intravitreous membrane; stage 2: focal traction, localized vascular changes, hyperemia, engorgement dilation, and blood vessel elevation; stage 3: localized detachment of medullary rays; stage 4: extensive retinal detachment, total medullary ray detachment, and peripapillary retinal detachment; and stage 5: total retinal detachment, retinal folds, and macular holes. B scans (A/B Scan, 10 MHz; Quantel Medical, Cournon d'Auvergne, France) and ocular photographs were taken on day 14 for analysis. 
Statistical Analysis
Data analysis was performed using the statistical software Prism 5 (GraphPad Software, Inc., San Diego, CA). All data are presented as the mean ± SEM. Differences were evaluated with ANOVA followed by Student-Newman-Keul's test for multiple comparisons. A P value less than 0.05 was considered to be a statistically significant difference. 
Results
Sema3A Inhibited VEGF165-Induced RPE Proliferation
A CCK-8 Proliferation Assay Kit was used to evaluate the proliferation effects of Sema3A in vitro, as stated in the Methods section. The results showed that Sema3A did not inhibit RPE proliferation under normal culture conditions (10% FBS) at different time points, even at high concentration (Sema3A 500 ng/mL) (compared with 10% FBS group) (Fig. 1). However, Sema3A can significantly inhibit exogenous VEGF165-induced RPE proliferation at both studied concentrations (250 and 500 ng/mL) and at varying time points compared with the VEGF165-treated group (Fig. 1). Because 250 ng/mL Sema3A can inhibit the proliferation effects significantly, we only showed the statistical results of 250 ng/mL Sema3A group in Figure 1
Figure 1
 
Effects of Sema3A on RPE proliferation. Left, middle, and right: The statistical results of RPE proliferation at different time points (24 [left], 48 [middle], 72 hours [right]). Semaphorin 3A did not affect RPE proliferation in general culture medium, but can inhibit RPE proliferation in VEGF165-stimulated conditions and was significantly different than the VEGF165-treated group. Each experiment was repeated in 5 wells and was duplicated at least three times. Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01.
Figure 1
 
Effects of Sema3A on RPE proliferation. Left, middle, and right: The statistical results of RPE proliferation at different time points (24 [left], 48 [middle], 72 hours [right]). Semaphorin 3A did not affect RPE proliferation in general culture medium, but can inhibit RPE proliferation in VEGF165-stimulated conditions and was significantly different than the VEGF165-treated group. Each experiment was repeated in 5 wells and was duplicated at least three times. Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01.
Sema3A Inhibited RPE Migration
Migration is an important step for RPE cells activity. We utilized the modified Boyden chamber assay, using VEGF165 as the chemotactic agent. As shown in Figure 2, the number of cells that passed through the membrane in Sema3A-treated (both 250 and 500 ng/mL) RPE groups was significantly lower than the number in the control group (10% FBS) (Figs. 2A, 2E). Additionally, in exogenous VEGF165-stimulated groups, Sema3A can inhibit the crossing of RPE (Figs. 2A, 2F). The above results showed that Sema3A effectively inhibited RPE migration. 
Figure 2
 
Effects of Sema3A on the migration of RPE. Cell nuclei were stained with DAPI and are shown in blue dots. Cells from five random view fields were counted, and the average was used for statistical analysis. (A) Statistical analysis showing Sema3A inhibits RPE migration both in general culture medium and under VEGF-stimulated conditions; (B) 0% FBS–treated group; (C) 10% FBS–culture group; (D) VEGF-treated group; (E) Sema3A (500 ng/mL)-treated group; and (F) Sema3A (500 ng/mL)- and VEGF-treated group. The data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.
Figure 2
 
Effects of Sema3A on the migration of RPE. Cell nuclei were stained with DAPI and are shown in blue dots. Cells from five random view fields were counted, and the average was used for statistical analysis. (A) Statistical analysis showing Sema3A inhibits RPE migration both in general culture medium and under VEGF-stimulated conditions; (B) 0% FBS–treated group; (C) 10% FBS–culture group; (D) VEGF-treated group; (E) Sema3A (500 ng/mL)-treated group; and (F) Sema3A (500 ng/mL)- and VEGF-treated group. The data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.
Sema3A-Induced RPE Cell Cycle Arrest but Not Apoptosis
A flow cytometry test (FITC Annexin V Apoptosis Detection Kit) was used to evaluate the effects of Sema3A on RPE cell cycle arrest (G2/M phase and S phase) and apoptosis (early apoptosis and late apoptosis). In our study, there was no significant difference between Sema3A-treated groups and control groups in the apoptosis test (Fig. 3A). However, Sema3A can induce RPE cell cycle arrest both in general culture medium and under VEGF165-induced conditions (Fig. 3B and Table 1). 
Figure 3
 
