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Inhibition of RACK1 ameliorates choroidal neovascularization formation in vitro and in vivo
Experimental and Molecular Pathology 100 (2016) 451–459
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Experimental and Molecular Pathology journal homepage: www.elsevier.com/locate/yexmp
Inhibition of RACK1 ameliorates choroidal neovascularization formation in vitro and in vivo Xiaojuan Liu a,c,1, Manhui Zhu b,c,1, Xiaowei Yang b,c, Ying Wang b,c, Bai Qin b,c, Chen Cui b,c, Hui Chen b,c,⁎,2, Aimin Sang b,c,⁎,2 a b c
Department of Pathogen Biology, Medical College, Nantong University, Nantong, Jiangsu 226001, China Department of Ophthalmology, Affiliated Hospital of Nantong University, Nantong, Jiangsu 226001, China Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Medical College, Nantong University, Nantong, Jiangsu 226001, China
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Article history: Received 17 February 2016 and in revised form 18 April 2016 Accepted 20 April 2016 Available online 22 April 2016 Keywords: Choroidal neovascularization (CNV) Age-related macular degeneration (AMD) Receptor for activated C-kinase 1 (RACK1) Vascular endothelial growth factor (VEGF)
a b s t r a c t Choroidal neovascularization (CNV) occurs as a result of age-related macular degeneration (AMD) and causes severe vision loss among elderly patients. The receptor for activated C-kinase 1 (RACK1) serves as a scaffold protein which is recently found to promote angiogenesis. However, the impact of RACK1 on the vascular endothelial growth factor (VEGF) expression in endothelial cells and subsequent choroidal angiogenesis formation remains to be elucidated. In this study, we found that RACK1 and VEGF expression increased, and reached the peak at 7 d in mouse CNV model by laser application. Furthermore, on RPE/choroid cryosections, RACK1 co-localized with CD31, suggesting that RACK1 was expressed in endothelial cells. In vitro, RF/6A cell hypoxia model showed that RACK1 expression was up-regulated in parallel with hypoxia-induced factor 1 (HIF-1α) and VEGF expression, reaching the peak at 6 h. Silencing of RACK1 suppressed the invasion and tube formation activity of RF/ 6A cells in ARPE-19 and RF/6A co-culture system, possibly through VEGF signal pathway. Overexpression of RACK1 showed the opposite effect. Intravitreal injection of anti-RACK1 monoclonal antibody predominantly decreased RACK1 and VEGF expression in mouse laser-induced CNV model. Meanwhile, anti-RACK1 monoclonal antibody intravitreal injection also decreased incidence of CNV and leakage area. These data indicated that RACK1 promoted CNV formation via VEGF pathway. Additionally, anti-RACK1 monoclonal antibody significantly decreased CNV in mouse model and may have therapeutic potential in human CNV. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the industrialized world (Wong et al., 2008). There are two forms of AMD, one is neovascular or wet AMD, the other is non-neovascular or dry AMD. Choroidal neovascularization (CNV) is characterized as wet AMD (Barbazetto et al., 2010), which refers to the growth of neovasculature derived from the choroid through breaks in Bruch's membrane into sub-retinal pigment epithelium (RPE) or sub-retinal spaces (Das and McGuire, 2003). In a study for individuals who are diagnosed with early or intermediate AMD at primary visit, approximately 10% develop CNV over a median follow-up period of 6.3 years (Clemons et al., 2005). Thus, there is an urgent need to clarify the pathophysiology of CNV and to identify viable treatment strategies that will arrest disease-induced vision loss.
⁎ Corresponding authors at: Department of Ophthalmology, Affiliated Hospital of Nantong University, Nantong, Jiangsu 226001, China. E-mail addresses: [email protected] (H. Chen), [email protected] (A. Sang). 1 Xiaojuan Liu and Manhui Zhu contributed equally to this work. 2 Hui Chen and Aimin Sang contributed equally to this work.
http://dx.doi.org/10.1016/j.yexmp.2016.04.004 0014-4800/© 2016 Elsevier Inc. All rights reserved.
Intravitreal injection of vascular endothelial growth factor (VEGF) antagonists is the major treatment for CNV currently. However, it is important to figure out that there is controversy regarding the safety of anti-VEGF intraocular injection (Elice et al., 2009; Eremina et al., 2008). These drugs are also known for their limited indications and insufficient efficacy. The cost, discomfort of, and time spent receiving monthly injections hinder the application of anti-VEGF therapy. Moreover, a portion of patients do not respond to the treatment well (Wickremasinghe et al., 2011). Thus, searching for the molecules to regulate VEGF expression and function is essential for the treatment development of AMD. Receptor for activated protein kinase C1 (RACK1) is upregulated in vascular endothelial cells during angiogenesis (Berns et al., 2000). Silencing of RACK1 dramatically attenuates tumor-associated angiogenesis (Wang et al., 2011a). RACK1, a 36 kDa protein containing 7 internal Trp-Asp 40 (WD40) repeats, is originally cloned as an anchoring protein for protein kinase C (PKC) (McCahill et al., 2002), stabilizing the active form of PKC and permitting its translocation to different intracellular sites (Ron et al., 1994; Ron and Mochly-Rosen, 1994). Recently, RACK1 is found to play a regulatory role in VEGF-VEGFR1-dependent cell migration via activation of PI3K/Akt and small GTPase Rac1 signaling
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pathways (Wang et al., 2011b). RACK1 silencing suppresses tumor cell growth and invasion in vitro (Wang et al., 2011a). However, whether RACK1 participates in CNV is unknown. Our research group has already successfully constructed adult C57BL/6J (B6) mouse CNV model and cell model of neovascularization (Peng et al., 2013; Wang et al., 2014). In the present study, we firstly detected the expression of RACK1 and VEGF in mouse CNV and cell hypoxia models. Then we investigated the role of RACK1 in CNV formation via RACK1 specific siRNA and monoclonal antibody to determine the function of RACK1 in CNV formation and whether intravitreal injection of anti-RACK1 monoclonal antibody could alleviate CNV formation. The data can supply novel molecular target for CNV treatment. 2. Materials and methods 2.1. Animals All experimental procedures were performed in accordance with the requirements of Animal Welfare committee of the Medical College of Nantong University. The research protocol for the use of animals has been approved by the Center for Laboratory Animals at Nantong University (Nantong, Jiangsu, China).
in 200 μl fresh RPMI 1640 medium with 0.5% FBS and were placed into the upper chamber. After 48 h, removing the non-invading cells in the upper chambers with a cotton swab, invaded cells on the lower surface of the porous membrane were fixed with 4% paraformaldehyde for 15 min, stained with 0.1% crystal violet for 30 min, and photographed under a light microscope (Olympus, Tokyo, Japan). Five random fields (× 200) were chosen in each insert, and the cell number was quantified manually. 2.5. Tube formation assay Treated and untreated ARPE-19 cells were plated in Transwells with 8 μm pore-size inserts, which were put in wells where RF/6A cells had been plated. Briefly, matrigel was thawed and laid into 24-well culture plates to a total volume of 200 μl in each well. Plates were stored at 37 °C for 30 min to form a gel layer. After gel polymerization, 2 × 104 RF/6A cells were seeded in each well and incubated with fresh RPMI 1640 medium supplemented with 0.5% FBS for 24 h at 37 °C in humidified air with 5% CO2. 2 × 104 ARPE-19 cells which had been subjected to transfection for 24 h were seeded into the upper chamber. The closed networks of tubes in each well were observed with an inverted phasecontrast microscope (Olympus, Tokyo, Japan). Incomplete networks were excluded. The experiments were performed in triplicate and five fields from each chamber were counted and averaged.
2.2. Cell culture 2.6. Laser-induced CNV Human retinal pigment epithelial cell line ARPE-19 and rhesus choroidal endothelial cell line RF/6A were purchased from American Type Culture Collection (ATCC, Manassas, VA) and were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) (Gibco, Rockville, MD) and 100 U/ml penicillin–streptomycin mixtures (Gibco) at 37 °C in 5% CO2. The culture medium was changed every 2 days. An in vitro RF/6A cell chemical hypoxia model was established by adding 300 μM cobalt chloride (CoCl2) to culture medium, and cells were harvested after 1, 3, 6, 12 and 24 h. Cells without CoCl2 treatment were regarded as normal control.
Adult C57BL/6J (B6) mice were anesthetized by intraperitoneal injection with 3% chloral hydrate, and pupils were dilated with topical administration of tropic amide phenylephrine eye drops (Santen, Osaka, Japan). Four burns were made by laser photocoagulation (647.1 nm; 50 mm spot size; 0.05 s duration; 360 mW) in each retina with a hand-held contact fundus lens (Ocular Instruments, Bellevue, WA) in 3, 6, 9, 12 o'clock positions between retinal vessels in a
2.3. Transfections A total of 2 × 106 RF6A cells per well were seeded in 6-well plates and allowed to grow overnight. Transfection of RACK1 siRNA (5′GCTGAAGACCAACCACATT-3′, Ribobio, Guangzhou, China) or FlagRACK1 was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the manufacturer's protocol. In brief, for 6-well plates, 4 μg DNA was mixed with 10 μl Lipofectamine 2000 at a final concentration of 2 μg DNA/ml, dissolved in RPMI 1640 without serum, the resulting complex was incubated at room temperature (RT) for 20 min to generate transfection mixture and was added to cells, which were then incubated for 4 to 6 h. Next, the cells were washed with RPMI 1640 and incubated in RPMI 1640 with 10% FBS for a further 48 h. Then the cells were collected for western blot analysis. 2.4. In vitro cell invasion assay To test the effect of RACK1 on invasion and tube formation of RF/6A cells, we applied ARPE-19 and RF/6A cell co-culture model. For invasion assay, RF/6A cells were plated in Costar Transwells (Costar, Corning, NY) with 8 μm pore-size inserts, which were put in 24-well plates where ARPE-19 cells was plated. Briefly, a total of 2 × 104 ARPE-19 cells were resuspended in 600 μl fresh RPMI 1640 medium supplemented with 10% FBS and transferred into the lower chamber. Cells were incubated for 24 h at 37 °C, then transfected with RACK1 siRNA or Flag-RACK1 before 8 μm pore-size inserts were placed in the wells. Cells with no treatment served as normal control. Matrigel (Sigma-Aldrich, Saint Louis, MO) 200 μl was added to 24-well plates and incubated at 37 °C for 30 min to form gels. A total of 2 × 104 RF/6A cells were resuspended
Fig. 1. Chemical hypoxia induces RACK1, HIF-1α and VEGF expression in RF/6A cells in vitro. (A) Western blots show RACK1, HIF-1α and VEGF expression. (B) Histogram shows densitometric analysis of the average levels to β-actin. *P b 0.05, compared with normal control. Results are mean ± SD, n = 3 in each group.
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peripapillary area of both eyes. Only burns that produced a bubble, indicating the rupture of the Bruch's membrane, were counted in the study. All mice were randomly divided into five groups based on days (d) following laser treatment (normal, 1 d, 3 d, 7 d, 14 d). For the western blot, each group contained 24 mice with laser treatment and 15 control mice without laser treatment belonged to control group. In control and post-laser 7 d groups, another 45 mice (ninety eyeballs) each were used for choroidal flat mount, immunofluorescence, and histopathology. 2.7. Western blot analysis To detect the protein level of molecules, we extracted mouse RPEchoroid complexes and retina from 3 mice at 1, 3, 7, 14 d after laser injury and in vitro cell lysates. Proteins and a molecular weight marker were separated by 10% SDS-PAGE, and transferred to a PVDF membrane. The membrane was then incubated with rabbit anti-RACK1 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-VEGF (1:500; Santa Cruz Biotechnology), mouse anti-HIF-1α (1:1000; Chemicon, Temecula, CA). The antibodies were incubated in blocking 5% skim milk over night at 4 °C and reacted with HRP-conjugated secondary antibodies (1:2000; Thermo Scientific, Rockford, IL) at 37 °C for 2 h. Extensive washes in 0.05% Tween-20 in TBS and followed by incubation with anti-β-actin (1:1000; Sigma-Aldrich, Saint Louis, MO).
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The blots were then incubated with chemifluorescent reagent ECL (Thermo Scientific, Rockford, IL) and exposed to X-ray film in the dark. The intensity of β-actin signal was used as an endogenous control and quantified the band optical density using Image J (National Institutes of Health, Bethesda, MD). 2.8. Immunofluorescence RACK1 tissue localization was examined on 8 μm cryosections (on day 7 after laser photocoagulation). The cryosections were blocked with 1% BSA for 4 h at RT, then incubated with rabbit RACK1 antibody (1:50; Santa Cruz Biotechnology) and mouse CD31 antibody (1:50, Abcam, Cambridge, MA) at 4 °C overnight. For CD31 staining, antigen retrieval was obtained through heated water bath at 97 °C for 10 min. Thereafter, the slides were stained with Alexa Fluor 488 goat antimouse IgG, Alexa Fluor 546 goat anti-rabbit IgG (1:200; Invitrogen, Carlsbad, CA) and Hoechst (1:2000; Sigma-Aldrich). The photomicrographs were taken by a digital high sensitivity camera (Hamamatsu, ORCA-ER C4742-95, Japan). 2.9. Intravitreal injection The intravitreal injection of 1 μl of anti-RACK1 monoclonal antibody (200 ng/ml) or vehicle (5% glucose solution, 5% GS) was given on day 1
Fig. 2. RACK1 silencing inhibits invasion and tube formation of RF/6A cells. (A) RACK1 and VEGF expression was detected by western blot in RF/6A cells following RACK1 siRNA transfection after 48 h. (B) Histogram shows densitometric analysis of the average levels for RACK1 and VEGF. β-actin was used as loading control. Cells transfected with scramble siRNA were used as negative control. (C) Representative photographs of invasive RF/6A cells (200× magnification). (D) The average number of invasive RF/6A cells per field. *P b 0.01, RACK1 siRNA versus Control. (E) Representative photographs of tube formation (40× magnification). (F) The average number of closed networks of tubes per field. *P b 0.01, RACK1 siRNA versus Control.