Effects of Sema3A on RPE apoptosis and cell cycle. (A) The statistical results of Sema3A on RPE apoptosis, which showed no significant difference between the experimental group and the controls. (B) The statistical data for the RPE cell cycle (G2/M phase + S phase) distribution of different groups. (B) Sema3A induces cell cycle arrest both in general culture medium and under VEGF-stimulated conditions. Data are presented as the mean ± SEM. Each experiment was repeated three times. *P < 0.05; **P < 0.01.
Figure 3
 
Effects of Sema3A on RPE apoptosis and cell cycle. (A) The statistical results of Sema3A on RPE apoptosis, which showed no significant difference between the experimental group and the controls. (B) The statistical data for the RPE cell cycle (G2/M phase + S phase) distribution of different groups. (B) Sema3A induces cell cycle arrest both in general culture medium and under VEGF-stimulated conditions. Data are presented as the mean ± SEM. Each experiment was repeated three times. *P < 0.05; **P < 0.01.
Table 1
 
Summary of Flow Cytomery Data of Cell Cycle Measured by PI
Table 1
 
Summary of Flow Cytomery Data of Cell Cycle Measured by PI
Group 24 h 48 h 72 h
Mean SEM Mean SEM Mean SEM
10% FBS 29.8 3.4 33.1 3.1 34.1 4.2
3A, 250 ng/mL 24.1 3.1 26.1 3.3 28.4 3.9
3A, 500 ng/mL 22.7 3.9 20.5 2.5 24.1 4
VEGF 36.4 4.1 38.2 4.4 32.1 2.6
VEGF+3A, 250 ng/mL 28.8 2.7 29.1 2.7 27.4 2.1
VEGF+3A, 500 ng/mL 26.2 1.9 26.7 3.0 26.0 1.8
Sema3A Inhibited VEGF165-Induced JNK and p38MAPK Signaling Pathway Phosphorylation
Immunoblot analysis of the JNK, p-JNK, p38MAPK, and p-p38MAPK signaling pathways revealed that Sema3A can activate the phosphorylation of JNK and p38MAPK in general culture medium, but was not significantly different compared with controls (Fig. 4); however, Sema3A inhibited the phosphorylation of JNK and p38MAPK in VEGF165-containing culture medium compared with the VEGF165-treated group (Figs. 4C, 4D). 
Figure 4
 
Effects of Sema3A on RPE cells JNK and p38MAPK signaling pathway phosphorylation. Immunoblot image (A, C) and statistical analysis (B, D) for JNK, p-JNK, p38MAPK, and p-p38MAPK signaling pathways. (B) Shows that Sema3A inhibits p-JNK both in general culture medium and under VEGF-stimulated conditions, and (D) shows that Sema3A inhibits p-p38MAPK in the presence of VEGF stimulation. Western blot analyses were repeated three times, and qualitatively similar results were obtained. Data are presented as the mean ± SEM. *P < 0.05.
Figure 4
 
Effects of Sema3A on RPE cells JNK and p38MAPK signaling pathway phosphorylation. Immunoblot image (A, C) and statistical analysis (B, D) for JNK, p-JNK, p38MAPK, and p-p38MAPK signaling pathways. (B) Shows that Sema3A inhibits p-JNK both in general culture medium and under VEGF-stimulated conditions, and (D) shows that Sema3A inhibits p-p38MAPK in the presence of VEGF stimulation. Western blot analyses were repeated three times, and qualitatively similar results were obtained. Data are presented as the mean ± SEM. *P < 0.05.
Expression Pattern of VEGFR2 and Nrp1 in RPE Cells Under Different Treatments
To identify the expression pattern of VEGFR2 and Nrp1 receptors in ARPE19 cells under different treatment strategies, both real-time PCR (Figs. 5A, 5B) and Western blot (Figs. 5C–E) analysis were performed. The results showed that both receptors were expressed in healthy RPE cells as reported by previous articles. 28,29 For the two receptors, the treatment of VEGF165, Sema3A, and both of the VEGF165 and Sema3A stimulation can induce the upregulation of VEGFR2 and Nrp1, and had significant difference comparing to 10% FBS treatment control group in RNA and protein level (Fig. 5). Although the costimulation by VEGF165 and Sema3A increase the expression of VEGFR2 and Nrp1 receptors in RPE cells, they had no significant difference comparing with other treatment groups in protein level in 48-hour treatment groups. 
Figure 5
 
Effects of Sema3A on VEGFR2, and NRP-1on RPE. (A, B) Statistical results of real-time PCR; (C, D) statistical results of Western blots; (E) the immunoblot image of the RPE cells under the treatment of different groups. All of the results were compared with control group (10% FBS–containing culture medium treatment group), and the results stated as the ratio fold. Real-time PCR analyses were repeated five times and Western blot analyses were repeated three times, and qualitatively similar results were obtained. Data are presented as the mean ± SEM. *P < 0.05 and **P < 0.01.
Figure 5
 
Effects of Sema3A on VEGFR2, and NRP-1on RPE. (A, B) Statistical results of real-time PCR; (C, D) statistical results of Western blots; (E) the immunoblot image of the RPE cells under the treatment of different groups. All of the results were compared with control group (10% FBS–containing culture medium treatment group), and the results stated as the ratio fold. Real-time PCR analyses were repeated five times and Western blot analyses were repeated three times, and qualitatively similar results were obtained. Data are presented as the mean ± SEM. *P < 0.05 and **P < 0.01.
Effects of Sema3A on VEGF165 and PEDF Secretion and Utilization
Sema3A-treated RPE showed no changes in VEGF165 and PEDF secretion in any group (Figure 6). While under VEGF165 treatment conditions, free VEGF165 detected in the Sema3A-treated group was much higher than in VEGF165-treated RPE (Figs. 6A–C), and the expression of PEDF was also higher than in the VEGF-treated group (Figs. 6D–F). The above results indicated that Sema3A did not affect the VEGF165 secretion in ARPE-19 cells under general culture condition; but rather inhibited the utilization ability of exogenous VEGF165's and impeding its functions. The mechanisms could be that Sema3A inhibit VEGF binding to VEGFR as previous reported. 9,14,17,30 32 Additionally, the elevation of PEDF in the Sema3A-treated group is mainly explained by the elevation of VEGF165, which tries to restore balance in the VEGF165/PEDF ratio, as previously reported. 33  
Figure 6
 
The concentration of VEGF165 and PEDF detected in the cell culture supernatant among different groups. (AC) Statistical results of free VEGF165 concentration measured by ELISA assay at different time points (24 [A], 48 [B], and 72 hours [C]). (DF) Statistical results of free PEDF concentration measured by ELISA assay at different time points (24 [D], 48 [E], and 72 hours [F]). Each experiment was repeated in 6 wells and was duplicated at least three times. Data are presented as the mean ± SEM. *P < 0.05 and **P < 0.01.
Figure 6
 
The concentration of VEGF165 and PEDF detected in the cell culture supernatant among different groups. (AC) Statistical results of free VEGF165 concentration measured by ELISA assay at different time points (24 [A], 48 [B], and 72 hours [C]). (DF) Statistical results of free PEDF concentration measured by ELISA assay at different time points (24 [D], 48 [E], and 72 hours [F]). Each experiment was repeated in 6 wells and was duplicated at least three times. Data are presented as the mean ± SEM. *P < 0.05 and **P < 0.01.
To further validate the above results, we also tested VEGF165 and PEDF in CoCl2-treated RPE cells. CoCl2 is a chemical hypoxic agent, which can induce the secretion of endogenous VEGF165 as reported. 24,25 As shown in Figure 7, in CoCl2-treated groups, Sema3A showed no effect on VEGF165 and PEDF secretion (compared with the CoCl2-treated group), however, Sema3A inhibited endogenous free VEGF165 utilization ability (Fig. 7). 
Figure 7
 
Effects of Sema3A on VEGF165 and PEDF concentration in CoCl2-treated RPE. (A) Statistical results of the ratio of free VEGF165 concentration comparing with the control group, and (B) the statistical results of the ration of PEDF concentration after CoCl2 treatment of RPE for 12 hours compared with the control group. There was no significant difference between the Sema3A-treated group and the control (CoCl2-treated group). All of the results were compared with control group (10% FBS–containing culture medium treatment group), and the results stated as the ratio fold. Each experiment was repeated in 6 wells and was duplicated at least three times. Data are presented as the mean ± SEM.
Figure 7
 
Effects of Sema3A on VEGF165 and PEDF concentration in CoCl2-treated RPE. (A) Statistical results of the ratio of free VEGF165 concentration comparing with the control group, and (B) the statistical results of the ration of PEDF concentration after CoCl2 treatment of RPE for 12 hours compared with the control group. There was no significant difference between the Sema3A-treated group and the control (CoCl2-treated group). All of the results were compared with control group (10% FBS–containing culture medium treatment group), and the results stated as the ratio fold. Each experiment was repeated in 6 wells and was duplicated at least three times. Data are presented as the mean ± SEM.
Sema3A Inhibited Cocultured HUVECs Tube Formation
The Matrigel assay is one of the most widely used methods to evaluate the angiogenic ability of endothelial cells in vitro. In our study, HUVECs had an impaired capacity to form a regular network at the Sema3A concentrations of 250 and 500 ng/mL (Fig. 8), both in the general culture medium and the VEGF165-containing medium, and the length of the angiogenesis network showed a significant difference compared with the controls (Fig. 8A). 
Figure 8
 