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and mice were killed on day 7 after laser treatment. Control group represented laser-induced CNV without any injection. 2.10. Choroidal flat mount On day 7 after laser coagulation, thirty eyes (6 eyes from 3 mice/ each group) were subject to choroidal flat mounts. The eyes were enucleated and immediately fixed in 4% paraformaldehyde (Guoyao Group of Chemical Reagents, Beijing, China) in pH 7.3 PBS for 1 h. Under a biopsy microscope, the anterior segments were wiped out, as well as the neurosensory retinas were detached and separated from the optic nerve head. The remaining eyecups were washed with cold ICC buffer (0.5% BSA, 0.2% Tween-20, and 0.1% Triton X100) in PBS. A 10 mg/ml solution of 40, 60-diamidino-2phenylindole (DAPI) (1:500), 1 mg/ml solution of Alexa Fluor 568 conjugated isolectin-B4 (1:100), and 0.2 U/ml solution of Alexa Fluor 488 conjugated phalloidin (1:100; Invitrogen-Molecular Probes, Eugene, OR) were prepared in ICC buffer. The eyecups were incubated with the above fluorescent dyes in a humidified chamber at 4 °C with gentle shaking for 4 h, followed by washing with cold ICC buffer. Four or five radial cuts were made toward the optic nerve head for flat mounting the sclera/choroid/RPE complexes together with the gel (Gel-mount; Biomedia Corp. Foster City, CA). The samples were covered and sealed for confocal microscopic analysis.
2.11. Confocal microscopy Multiplane z-series images were collected using a confocal microscope (SP2; Leica, Exton, PA) with a 63×, 1.25 numerical aperture and oil-immersion objective. All images were collected at a 1024 × 1024 pixel resolution and a depth of 8 bits per channel. Voxel dimensions were 0.3662 μm for x- and y-axis and 0.4884 μm for z-axis. Fluorescent signals for DAPI (400–500 nm), Alexa Fluor 488 (500–550 nm), and Alexa Fluor 568 (560–660 nm) were collected using a sequential scan mode. 2.12. Fundus fluorescein angiography To confirm the inhibitory effect of anti-RACK1 monoclonal antibody on CNV formation, fluorescein angiography was performed on day 7 after laser photocoagulation. The development of CNV was evaluated using a digital fundus camera connected to a slit lamp delivery system (Kanghua, Chongqing, China), captured 3 min after 0.3 ml of 2% fluorescein sodium (Alcon Laboratories, Irvine, CA) was injected into the intraperitoneal cavity of mice as previously described (Wang et al., 2014). Angiograms were graded as follows: no staining, Score 0; slightly stained, score 1; moderately stained, score 2; strongly stained, score 3 (Takehana et al., 1999). The area of CNV lesion was measured three times and averaged using Image J (USA National Institutes of Health, Bethesda, MD) (Xie et al., 2011).
Fig. 3. RACK1 overexpression enhances RF/6A cell invasion and tube formation. (A) RACK1 and VEGF expression was detected by western blot in RF/6A cells following Flag-RACK1 transfection after 48 h. (B) Histogram shows densitometric analysis of the average levels for RACK1 and VEGF. β-actin was used as loading control. Cells transfected with Flag were used as negative control. (C) Representative photographs of invasive RF/6A cells (200× magnification). (D) The average number of invasive RF/6A cells per field. *P b 0.01, Flag-RACK1 versus Control. (E) Representative photographs of tube formation (40× magnification). (F) The average number of closed networks of tubes per field. *P b 0.01, Flag-RACK1 versus Control.
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laser photocoagulation. The sections were cover slipped by mounting medium. Serial sections were examined, and the samples that contained the thickest or widest lesions among the set of specimens that was obtained for each instance of CNV were estimated. Slices with HE staining were examined using a light microscope (Olympus, Tokyo, Japan). IPP 6.0 was used to calculate the maximum thicknesses and lengths of each CNV lesion. 2.14. Statistical analysis All values were presented as means ± standard deviation (SD). One-way ANOVA was used for statistical comparisons of multiple groups. Descriptive statistics were performed using Stata statistical software version 11.0. P b 0.05 was considered statistically significant. Each experiment consisted of at least three replicates. 3. Results 3.1. HIF-1α, VEGF and RACK1 expression is up-regulated in RF/6A cells under hypoxic condition To further explore the functions of RACK1 in CNV, we examined the expression of RACK1, VEGF and HIF-1α in RF/6A cells after CoCl2 exposure. VEGF is a target gene of HIF-1α (Medici and Olsen, 2012; Qi et al., 2014). As expected, western blot showed that the expression of HIF-1α, VEGF and RACK1 was up-regulated in similar time-dependent manner under hypoxia condition (Fig. 1). 3.2. RACK1 silencing inhibits RF/6A invasion and tube formation
Fig. 4. RACK1, VEGF expression is up-regulated after CNV formation. The mouse CNV model was performed by laser photocoagulation. (A) Leakage area of CNV was examined by FFA. (B) RACK1 and VEGF protein level was detected by western blot. β-actin was used as loading control. (C) Histogram shows densitometric analysis of the average levels for RACK1 and VEGF to β-actin. *P b 0.05; significantly different from normal control.
RACK1 siRNAs were transfected in RF/6A cells after hypoxia. The results showed that RACK1 expression was down-regulated by 51 ± 8% (Fig. 2A and B). Accordingly, VEGF expression was inhibited by RACK1 siRNA transfection. When RACK1 expression was inhibited, RF/6A cell invasion and tube formation capability was reduced (Fig. 2C, D, E and F), showing that RACK1 may participate in CNV formation.
2.13. Histopathology
3.3. RACK1 overexpression promotes RF/6A invasion and tube formation
Eight eyes were enucleated in each group, and 8 μm cryosections were prepared for hematoxylin and eosin (HE) staining on day 7 after
Flag-RACK1 was constructed and transfected into RF/6A cells. RACK1 and VEGF expression in Flag-RACK1 transfection group markedly
Fig. 5. RACK1 is localized in endothelial cells. Cellular localization of RACK1 in retina/choroid cryosections was determined by double immunostaining accompanied with endothelial marker CD31. Scale bar = 50 μm.
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increased (1.93 ± 0.09 fold and 1.81 ± 0.12 fold, respectively) compared to control and Flag transfection groups (Fig. 3A and B). The transwell assay showed that up-regulation of RACK1 elevated invasion and tube formation ability of RF/6A cells (Fig. 3C, D, E, and F). 3.4. RACK1, VEGF expression increases after laser photocoagulation To investigate the expression of RACK1 in CNV, we extracted protein of RPE-choroid complexes and retinas for western blot analysis. After laser injury, RACK1 expression was up-regulated, reached the peak at 7 d, showing a similar change tendency with VEGF (Fig. 4). 3.5. RACK1 is localized in vascular endothelium inside CNV To identify the cellular localization of RACK1 inside CNV site, we performed double immunostaining of RACK1 with CD31, a marker for endothelial cells. Inside CNV, RACK1 was localized in vascular endothelium (Fig. 5). 3.6. Anti-RACK1 monoclonal antibody alleviates the leakage of CNV To further investigate the functions of RACK1 in CNV formation, we used intravitreal injection of RAK1 monoclonal antibody in mouse CNV model. Fluorescein angiogram assay showed that the leakage area of CNV was smaller in anti-RACK1 injection group than in vehicle injection group on day 7 after laser photocoagulation (Fig. 6A). RACK1 monoclonal antibody decreased the number of spots with strong dye staining (score 2 or more) and increased the number of spots with weak staining (score 0 or 1) (Fig. 6B). 3.7. Anti-RACK1 monoclonal antibody suppresses RACK1 and VEGF expression Then we explored RACK1 and VEGF expression following antiRACK1 monoclonal antibody intravitreal injection with laser injury.