Effects of Sema3A on RPE tube formation. The length of tube branches per view field was measured. (A) The statistical analysis. Microphotographs are representative of tube-like structure generation after 10 hours of incubation. (B) Ten percent FBS–treated group; (C) CoCl2-treated group; (D) Sema3A 500 ng/ml–treated group; (E) Sema3A 250 ng/mL in CoCl2-treated group; and (F) Sema3A 500 ng/mL in CoCl2-treated group. Dulbecco's modified Eagle's medium + 10% FBS–treated group were set to 100%. Data are presented as the mean ± SEM. Each experiment was repeated at least three separate times. *P < 0.05.
Figure 8
 
Effects of Sema3A on RPE tube formation. The length of tube branches per view field was measured. (A) The statistical analysis. Microphotographs are representative of tube-like structure generation after 10 hours of incubation. (B) Ten percent FBS–treated group; (C) CoCl2-treated group; (D) Sema3A 500 ng/ml–treated group; (E) Sema3A 250 ng/mL in CoCl2-treated group; and (F) Sema3A 500 ng/mL in CoCl2-treated group. Dulbecco's modified Eagle's medium + 10% FBS–treated group were set to 100%. Data are presented as the mean ± SEM. Each experiment was repeated at least three separate times. *P < 0.05.
Sema3A Inhibited Experimental PVR in Rabbits
The development of PVR in each group was summarized in Table 2. Intravitreous injection of cultured ARPE19 cells alone induced extensive retinal detachment in 100% of rabbits (stages 4 and 5) (8/8). Injection of cultured RPE cells with 100 ng Sema3A resulted in localized retinal detachment in 75% (6/8) and extensive retinal detachment in 25% of rabbits (2/8). The injection of cultured RPE cells with 200 ng Sema3A resulted in focal traction, localized vascular changes in 62.5% (5/8), localized retinal detachment in 25% (2/8), and extensive retinal detachment in 12.5% of rabbits (1/8) (Fig. 9). 
Figure 9
 
Effects of Sema3A on experimental PVR in rabbit eyes. B-scan photographs and gross pathology photographs at 14 days after injection are shown in this figure. Photograph of a healthy vitreous body in vivo (A), RPE-injected eye (B), RPE with 100 ng Sema3A–injected eye (C), and RPE with 200 ng Sema3A-injected eye (D). Photograph from a healthy dissected, enucleated eye (E), RPE-injected eye (F), RPE with 100 ng Sema3A–injected eye (G), and RPE with 200 ng Sema3A–injected eye (H). Injection of RPE cells with Sema3A (100 ng) and Sema3A (200 ng) showed reduced PVR.
Figure 9
 
Effects of Sema3A on experimental PVR in rabbit eyes. B-scan photographs and gross pathology photographs at 14 days after injection are shown in this figure. Photograph of a healthy vitreous body in vivo (A), RPE-injected eye (B), RPE with 100 ng Sema3A–injected eye (C), and RPE with 200 ng Sema3A-injected eye (D). Photograph from a healthy dissected, enucleated eye (E), RPE-injected eye (F), RPE with 100 ng Sema3A–injected eye (G), and RPE with 200 ng Sema3A–injected eye (H). Injection of RPE cells with Sema3A (100 ng) and Sema3A (200 ng) showed reduced PVR.
Table 2
 