Fig. 7. RACK1 monoclonal antibody intravitreal injection down-regulates RACK1 and VEGF expression. (A) Western blot analysis showed that RACK1 and VEGF expression in the RPE-choroid complex and retinal tissues at day 7 after laser coagulation. (B) Quantification graphs for RACK1 and VEGF. Data of relative protein level to β-actin are presented as mean ± SD. *P b 0.01, anti-RACK1 versus vehicle.
RACK1 protein level in RPE-choroid complexes and retina was reduced dramatically in anti-RACK1 injection group compared with control and vehicle injection groups. Furthermore, VEGF expression showed a similar decreasing change with RACK1 (Fig. 7A and B), suggesting that RACK1 might promote CNV formation via up-regulating VEGF expression.
Fig. 6. RACK1 monoclonal antibody intravitreal injection reduces leakage area of CNV. (A) Hyper fluorescent leakage surrounding the laser spots was relative weak leakage (grade 1) in anti-RACK1 injection mice retina. (B) The intensity of FFA leakage grade in control, vehicle and anti-RACK1 injection groups. (C) Comparison of semi-quantitative leakage score between vehicle and anti-RACK1 injection mice. The mean leakage score of CNV in control group was set at 100. *P b 0.01.
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3.8. Anti-RACK1 monoclonal antibody reduces the areas of CNV Phalloidin labeling examined actin bundles of RPE cells, forming a tightly packed, uniform hexagonal monolayer (Fig. 8B). DAPI labeling identified the nuclei (Fig. 8C, G, and K). At 7 d, a well-defined radial array of isolectin labeled vessels was visible inside CNV in control and vehicle groups (Fig. 8A and E). The merged images showed that phalloidin labeled RPE cells covered the area of CNV (Fig. 8D, H, and L). Compared to control and vehicle injection groups, the areas of CNV were smaller and the vessel tubes were fewer in anti-RACK1 injection group (Fig. 8A, E, I). On day 7 after laser photocoagulation, histopathology analysis showed that anti-RACK1 monoclonal antibody treatment significantly decreased the thickness and length of retinal lesions caused by CNV (Fig. 9).
4. Discussion Our study demonstrated that up-regulation of RACK1 promoted invasion and tube formation capability of RF/6A cells to boost angiogenesis. Treatment with anti-RACK1 monoclonal antibody significantly decreased the dimensions of laser-induced CNV in mouse model, suggesting that RACK1 might be an important molecular target for antiangiogenesis therapies.
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Many growth factors such as VEGF, placental growth factor (PIGF), platelet-derived growth factor β (PDGF-β), and pigment epitheliumderived factor (PEDF) (Campa et al., 2010), are involved in different stages of CNV. Among these growth factors, VEGF is the most important (Nagai et al., 2014). Other mediators related to VEGF upregulation such as transforming growth factor beta (TGF-β), angiostatin and hypoxiainducible factor (Novack, 2008), are targets for the treatment of CNV. The RPE with its highly organized basement membrane, known as Bruch's membrane, is essential for normal functioning of the retina (Lakkaraju, 2012; Strauss, 2005). A break of Bruch's membrane is necessary for CNV to develop. In our experiments, we used ARPE-19 and RF/ 6A cells to imitate in vitro neovascularization. Anti-RACK1 monoclonal antibody was conducted on laser-induced CNV mouse model, which is well established for investigating ocular angiogenesis (Du et al., 2013; Grossniklaus et al., 2010; Li et al., 2015). RACK1 is found to co-localize with activated PKCβI and PKCβII, suggesting specific binding of RACK1 to PKCβ (Ron et al., 1995). VEGFinduced tumor angiogenesis and tumor growth in vivo are PKCβ dependent (Yoshiji et al., 1999). Inhibition of PKCβ significantly suppresses VEGF-induced neovascularization in a mouse model of hepatocellular carcinoma cells (Yoshiji et al., 1999). Furthermore, RACK1 is massively induced in subconfluent and contact-inhibited bovine endothelial cells, during angiogenesis in vitro, active phases of the murine ovarian cycle, human tumor angiogenesis, and in cancer cells in vivo as assessed by quantitative PCR and in situ hybridization (Berns et al., 2000). Our
Fig. 8. RACK1 monoclonal antibody intravitreal injection inhibits CNV formation. Representative images of flat mount preparations from control and RACK1 antibody injection groups 7 d after laser photocoagulation. (A–H) The area of the vessels in control and PBS injection groups. (I–L) The area of the vessels was smaller in anti-RACK1 injection mice compared with control mice. Mouse RPE-choroid flat mount preparations from laser-injured regions were fluorescently labeled with F-actin marker phalloidin (red), endothelial and microglia cell marker isolectin-B4 (green), and nuclear marker DAPI (blue; merged images) (63× magnification). Scale bars = 100 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 9. RACK1 monoclonal antibody intravitreal injection inhibits CNV formation. HE stains images of CNV. (A) Normal mice retinal and choroid structure. (B–D) Each photograph showed the central area of CNV inside mice retinal and choroid in control, vehicle or anti-RACK1 groups. Scale bar = 100 μm (RPE: retinal pigment epithelium; OS: outer segment; IS: inner segment; ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; GC: ganglion cell layer). (E) Statistical analysis of data presented from B to D, n = 12–16 spots *P b 0.01. Black dash lines represented the edge of CNV.
study also reveals that RACK1 expression is up-regulated during CNV process, in line with the previous researches. We explore the effect of RACK1 on neovascularization both in vitro and in vivo, showing that silencing of RACK1 reduces invasion and tube formation of RF/6A cells. Additionally, anti-RACK1 monoclonal antibody can reduce the leakage of CNV lesions. The data demonstrate that RACK1 promotes CNV formation. The mechanism of promoting angiogenesis role of RACK1 could be that RACK1 participates in VEGF/Flt1triggered vascular endothelial cell migration (Wang et al., 2011b). RACK1 is associated with tumor angiogenesis in a variety of researches (Dirkx et al., 2006; Leek et al., 1999). It is necessary to investigate the mechanism underlying angiogenesis function of RACK1 in more detail. Our findings reveal the promotion function of RACK1 in CNV formation. We anticipate that targeted therapy toward RACK1 might be promises to interventions of pathological processes in CNV. Further investigations are necessary to clarify the potential mechanisms of RACK1 participation in CNV process. Conflicts of interest The authors declare that they have no conflict of interest. Acknowledgments The study was supported by National Natural Science Foundation of China (No. 81401365); Nantong science and technology project (MS12015056); a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). References Barbazetto, I.A., et al., 2010. Incidence of new choroidal neovascularization in fellow eyes of patients treated in the MARINA and ANCHOR trials. Am J. Ophthalmol. 149, 939–946, e1.
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X. Liu et al. / Experimental and Molecular Pathology 100 (2016) 451–459 Ron, D., et al., 1994. Cloning of an intracellular receptor for protein kinase C: a homolog of the beta subunit of G proteins. Proc. Natl. Acad. Sci. U. S. A. 91, 839–843. Ron, D., et al., 1995. C2 region-derived peptides inhibit translocation and function of beta protein kinase C in vivo. J. Biol. Chem. 270, 24180–24187. Strauss, O., 2005. The retinal pigment epithelium in visual function. Physiol. Rev. 85, 845–881. Takehana, Y., et al., 1999. Suppression of laser-induced choroidal neovascularization by oral tranilast in the rat. Invest. Ophthalmol. Vis. Sci. 40, 459–466. Wang, F., et al., 2011a. Downregulation of receptor for activated C-kinase 1 (RACK1) suppresses tumor growth by inhibiting tumor cell proliferation and tumor-associated angiogenesis. Cancer Sci. 102, 2007–2013. Wang, F., et al., 2011b. RACK1 regulates VEGF/Flt1-mediated cell migration via activation of a PI3K/Akt pathway. J. Biol. Chem. 286, 9097–9106.
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Contents lists available at ScienceDirect
Experimental and Molecular Pathology journal homepage: www.elsevier.com/locate/yexmp
Inhibition of RACK1 ameliorates choroidal neovascularization formation in vitro and in vivo Xiaojuan Liu a,c,1, Manhui Zhu b,c,1, Xiaowei Yang b,c, Ying Wang b,c, Bai Qin b,c, Chen Cui b,c, Hui Chen b,c,⁎,2, Aimin Sang b,c,⁎,2 a b c
Department of Pathogen Biology, Medical College, Nantong University, Nantong, Jiangsu 226001, China Department of Ophthalmology, Affiliated Hospital of Nantong University, Nantong, Jiangsu 226001, China Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Medical College, Nantong University, Nantong, Jiangsu 226001, China
a r t i c l e
i n f o
Article history: Received 17 February 2016 and in revised form 18 April 2016 Accepted 20 April 2016 Available online 22 April 2016 Keywords: Choroidal neovascularization (CNV) Age-related macular degeneration (AMD) Receptor for activated C-kinase 1 (RACK1) Vascular endothelial growth factor (VEGF)
a b s t r a c t Choroidal neovascularization (CNV) occurs as a result of age-related macular degeneration (AMD) and causes severe vision loss among elderly patients. The receptor for activated C-kinase 1 (RACK1) serves as a scaffold protein which is recently found to promote angiogenesis. However, the impact of RACK1 on the vascular endothelial growth factor (VEGF) expression in endothelial cells and subsequent choroidal angiogenesis formation remains to be elucidated. In this study, we found that RACK1 and VEGF expression increased, and reached the peak at 7 d in mouse CNV model by laser application. Furthermore, on RPE/choroid cryosections, RACK1 co-localized with CD31, suggesting that RACK1 was expressed in endothelial cells. In vitro, RF/6A cell hypoxia model showed that RACK1 expression was up-regulated in parallel with hypoxia-induced factor 1 (HIF-1α) and VEGF expression, reaching the peak at 6 h. Silencing of RACK1 suppressed the invasion and tube formation activity of RF/ 6A cells in ARPE-19 and RF/6A co-culture system, possibly through VEGF signal pathway. Overexpression of RACK1 showed the opposite effect. Intravitreal injection of anti-RACK1 monoclonal antibody predominantly decreased RACK1 and VEGF expression in mouse laser-induced CNV model. Meanwhile, anti-RACK1 monoclonal antibody intravitreal injection also decreased incidence of CNV and leakage area. These data indicated that RACK1 promoted CNV formation via VEGF pathway. Additionally, anti-RACK1 monoclonal antibody significantly decreased CNV in mouse model and may have therapeutic potential in human CNV. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the industrialized world (Wong et al., 2008). There are two forms of AMD, one is neovascular or wet AMD, the other is non-neovascular or dry AMD. Choroidal neovascularization (CNV) is characterized as wet AMD (Barbazetto et al., 2010), which refers to the growth of neovasculature derived from the choroid through breaks in Bruch's membrane into sub-retinal pigment epithelium (RPE) or sub-retinal spaces (Das and McGuire, 2003). In a study for individuals who are diagnosed with early or intermediate AMD at primary visit, approximately 10% develop CNV over a median follow-up period of 6.3 years (Clemons et al., 2005). Thus, there is an urgent need to clarify the pathophysiology of CNV and to identify viable treatment strategies that will arrest disease-induced vision loss.
⁎ Corresponding authors at: Department of Ophthalmology, Affiliated Hospital of Nantong University, Nantong, Jiangsu 226001, China. E-mail addresses: [email protected] (H. Chen), [email protected] (A. Sang). 1 Xiaojuan Liu and Manhui Zhu contributed equally to this work. 2 Hui Chen and Aimin Sang contributed equally to this work.
http://dx.doi.org/10.1016/j.yexmp.2016.04.004 0014-4800/© 2016 Elsevier Inc. All rights reserved.
Intravitreal injection of vascular endothelial growth factor (VEGF) antagonists is the major treatment for CNV currently. However, it is important to figure out that there is controversy regarding the safety of anti-VEGF intraocular injection (Elice et al., 2009; Eremina et al., 2008). These drugs are also known for their limited indications and insufficient efficacy. The cost, discomfort of, and time spent receiving monthly injections hinder the application of anti-VEGF therapy. Moreover, a portion of patients do not respond to the treatment well (Wickremasinghe et al., 2011). Thus, searching for the molecules to regulate VEGF expression and function is essential for the treatment development of AMD. Receptor for activated protein kinase C1 (RACK1) is upregulated in vascular endothelial cells during angiogenesis (Berns et al., 2000). Silencing of RACK1 dramatically attenuates tumor-associated angiogenesis (Wang et al., 2011a). RACK1, a 36 kDa protein containing 7 internal Trp-Asp 40 (WD40) repeats, is originally cloned as an anchoring protein for protein kinase C (PKC) (McCahill et al., 2002), stabilizing the active form of PKC and permitting its translocation to different intracellular sites (Ron et al., 1994; Ron and Mochly-Rosen, 1994). Recently, RACK1 is found to play a regulatory role in VEGF-VEGFR1-dependent cell migration via activation of PI3K/Akt and small GTPase Rac1 signaling
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pathways (Wang et al., 2011b). RACK1 silencing suppresses tumor cell growth and invasion in vitro (Wang et al., 2011a). However, whether RACK1 participates in CNV is unknown. Our research group has already successfully constructed adult C57BL/6J (B6) mouse CNV model and cell model of neovascularization (Peng et al., 2013; Wang et al., 2014). In the present study, we firstly detected the expression of RACK1 and VEGF in mouse CNV and cell hypoxia models. Then we investigated the role of RACK1 in CNV formation via RACK1 specific siRNA and monoclonal antibody to determine the function of RACK1 in CNV formation and whether intravitreal injection of anti-RACK1 monoclonal antibody could alleviate CNV formation. The data can supply novel molecular target for CNV treatment. 2. Materials and methods 2.1. Animals All experimental procedures were performed in accordance with the requirements of Animal Welfare committee of the Medical College of Nantong University. The research protocol for the use of animals has been approved by the Center for Laboratory Animals at Nantong University (Nantong, Jiangsu, China).
in 200 μl fresh RPMI 1640 medium with 0.5% FBS and were placed into the upper chamber. After 48 h, removing the non-invading cells in the upper chambers with a cotton swab, invaded cells on the lower surface of the porous membrane were fixed with 4% paraformaldehyde for 15 min, stained with 0.1% crystal violet for 30 min, and photographed under a light microscope (Olympus, Tokyo, Japan). Five random fields (× 200) were chosen in each insert, and the cell number was quantified manually. 2.5. Tube formation assay Treated and untreated ARPE-19 cells were plated in Transwells with 8 μm pore-size inserts, which were put in wells where RF/6A cells had been plated. Briefly, matrigel was thawed and laid into 24-well culture plates to a total volume of 200 μl in each well. Plates were stored at 37 °C for 30 min to form a gel layer. After gel polymerization, 2 × 104 RF/6A cells were seeded in each well and incubated with fresh RPMI 1640 medium supplemented with 0.5% FBS for 24 h at 37 °C in humidified air with 5% CO2. 2 × 104 ARPE-19 cells which had been subjected to transfection for 24 h were seeded into the upper chamber. The closed networks of tubes in each well were observed with an inverted phasecontrast microscope (Olympus, Tokyo, Japan). Incomplete networks were excluded. The experiments were performed in triplicate and five fields from each chamber were counted and averaged.