Summary of the Stage of PVR Formation in Different Treatment Groups
Table 2
 
Summary of the Stage of PVR Formation in Different Treatment Groups
Vitreous Injection PVR Formation, Stage 0–5
0 1 2 3 4 5
Normal control, n = 3 3 0 0 0 0 0
RPE cells only, n = 8 0 0 0 0 2 6
RPE + 100 ng Sema3A, n = 8 0 0 0 6 2 0
RPE + 200 ng Sema3A, n = 8 0 0 5 2 1 0
Discussion
The retinal pigment epithelium layer, localized between photoreceptor and choriocapillaris layers in the eyeball, plays important roles for the functions of the retina. 1,2,34 A failure of any one of the RPE's functions, such as secretion, phagocytosis, and so on, can lead to degeneration, abnormal proliferation of the retina and loss of visual function. 1 In healthy, developed eyes, RPE does not undergo mitosis; however, under some pathologic conditions, such as retinal detachment and ocular trauma, RPE cells become proliferative and migratory, which is known as proliferative vitreoretinopathy. 18 Another important dysfunction of RPE is abnormal secretion. As is known, the RPE produces and secretes a variety of growth factors to maintain retinal function, including VEGF, tissue inhibitor of matrix metalloprotease, fibroblast growth factors, platelet-derived growth factor, and PEDF. 1 Dysregulation of these factors can contribute to the pathogenesis of retinal diseases, especially retinal endothelial cell proliferation, which leads to neovascularization, such as in AMD, DR, and PVR. 1 Thus, inhibition of pathologic proliferation and restoration of the balance of growth factor secretion or inhibition of its malfunction are essential research topics. 
The neuropilins (Nrps) are multifunctional transmembrane receptors for several members of VEGF family, including VEGF165, VEGF164, and so on. 12,31 However, recent findings indicated that they have a much broader spectrum of ligands, TGF-β, platelet derived growth factor (PDGF), and integrins. 15 Sema3A is an alternative ligand for Nrp1 and activates signal transduction mediated by PlexA1-4, which associates with Nrp1. Much evidence indicates that Sema3A can affect the behavior of tumor cells by inhibiting the interaction of VEGF with Nrp1 and can inhibit tumor progression through the inhibition of tumor angiogenesis, metastasis, and tumor cell survival 32 and the guidance of axonal pathfinding during the development of the nervous system. 9 Moreover, HRPE cells and the ARPE19 cell line have already been reported to highly express Sema3A and Nrp1, but the relation between Sema3A and RPE is unclear. 28,35,36 In this study, we try to address the role of Sema3A in RPE behavior. 
In the present study, we showed the following evidence: (1) Sema3A inhibited activities of RPE, such as proliferation, migration, cell cycle, and inhibited the proliferative effects of RPE, both in vitro and in vivo, Sema3A could be a treatment alternative for RPE proliferative disease, such as PVR, (2) Sema3A did not affect the secretion of VEGF165 but can inhibit VEGF165-induced RPE activity by inhibiting JNK and p38MAPK phosphorylation, at least partially, through impeding the utilization of VEGF165, (3) Sema3A has a VEGFR-independent effect on RPE rather than impacted the VEGF165 function, such as inhibit migration, cell cycle arrest in normal culture condition, and (4) both VEGF165 and Sema3A can induce the upregulation of VEGFR2 and NRP1 receptors, while under the treatment of both stimulation agents, the receptors had no significant changes in protein level compared with separated treatment groups. 
Here, we presented our results and discussion in two parts: RPE behavior under normal culture conditions and in the presence of VEGF165 stimulation. As we showed in the results, Sema3A inhibited RPE migration and induces cell cycle arrest in general culture medium. These results are consistent with our previous study that used HUVEC as the target (unpublished data) and with other previous studies showing that Sema3A impairs cell adhesion and migration by negatively regulating integrin-mediated adhesion, which is followed by a collapse of the actin cytoskeleton. 10,37 Although the previous study showed that Sema3A promotes apoptosis of endothelial cells, 19 we only showed that Sema3A-induced cell cycle arrest, not apoptosis. This could be because the cells we used are immortalized cell lines, whereas the other studies used primary cells. 19 Although previous reports stated that Sema3A significantly induced the signaling pathways JNK and p38MAPK, 30,38 in our results, the phosphorylation of these two signaling pathways was activated but not significantly different compared with the control. These results explain why Sema3A did not inhibit proliferation in general culture medium. However, Sema3A can still inhibit RPE migration, which is important in proliferative eye diseases, and this is VEGFR-independent effects, which are affected by Sema3A. 
For the second part of our study, we investigated the effects of Sema3A on RPE when cocultured in the presence of the most potent angiogenesis factor, VEGF165. According to our results (ELISA results), Sema3A did not affect VEGF165 or PEDF secretion. In the presence of exogenous and endogenous VEGF165, Sema3A inhibited VEGF165-induced RPE behavior, including proliferation, migration, cell cycle arrest, JNK and p38MAPK phosphorylation, and cocultured HUVEC tube formation. In the present study, phosphorylation of JNK and p38MAPK was not completely inhibited by Sema3A. This could be explained by that JNK and p38MAPK signal pathways can be induced by a lot of growth factors, inflammatory factors and stress, such as epithelium growth factor, TGF-beta, interlukin-1, and so on. And several studies have demonstrated that VEGF induces the upregulation of several kinds of angiogenesis and inflammation factors. 39,40 In our study, Sema3A only inhibited the VEGF165 induced JNK and p38MAPK phosphorylation partially, but not all of the upregulated signal pathways induced by all of the factors, thus, the phosphorylation of JNK and p38MAPK are only partially inhibited. Besides the behavior study of the RPE cells, we also detected the expression pattern of the two crucial receptors, VEGFR2 and Nrp1, which are important in the function of VEGF165. In the present study, the results showed that both VEGF165 and Sema3A can induce the upregulation of these two receptors. While under the treatment of both stimulation factors, the two receptors remained higher comparing with the untreated group, but had no significant difference compared with the VEGF165- and Sema3A-treated group. As the ligands for VEGFR2 and Nrp1, exogenous application of VEGF165 and Sema3A can induce the increase of the receptors is as expected. While under the costimulation, although the protein level of the two receptors is slightly higher than separately treatment groups, there was no significant difference between the control groups. The comparable elevation of the two receptors can be explained by maintaining the balance of the two receptors, and restrict the utilization of VEGF165. Based on the previously published articles and our results, the mechanism of the Sema3A inhibit the function of VEGF165 most likely includes competition of Sema3A with VEGF165 for their b1 binding site on Nrp1. 41,42 As we stated before, VEGF family members mediate their downstream effects by binding to neuropilins and forming complexes with VEGF receptors, which is analogous to the Sema3-neuropilin-plexin complex. 43 While Nrp1 is not required for VEGF165 function, it can enhance the signaling of VEGF165 through VEGFR2, one of its receptor tyrosine kinases, 43 which was supported by our Western blot analysis, showing that VEGFR2 downstream signaling through JNK and p38MAPK phosphorylation was downregulated. Also, in spite of the upregulation of VEGFR2 under the treatment of VEGF165 and Sema3A, there is no increase of phosphorylation of JNK and p38MAPK, which could be the results of simultaneously increase of Nrp1 receptor, which blocks the utilization of VEGF165. Another study by Guttmann-Raviv et al. 19 also suggested that Sema3A can induce inhibitory effects on signaling mediated directly by Nrp1. And recent findings indicate that the range of growth factors that bind to Nrp1 is not limited to semaphorin and VEGF165; it also includes others, such as VEGF121, 44 placental growth factor, 45 hepatocyte growth factor, 46 and fibroblast growth factor 2. 47 All of these growth factors play important roles in proliferative and neovascular eye diseases. Thus, Sema3A can compete with these factors for binding to Nrp1 and inhibit their signal pathways, just as it inhibited VEGF165 function. Besides the in vitro study, we also demonstrated that Sema3A can inhibit PVR formation in a dose-dependent manner, which provide new information for clinical PVR treatment. In our study, we also used the coculture method to explore the effect of Sema3A on tube formation. As expected, Sema3A inhibited HUVECs tube formation in the presence of both endogenous and exogenous VEGF165 and we further confirmed that Sema3A can inhibit the function of VEGF165 not only in RPE cells, but also in HUVECs. Although the previous studies and our results showed the mechanisms of Sema3A on the function of VEGF165-induced RPE cells activities in a certain extent, more experiments need to be done to demonstrate that the affinities of sema3A and VEGF to Nrp1 and VEGFR2, to make clear spectrum on the binding studies. 
In summary, we comprehensively studied the effects of Sema3A on RPE. This study demonstrated for the first time that Sema3A, a chemorepellent guidance protein, can inhibit RPE activities under general culture conditions and in the presence of exogenous and endogenous VEGF165. These activities include proliferation, migration, cell cycle arrest, and JNK and p38MAPK signaling pathway phosphorylation via VEGF165. And Sema3A can inhibit proliferation of RPE in vivo. The encouraging results provide a useful therapeutic strategy and an adjunctive treatment strategy for the treatment of retinal proliferative diseases. 
Acknowledgments
We thank Xin Yu, PhD, for her help with flow cytometry detection. 
Supported by grants from the Peking University People's Hospital Research and Development Fund (RDB2012-24 [YB]), the National Basic Research Program of China (973 Program, 2011CB510200), and the National Natural Science Foundation of China (81200690 [YB]). 
Disclosure: Y. Bai, None; W. Yu, None; N. Han, None; F. Yang, None; Y. Sun, None; L. Zhang, None; M. Zhao, None; L. Huang, None; A. Zhou, None; F. Wang, None; X. Li, None 
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Footnotes
 YB and WY contributed equally to the work presented here and should therefore be regarded as equivalent authors.
Figure 1
 