2.2. Cell culture 2.6. Laser-induced CNV Human retinal pigment epithelial cell line ARPE-19 and rhesus choroidal endothelial cell line RF/6A were purchased from American Type Culture Collection (ATCC, Manassas, VA) and were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) (Gibco, Rockville, MD) and 100 U/ml penicillin–streptomycin mixtures (Gibco) at 37 °C in 5% CO2. The culture medium was changed every 2 days. An in vitro RF/6A cell chemical hypoxia model was established by adding 300 μM cobalt chloride (CoCl2) to culture medium, and cells were harvested after 1, 3, 6, 12 and 24 h. Cells without CoCl2 treatment were regarded as normal control.
Adult C57BL/6J (B6) mice were anesthetized by intraperitoneal injection with 3% chloral hydrate, and pupils were dilated with topical administration of tropic amide phenylephrine eye drops (Santen, Osaka, Japan). Four burns were made by laser photocoagulation (647.1 nm; 50 mm spot size; 0.05 s duration; 360 mW) in each retina with a hand-held contact fundus lens (Ocular Instruments, Bellevue, WA) in 3, 6, 9, 12 o'clock positions between retinal vessels in a
2.3. Transfections A total of 2 × 106 RF6A cells per well were seeded in 6-well plates and allowed to grow overnight. Transfection of RACK1 siRNA (5′GCTGAAGACCAACCACATT-3′, Ribobio, Guangzhou, China) or FlagRACK1 was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) following the manufacturer's protocol. In brief, for 6-well plates, 4 μg DNA was mixed with 10 μl Lipofectamine 2000 at a final concentration of 2 μg DNA/ml, dissolved in RPMI 1640 without serum, the resulting complex was incubated at room temperature (RT) for 20 min to generate transfection mixture and was added to cells, which were then incubated for 4 to 6 h. Next, the cells were washed with RPMI 1640 and incubated in RPMI 1640 with 10% FBS for a further 48 h. Then the cells were collected for western blot analysis. 2.4. In vitro cell invasion assay To test the effect of RACK1 on invasion and tube formation of RF/6A cells, we applied ARPE-19 and RF/6A cell co-culture model. For invasion assay, RF/6A cells were plated in Costar Transwells (Costar, Corning, NY) with 8 μm pore-size inserts, which were put in 24-well plates where ARPE-19 cells was plated. Briefly, a total of 2 × 104 ARPE-19 cells were resuspended in 600 μl fresh RPMI 1640 medium supplemented with 10% FBS and transferred into the lower chamber. Cells were incubated for 24 h at 37 °C, then transfected with RACK1 siRNA or Flag-RACK1 before 8 μm pore-size inserts were placed in the wells. Cells with no treatment served as normal control. Matrigel (Sigma-Aldrich, Saint Louis, MO) 200 μl was added to 24-well plates and incubated at 37 °C for 30 min to form gels. A total of 2 × 104 RF/6A cells were resuspended
Fig. 1. Chemical hypoxia induces RACK1, HIF-1α and VEGF expression in RF/6A cells in vitro. (A) Western blots show RACK1, HIF-1α and VEGF expression. (B) Histogram shows densitometric analysis of the average levels to β-actin. *P b 0.05, compared with normal control. Results are mean ± SD, n = 3 in each group.
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peripapillary area of both eyes. Only burns that produced a bubble, indicating the rupture of the Bruch's membrane, were counted in the study. All mice were randomly divided into five groups based on days (d) following laser treatment (normal, 1 d, 3 d, 7 d, 14 d). For the western blot, each group contained 24 mice with laser treatment and 15 control mice without laser treatment belonged to control group. In control and post-laser 7 d groups, another 45 mice (ninety eyeballs) each were used for choroidal flat mount, immunofluorescence, and histopathology. 2.7. Western blot analysis To detect the protein level of molecules, we extracted mouse RPEchoroid complexes and retina from 3 mice at 1, 3, 7, 14 d after laser injury and in vitro cell lysates. Proteins and a molecular weight marker were separated by 10% SDS-PAGE, and transferred to a PVDF membrane. The membrane was then incubated with rabbit anti-RACK1 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-VEGF (1:500; Santa Cruz Biotechnology), mouse anti-HIF-1α (1:1000; Chemicon, Temecula, CA). The antibodies were incubated in blocking 5% skim milk over night at 4 °C and reacted with HRP-conjugated secondary antibodies (1:2000; Thermo Scientific, Rockford, IL) at 37 °C for 2 h. Extensive washes in 0.05% Tween-20 in TBS and followed by incubation with anti-β-actin (1:1000; Sigma-Aldrich, Saint Louis, MO).
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The blots were then incubated with chemifluorescent reagent ECL (Thermo Scientific, Rockford, IL) and exposed to X-ray film in the dark. The intensity of β-actin signal was used as an endogenous control and quantified the band optical density using Image J (National Institutes of Health, Bethesda, MD). 2.8. Immunofluorescence RACK1 tissue localization was examined on 8 μm cryosections (on day 7 after laser photocoagulation). The cryosections were blocked with 1% BSA for 4 h at RT, then incubated with rabbit RACK1 antibody (1:50; Santa Cruz Biotechnology) and mouse CD31 antibody (1:50, Abcam, Cambridge, MA) at 4 °C overnight. For CD31 staining, antigen retrieval was obtained through heated water bath at 97 °C for 10 min. Thereafter, the slides were stained with Alexa Fluor 488 goat antimouse IgG, Alexa Fluor 546 goat anti-rabbit IgG (1:200; Invitrogen, Carlsbad, CA) and Hoechst (1:2000; Sigma-Aldrich). The photomicrographs were taken by a digital high sensitivity camera (Hamamatsu, ORCA-ER C4742-95, Japan). 2.9. Intravitreal injection The intravitreal injection of 1 μl of anti-RACK1 monoclonal antibody (200 ng/ml) or vehicle (5% glucose solution, 5% GS) was given on day 1
Fig. 2. RACK1 silencing inhibits invasion and tube formation of RF/6A cells. (A) RACK1 and VEGF expression was detected by western blot in RF/6A cells following RACK1 siRNA transfection after 48 h. (B) Histogram shows densitometric analysis of the average levels for RACK1 and VEGF. β-actin was used as loading control. Cells transfected with scramble siRNA were used as negative control. (C) Representative photographs of invasive RF/6A cells (200× magnification). (D) The average number of invasive RF/6A cells per field. *P b 0.01, RACK1 siRNA versus Control. (E) Representative photographs of tube formation (40× magnification). (F) The average number of closed networks of tubes per field. *P b 0.01, RACK1 siRNA versus Control.