Effects of Sema3A on RPE proliferation. Left, middle, and right: The statistical results of RPE proliferation at different time points (24 [left], 48 [middle], 72 hours [right]). Semaphorin 3A did not affect RPE proliferation in general culture medium, but can inhibit RPE proliferation in VEGF165-stimulated conditions and was significantly different than the VEGF165-treated group. Each experiment was repeated in 5 wells and was duplicated at least three times. Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01.
Figure 1
 
Effects of Sema3A on RPE proliferation. Left, middle, and right: The statistical results of RPE proliferation at different time points (24 [left], 48 [middle], 72 hours [right]). Semaphorin 3A did not affect RPE proliferation in general culture medium, but can inhibit RPE proliferation in VEGF165-stimulated conditions and was significantly different than the VEGF165-treated group. Each experiment was repeated in 5 wells and was duplicated at least three times. Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01.
Figure 2
 
Effects of Sema3A on the migration of RPE. Cell nuclei were stained with DAPI and are shown in blue dots. Cells from five random view fields were counted, and the average was used for statistical analysis. (A) Statistical analysis showing Sema3A inhibits RPE migration both in general culture medium and under VEGF-stimulated conditions; (B) 0% FBS–treated group; (C) 10% FBS–culture group; (D) VEGF-treated group; (E) Sema3A (500 ng/mL)-treated group; and (F) Sema3A (500 ng/mL)- and VEGF-treated group. The data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.
Figure 2
 
Effects of Sema3A on the migration of RPE. Cell nuclei were stained with DAPI and are shown in blue dots. Cells from five random view fields were counted, and the average was used for statistical analysis. (A) Statistical analysis showing Sema3A inhibits RPE migration both in general culture medium and under VEGF-stimulated conditions; (B) 0% FBS–treated group; (C) 10% FBS–culture group; (D) VEGF-treated group; (E) Sema3A (500 ng/mL)-treated group; and (F) Sema3A (500 ng/mL)- and VEGF-treated group. The data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.
Figure 3
 
Effects of Sema3A on RPE apoptosis and cell cycle. (A) The statistical results of Sema3A on RPE apoptosis, which showed no significant difference between the experimental group and the controls. (B) The statistical data for the RPE cell cycle (G2/M phase + S phase) distribution of different groups. (B) Sema3A induces cell cycle arrest both in general culture medium and under VEGF-stimulated conditions. Data are presented as the mean ± SEM. Each experiment was repeated three times. *P < 0.05; **P < 0.01.
Figure 3
 
Effects of Sema3A on RPE apoptosis and cell cycle. (A) The statistical results of Sema3A on RPE apoptosis, which showed no significant difference between the experimental group and the controls. (B) The statistical data for the RPE cell cycle (G2/M phase + S phase) distribution of different groups. (B) Sema3A induces cell cycle arrest both in general culture medium and under VEGF-stimulated conditions. Data are presented as the mean ± SEM. Each experiment was repeated three times. *P < 0.05; **P < 0.01.
Figure 4
 
Effects of Sema3A on RPE cells JNK and p38MAPK signaling pathway phosphorylation. Immunoblot image (A, C) and statistical analysis (B, D) for JNK, p-JNK, p38MAPK, and p-p38MAPK signaling pathways. (B) Shows that Sema3A inhibits p-JNK both in general culture medium and under VEGF-stimulated conditions, and (D) shows that Sema3A inhibits p-p38MAPK in the presence of VEGF stimulation. Western blot analyses were repeated three times, and qualitatively similar results were obtained. Data are presented as the mean ± SEM. *P < 0.05.
Figure 4
 
Effects of Sema3A on RPE cells JNK and p38MAPK signaling pathway phosphorylation. Immunoblot image (A, C) and statistical analysis (B, D) for JNK, p-JNK, p38MAPK, and p-p38MAPK signaling pathways. (B) Shows that Sema3A inhibits p-JNK both in general culture medium and under VEGF-stimulated conditions, and (D) shows that Sema3A inhibits p-p38MAPK in the presence of VEGF stimulation. Western blot analyses were repeated three times, and qualitatively similar results were obtained. Data are presented as the mean ± SEM. *P < 0.05.
Figure 5
 
Effects of Sema3A on VEGFR2, and NRP-1on RPE. (A, B) Statistical results of real-time PCR; (C, D) statistical results of Western blots; (E) the immunoblot image of the RPE cells under the treatment of different groups. All of the results were compared with control group (10% FBS–containing culture medium treatment group), and the results stated as the ratio fold. Real-time PCR analyses were repeated five times and Western blot analyses were repeated three times, and qualitatively similar results were obtained. Data are presented as the mean ± SEM. *P < 0.05 and **P < 0.01.
Figure 5
 
Effects of Sema3A on VEGFR2, and NRP-1on RPE. (A, B) Statistical results of real-time PCR; (C, D) statistical results of Western blots; (E) the immunoblot image of the RPE cells under the treatment of different groups. All of the results were compared with control group (10% FBS–containing culture medium treatment group), and the results stated as the ratio fold. Real-time PCR analyses were repeated five times and Western blot analyses were repeated three times, and qualitatively similar results were obtained. Data are presented as the mean ± SEM. *P < 0.05 and **P < 0.01.
Figure 6
 
The concentration of VEGF165 and PEDF detected in the cell culture supernatant among different groups. (AC) Statistical results of free VEGF165 concentration measured by ELISA assay at different time points (24 [A], 48 [B], and 72 hours [C]). (DF) Statistical results of free PEDF concentration measured by ELISA assay at different time points (24 [D], 48 [E], and 72 hours [F]). Each experiment was repeated in 6 wells and was duplicated at least three times. Data are presented as the mean ± SEM. *P < 0.05 and **P < 0.01.
Figure 6
 
The concentration of VEGF165 and PEDF detected in the cell culture supernatant among different groups. (AC) Statistical results of free VEGF165 concentration measured by ELISA assay at different time points (24 [A], 48 [B], and 72 hours [C]). (DF) Statistical results of free PEDF concentration measured by ELISA assay at different time points (24 [D], 48 [E], and 72 hours [F]). Each experiment was repeated in 6 wells and was duplicated at least three times. Data are presented as the mean ± SEM. *P < 0.05 and **P < 0.01.
Figure 7
 