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and mice were killed on day 7 after laser treatment. Control group represented laser-induced CNV without any injection. 2.10. Choroidal flat mount On day 7 after laser coagulation, thirty eyes (6 eyes from 3 mice/ each group) were subject to choroidal flat mounts. The eyes were enucleated and immediately fixed in 4% paraformaldehyde (Guoyao Group of Chemical Reagents, Beijing, China) in pH 7.3 PBS for 1 h. Under a biopsy microscope, the anterior segments were wiped out, as well as the neurosensory retinas were detached and separated from the optic nerve head. The remaining eyecups were washed with cold ICC buffer (0.5% BSA, 0.2% Tween-20, and 0.1% Triton X100) in PBS. A 10 mg/ml solution of 40, 60-diamidino-2phenylindole (DAPI) (1:500), 1 mg/ml solution of Alexa Fluor 568 conjugated isolectin-B4 (1:100), and 0.2 U/ml solution of Alexa Fluor 488 conjugated phalloidin (1:100; Invitrogen-Molecular Probes, Eugene, OR) were prepared in ICC buffer. The eyecups were incubated with the above fluorescent dyes in a humidified chamber at 4 °C with gentle shaking for 4 h, followed by washing with cold ICC buffer. Four or five radial cuts were made toward the optic nerve head for flat mounting the sclera/choroid/RPE complexes together with the gel (Gel-mount; Biomedia Corp. Foster City, CA). The samples were covered and sealed for confocal microscopic analysis.
2.11. Confocal microscopy Multiplane z-series images were collected using a confocal microscope (SP2; Leica, Exton, PA) with a 63×, 1.25 numerical aperture and oil-immersion objective. All images were collected at a 1024 × 1024 pixel resolution and a depth of 8 bits per channel. Voxel dimensions were 0.3662 μm for x- and y-axis and 0.4884 μm for z-axis. Fluorescent signals for DAPI (400–500 nm), Alexa Fluor 488 (500–550 nm), and Alexa Fluor 568 (560–660 nm) were collected using a sequential scan mode. 2.12. Fundus fluorescein angiography To confirm the inhibitory effect of anti-RACK1 monoclonal antibody on CNV formation, fluorescein angiography was performed on day 7 after laser photocoagulation. The development of CNV was evaluated using a digital fundus camera connected to a slit lamp delivery system (Kanghua, Chongqing, China), captured 3 min after 0.3 ml of 2% fluorescein sodium (Alcon Laboratories, Irvine, CA) was injected into the intraperitoneal cavity of mice as previously described (Wang et al., 2014). Angiograms were graded as follows: no staining, Score 0; slightly stained, score 1; moderately stained, score 2; strongly stained, score 3 (Takehana et al., 1999). The area of CNV lesion was measured three times and averaged using Image J (USA National Institutes of Health, Bethesda, MD) (Xie et al., 2011).
Fig. 3. RACK1 overexpression enhances RF/6A cell invasion and tube formation. (A) RACK1 and VEGF expression was detected by western blot in RF/6A cells following Flag-RACK1 transfection after 48 h. (B) Histogram shows densitometric analysis of the average levels for RACK1 and VEGF. β-actin was used as loading control. Cells transfected with Flag were used as negative control. (C) Representative photographs of invasive RF/6A cells (200× magnification). (D) The average number of invasive RF/6A cells per field. *P b 0.01, Flag-RACK1 versus Control. (E) Representative photographs of tube formation (40× magnification). (F) The average number of closed networks of tubes per field. *P b 0.01, Flag-RACK1 versus Control.
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laser photocoagulation. The sections were cover slipped by mounting medium. Serial sections were examined, and the samples that contained the thickest or widest lesions among the set of specimens that was obtained for each instance of CNV were estimated. Slices with HE staining were examined using a light microscope (Olympus, Tokyo, Japan). IPP 6.0 was used to calculate the maximum thicknesses and lengths of each CNV lesion. 2.14. Statistical analysis All values were presented as means ± standard deviation (SD). One-way ANOVA was used for statistical comparisons of multiple groups. Descriptive statistics were performed using Stata statistical software version 11.0. P b 0.05 was considered statistically significant. Each experiment consisted of at least three replicates. 3. Results 3.1. HIF-1α, VEGF and RACK1 expression is up-regulated in RF/6A cells under hypoxic condition To further explore the functions of RACK1 in CNV, we examined the expression of RACK1, VEGF and HIF-1α in RF/6A cells after CoCl2 exposure. VEGF is a target gene of HIF-1α (Medici and Olsen, 2012; Qi et al., 2014). As expected, western blot showed that the expression of HIF-1α, VEGF and RACK1 was up-regulated in similar time-dependent manner under hypoxia condition (Fig. 1). 3.2. RACK1 silencing inhibits RF/6A invasion and tube formation
Fig. 4. RACK1, VEGF expression is up-regulated after CNV formation. The mouse CNV model was performed by laser photocoagulation. (A) Leakage area of CNV was examined by FFA. (B) RACK1 and VEGF protein level was detected by western blot. β-actin was used as loading control. (C) Histogram shows densitometric analysis of the average levels for RACK1 and VEGF to β-actin. *P b 0.05; significantly different from normal control.
RACK1 siRNAs were transfected in RF/6A cells after hypoxia. The results showed that RACK1 expression was down-regulated by 51 ± 8% (Fig. 2A and B). Accordingly, VEGF expression was inhibited by RACK1 siRNA transfection. When RACK1 expression was inhibited, RF/6A cell invasion and tube formation capability was reduced (Fig. 2C, D, E and F), showing that RACK1 may participate in CNV formation.
2.13. Histopathology
3.3. RACK1 overexpression promotes RF/6A invasion and tube formation
Eight eyes were enucleated in each group, and 8 μm cryosections were prepared for hematoxylin and eosin (HE) staining on day 7 after
Flag-RACK1 was constructed and transfected into RF/6A cells. RACK1 and VEGF expression in Flag-RACK1 transfection group markedly
Fig. 5. RACK1 is localized in endothelial cells. Cellular localization of RACK1 in retina/choroid cryosections was determined by double immunostaining accompanied with endothelial marker CD31. Scale bar = 50 μm.
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increased (1.93 ± 0.09 fold and 1.81 ± 0.12 fold, respectively) compared to control and Flag transfection groups (Fig. 3A and B). The transwell assay showed that up-regulation of RACK1 elevated invasion and tube formation ability of RF/6A cells (Fig. 3C, D, E, and F). 3.4. RACK1, VEGF expression increases after laser photocoagulation To investigate the expression of RACK1 in CNV, we extracted protein of RPE-choroid complexes and retinas for western blot analysis. After laser injury, RACK1 expression was up-regulated, reached the peak at 7 d, showing a similar change tendency with VEGF (Fig. 4). 3.5. RACK1 is localized in vascular endothelium inside CNV To identify the cellular localization of RACK1 inside CNV site, we performed double immunostaining of RACK1 with CD31, a marker for endothelial cells. Inside CNV, RACK1 was localized in vascular endothelium (Fig. 5). 3.6. Anti-RACK1 monoclonal antibody alleviates the leakage of CNV To further investigate the functions of RACK1 in CNV formation, we used intravitreal injection of RAK1 monoclonal antibody in mouse CNV model. Fluorescein angiogram assay showed that the leakage area of CNV was smaller in anti-RACK1 injection group than in vehicle injection group on day 7 after laser photocoagulation (Fig. 6A). RACK1 monoclonal antibody decreased the number of spots with strong dye staining (score 2 or more) and increased the number of spots with weak staining (score 0 or 1) (Fig. 6B). 3.7. Anti-RACK1 monoclonal antibody suppresses RACK1 and VEGF expression Then we explored RACK1 and VEGF expression following antiRACK1 monoclonal antibody intravitreal injection with laser injury.