Effects of Sema3A on VEGF165 and PEDF concentration in CoCl2-treated RPE. (A) Statistical results of the ratio of free VEGF165 concentration comparing with the control group, and (B) the statistical results of the ration of PEDF concentration after CoCl2 treatment of RPE for 12 hours compared with the control group. There was no significant difference between the Sema3A-treated group and the control (CoCl2-treated group). All of the results were compared with control group (10% FBS–containing culture medium treatment group), and the results stated as the ratio fold. Each experiment was repeated in 6 wells and was duplicated at least three times. Data are presented as the mean ± SEM.
Figure 7
 
Effects of Sema3A on VEGF165 and PEDF concentration in CoCl2-treated RPE. (A) Statistical results of the ratio of free VEGF165 concentration comparing with the control group, and (B) the statistical results of the ration of PEDF concentration after CoCl2 treatment of RPE for 12 hours compared with the control group. There was no significant difference between the Sema3A-treated group and the control (CoCl2-treated group). All of the results were compared with control group (10% FBS–containing culture medium treatment group), and the results stated as the ratio fold. Each experiment was repeated in 6 wells and was duplicated at least three times. Data are presented as the mean ± SEM.
Figure 8
 
Effects of Sema3A on RPE tube formation. The length of tube branches per view field was measured. (A) The statistical analysis. Microphotographs are representative of tube-like structure generation after 10 hours of incubation. (B) Ten percent FBS–treated group; (C) CoCl2-treated group; (D) Sema3A 500 ng/ml–treated group; (E) Sema3A 250 ng/mL in CoCl2-treated group; and (F) Sema3A 500 ng/mL in CoCl2-treated group. Dulbecco's modified Eagle's medium + 10% FBS–treated group were set to 100%. Data are presented as the mean ± SEM. Each experiment was repeated at least three separate times. *P < 0.05.
Figure 8
 
Effects of Sema3A on RPE tube formation. The length of tube branches per view field was measured. (A) The statistical analysis. Microphotographs are representative of tube-like structure generation after 10 hours of incubation. (B) Ten percent FBS–treated group; (C) CoCl2-treated group; (D) Sema3A 500 ng/ml–treated group; (E) Sema3A 250 ng/mL in CoCl2-treated group; and (F) Sema3A 500 ng/mL in CoCl2-treated group. Dulbecco's modified Eagle's medium + 10% FBS–treated group were set to 100%. Data are presented as the mean ± SEM. Each experiment was repeated at least three separate times. *P < 0.05.
Figure 9
 
Effects of Sema3A on experimental PVR in rabbit eyes. B-scan photographs and gross pathology photographs at 14 days after injection are shown in this figure. Photograph of a healthy vitreous body in vivo (A), RPE-injected eye (B), RPE with 100 ng Sema3A–injected eye (C), and RPE with 200 ng Sema3A-injected eye (D). Photograph from a healthy dissected, enucleated eye (E), RPE-injected eye (F), RPE with 100 ng Sema3A–injected eye (G), and RPE with 200 ng Sema3A–injected eye (H). Injection of RPE cells with Sema3A (100 ng) and Sema3A (200 ng) showed reduced PVR.
Figure 9
 
Effects of Sema3A on experimental PVR in rabbit eyes. B-scan photographs and gross pathology photographs at 14 days after injection are shown in this figure. Photograph of a healthy vitreous body in vivo (A), RPE-injected eye (B), RPE with 100 ng Sema3A–injected eye (C), and RPE with 200 ng Sema3A-injected eye (D). Photograph from a healthy dissected, enucleated eye (E), RPE-injected eye (F), RPE with 100 ng Sema3A–injected eye (G), and RPE with 200 ng Sema3A–injected eye (H). Injection of RPE cells with Sema3A (100 ng) and Sema3A (200 ng) showed reduced PVR.
Table 1
 
Summary of Flow Cytomery Data of Cell Cycle Measured by PI
Table 1
 
Summary of Flow Cytomery Data of Cell Cycle Measured by PI
Group 24 h 48 h 72 h
Mean SEM Mean SEM Mean SEM
10% FBS 29.8 3.4 33.1 3.1 34.1 4.2
3A, 250 ng/mL 24.1 3.1 26.1 3.3 28.4 3.9
3A, 500 ng/mL 22.7 3.9 20.5 2.5 24.1 4
VEGF 36.4 4.1 38.2 4.4 32.1 2.6
VEGF+3A, 250 ng/mL 28.8 2.7 29.1 2.7 27.4 2.1
VEGF+3A, 500 ng/mL 26.2 1.9 26.7 3.0 26.0 1.8
Table 2
 
Summary of the Stage of PVR Formation in Different Treatment Groups
Table 2
 
Summary of the Stage of PVR Formation in Different Treatment Groups
Vitreous Injection PVR Formation, Stage 0–5
0 1 2 3 4 5
Normal control, n = 3 3 0 0 0 0 0
RPE cells only, n = 8 0 0 0 0 2 6
RPE + 100 ng Sema3A, n = 8 0 0 0 6 2 0
RPE + 200 ng Sema3A, n = 8 0 0 5 2 1 0
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