Fig. 7. RACK1 monoclonal antibody intravitreal injection down-regulates RACK1 and VEGF expression. (A) Western blot analysis showed that RACK1 and VEGF expression in the RPE-choroid complex and retinal tissues at day 7 after laser coagulation. (B) Quantification graphs for RACK1 and VEGF. Data of relative protein level to β-actin are presented as mean ± SD. *P b 0.01, anti-RACK1 versus vehicle.
RACK1 protein level in RPE-choroid complexes and retina was reduced dramatically in anti-RACK1 injection group compared with control and vehicle injection groups. Furthermore, VEGF expression showed a similar decreasing change with RACK1 (Fig. 7A and B), suggesting that RACK1 might promote CNV formation via up-regulating VEGF expression.
Fig. 6. RACK1 monoclonal antibody intravitreal injection reduces leakage area of CNV. (A) Hyper fluorescent leakage surrounding the laser spots was relative weak leakage (grade 1) in anti-RACK1 injection mice retina. (B) The intensity of FFA leakage grade in control, vehicle and anti-RACK1 injection groups. (C) Comparison of semi-quantitative leakage score between vehicle and anti-RACK1 injection mice. The mean leakage score of CNV in control group was set at 100. *P b 0.01.
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3.8. Anti-RACK1 monoclonal antibody reduces the areas of CNV Phalloidin labeling examined actin bundles of RPE cells, forming a tightly packed, uniform hexagonal monolayer (Fig. 8B). DAPI labeling identified the nuclei (Fig. 8C, G, and K). At 7 d, a well-defined radial array of isolectin labeled vessels was visible inside CNV in control and vehicle groups (Fig. 8A and E). The merged images showed that phalloidin labeled RPE cells covered the area of CNV (Fig. 8D, H, and L). Compared to control and vehicle injection groups, the areas of CNV were smaller and the vessel tubes were fewer in anti-RACK1 injection group (Fig. 8A, E, I). On day 7 after laser photocoagulation, histopathology analysis showed that anti-RACK1 monoclonal antibody treatment significantly decreased the thickness and length of retinal lesions caused by CNV (Fig. 9).
4. Discussion Our study demonstrated that up-regulation of RACK1 promoted invasion and tube formation capability of RF/6A cells to boost angiogenesis. Treatment with anti-RACK1 monoclonal antibody significantly decreased the dimensions of laser-induced CNV in mouse model, suggesting that RACK1 might be an important molecular target for antiangiogenesis therapies.
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Many growth factors such as VEGF, placental growth factor (PIGF), platelet-derived growth factor β (PDGF-β), and pigment epitheliumderived factor (PEDF) (Campa et al., 2010), are involved in different stages of CNV. Among these growth factors, VEGF is the most important (Nagai et al., 2014). Other mediators related to VEGF upregulation such as transforming growth factor beta (TGF-β), angiostatin and hypoxiainducible factor (Novack, 2008), are targets for the treatment of CNV. The RPE with its highly organized basement membrane, known as Bruch's membrane, is essential for normal functioning of the retina (Lakkaraju, 2012; Strauss, 2005). A break of Bruch's membrane is necessary for CNV to develop. In our experiments, we used ARPE-19 and RF/ 6A cells to imitate in vitro neovascularization. Anti-RACK1 monoclonal antibody was conducted on laser-induced CNV mouse model, which is well established for investigating ocular angiogenesis (Du et al., 2013; Grossniklaus et al., 2010; Li et al., 2015). RACK1 is found to co-localize with activated PKCβI and PKCβII, suggesting specific binding of RACK1 to PKCβ (Ron et al., 1995). VEGFinduced tumor angiogenesis and tumor growth in vivo are PKCβ dependent (Yoshiji et al., 1999). Inhibition of PKCβ significantly suppresses VEGF-induced neovascularization in a mouse model of hepatocellular carcinoma cells (Yoshiji et al., 1999). Furthermore, RACK1 is massively induced in subconfluent and contact-inhibited bovine endothelial cells, during angiogenesis in vitro, active phases of the murine ovarian cycle, human tumor angiogenesis, and in cancer cells in vivo as assessed by quantitative PCR and in situ hybridization (Berns et al., 2000). Our
Fig. 8. RACK1 monoclonal antibody intravitreal injection inhibits CNV formation. Representative images of flat mount preparations from control and RACK1 antibody injection groups 7 d after laser photocoagulation. (A–H) The area of the vessels in control and PBS injection groups. (I–L) The area of the vessels was smaller in anti-RACK1 injection mice compared with control mice. Mouse RPE-choroid flat mount preparations from laser-injured regions were fluorescently labeled with F-actin marker phalloidin (red), endothelial and microglia cell marker isolectin-B4 (green), and nuclear marker DAPI (blue; merged images) (63× magnification). Scale bars = 100 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 9. RACK1 monoclonal antibody intravitreal injection inhibits CNV formation. HE stains images of CNV. (A) Normal mice retinal and choroid structure. (B–D) Each photograph showed the central area of CNV inside mice retinal and choroid in control, vehicle or anti-RACK1 groups. Scale bar = 100 μm (RPE: retinal pigment epithelium; OS: outer segment; IS: inner segment; ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; GC: ganglion cell layer). (E) Statistical analysis of data presented from B to D, n = 12–16 spots *P b 0.01. Black dash lines represented the edge of CNV.
study also reveals that RACK1 expression is up-regulated during CNV process, in line with the previous researches. We explore the effect of RACK1 on neovascularization both in vitro and in vivo, showing that silencing of RACK1 reduces invasion and tube formation of RF/6A cells. Additionally, anti-RACK1 monoclonal antibody can reduce the leakage of CNV lesions. The data demonstrate that RACK1 promotes CNV formation. The mechanism of promoting angiogenesis role of RACK1 could be that RACK1 participates in VEGF/Flt1triggered vascular endothelial cell migration (Wang et al., 2011b). RACK1 is associated with tumor angiogenesis in a variety of researches (Dirkx et al., 2006; Leek et al., 1999). It is necessary to investigate the mechanism underlying angiogenesis function of RACK1 in more detail. Our findings reveal the promotion function of RACK1 in CNV formation. We anticipate that targeted therapy toward RACK1 might be promises to interventions of pathological processes in CNV. Further investigations are necessary to clarify the potential mechanisms of RACK1 participation in CNV process. Conflicts of interest The authors declare that they have no conflict of interest. Acknowledgments The study was supported by National Natural Science Foundation of China (No. 81401365); Nantong science and technology project (MS12015056); a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). References Barbazetto, I.A., et al., 2010. Incidence of new choroidal neovascularization in fellow eyes of patients treated in the MARINA and ANCHOR trials. Am J. Ophthalmol. 149, 939–946, e1.
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