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Blockage of tissue factor ameliorates the lesion of laser-induced choroidal neovascularization in mice
Experimental Eye Research 127 (2014) 117e123
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Blockage of tissue factor ameliorates the lesion of laser-induced choroidal neovascularization in mice Li Wang a, Zhongkai Yang b, Ying Yu a, Chen Cui a, Huaijin Guan a, Hui Chen a, * a b
Eye Institute, Affiliated Hospital of Nantong University, 20 Xisi Road, Nantong 226001, Jiangsu, China Department of Ophthalmology, Xinhua Hospital, Yili, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 February 2014 Accepted in revised form 8 July 2014 Available online 22 July 2014
Choroidal neovascularization (CNV) occurs as a result of age-related macular degeneration (AMD) and causes severe vision loss among elderly patients. High expression of tissue factor (TF) was found in the retinas of AMD patients. In this study, we used anti-TF monoclonal antibody to test the effect of the TF blockage on experimental CNV induced by laser photocoagulation of retina in mice. Anti-TF monoclonal antibody or vehicle was administered intravitreally at day 1, 2 or 3 after laser application. We found that TF expression increased, and reached the peak at the 3rd week after the after laser application. Anti-TF monoclonal antibody can predominantly decrease the expression of TF, VEGF and F4/80, and reduced the area of CNV. Anti-TF monoclonal antibody also decreased incidence of CNV and leakage area. There were no pathological changes in the liver, heart, brain or kidney tissue. We conclude that TF plays an important role in CNV and anti-TF monoclonal antibody significantly decreases CNV in mouse model and anti-TF monoclonal antibody may have therapeutic potential in AMD. © 2014 Elsevier Ltd. All rights reserved.
Keywords: tissue factor choroidal neovascularization anti-TF monoclonal antibody mouse
1. Introduction Age-related macular degeneration (AMD) is the leading cause of blindness in most developed countries (Smith et al., 2001). Choroidal neovascularization (CNV) beneath the macula, which occurs at the end stage of the disease and is characterized as the ‘‘wet form’’ of AMD, causes severe central vision loss and it has serious effects on the quality of life in patients (Calabrese et al., 2011; Swaroop et al., 2009). CNV is pathological angiogenic process that arises from the choroid into the Sub-retinal pigment epithelium (RPE) or subretinal spaces (Yoshida et al., 2003; Campochiaro, 2000). Nevertheless, the pathogenic mechanisms underlying CNV are complex and still largely unknown, the current therapeutic options (VEGF trap (Bakall et al., 2013; Heier et al., 2012) and anti-VEGF antibodies (Wickremasinghe et al., 2011)) for CNV are effective but a portion of patients do not respond to the treatment well. In addition, current anti-VEGF drugs can increase the risks of RPE tears (Arias et al., 2007), cardiovascular disease (Valmaggia et al., 2008), nervous system disease (transient ischemic attack) (Spaide et al., 2009) and other systemic adverse events.
* Corresponding author. Tel.: þ86 13809082586; fax: þ86 513 85519820. E-mail address: [email protected] (H. Chen). http://dx.doi.org/10.1016/j.exer.2014.07.006 0014-4835/© 2014 Elsevier Ltd. All rights reserved.
Some studies showed that TF is one of the key mediators of physiological and pathologic angiogenesis (Cho et al., 2011; Belting et al., 2004; Rak et al., 2006; Ahamed and Ruf, 2004; Wang and Zou, 2012). TF is also known as coagulation factor III, which is a cellsurface glycoprotein receptor which initiates the blood coagulation cascade after vessel injury and implicated in various pathological processes (Harlos et al., 1994). TF can regulate the angiogenesis factor (Pendurthi et al., 2000), for instance, early growth response gene 1 (Egr-1) and vascular endothelial growth factor (VEGF) (Rong et al., 2006). Binding of FVIIa to TF directly causes protease-activated receptor (PAR)-2 and results in phosphorylation of the TF (Uusitalo-Jarvinen et al., 2007), subsequently promoting pathological angiogenesis. The lack of negative regulatory control by the TF cytoplasmic domain is also a pathway by which pro-angiogenic signaling of PAR-2 can be turned on (Bluff et al., 2008). A strong link exists between tumor VEGF and endothelial TF in vivo angiogenic conditions. VEGF and TF can upregulate each other (Croce and Libby, 2007). TF is over expressed in AMD tissue and it is triggered by inflammatory factors High expression of TF was found in RPE cells and macrophages by immunostaining of CNV biopsy (Grossniklaus et al., 2002), but not in the normal blood vessels (Belting et al., 2004). Our previous studies found that anti-TF siRNA can inhibit cell proliferation, cell migration, and tube formation in an in vitro model of neovascularization (Peng et al., 2013). Some studies
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2.4. Choroidal flat mount
showed that the frequency of leakage in CNV lesions was significantly reduced using fVII-verteporfin, a ligand that binds with exceptional affinity and specificity to its receptor TF (Lu et al., 2009). Earlier studies showed that the Icon (Tezel et al., 2007), which is composed of factor VII(fVII), conjugated to the Fc domain of an IgG1 Ig, the TFeIcon complex is strongly efficacious in preventing CNV formation, and the Icon functions as an anti-TF antibody (Bora et al., 2003). Anti-TF monoclonal antibody can significantly inhibit the growth of pulmonary metastases angiogenesis (Mueller et al., 1992) experimental lung metastasis of invasive breast cancer angiogenesis (Ngo et al., 2007) as well attenuated tumor growth and angiogenesis (Ruf et al., 2011). These results suggest that anti-TF monoclonal antibody may have beneficial and potential effects on CNV. In this paper, we tested first the efficacy of anti-TF monoclonal antibody on laser-induced mouse model of CNV.
Three mice per group were killed at 7 days after laser injury, and the area of the CNV lesions was quantified using the choroidal flat mount technique (Nagai et al., 2006). Briefly, eyes were enucleated and immediately fixed with 4% paraformaldehyde for 30 min at 4 C or ice. The anterior segment and retina of each eye were removed to obtain the RPE-choroidal complex, and the complex were washed three times in PBS (136.9 mMNaCl, 8.1 mMNa2HPO412H2O, 1.46 mM Na2H2PO2.2H2O). The RPE-choroidal complex then incubated overnight at 4 C with fluorescein isothiocyanate-conjugated isolectin B4 (1:150; Vector Laboratories, Burlingame, CA). The fluorescence microscope (DM400 B, Leica, Germany) with wavelength (488 nm) laser was used to observe CNV. The CNV-related fluorescence area was measured with Image J (USA National Institutes of Health, Bethesda, MD).
2. Methods
2.5. Fundus fluorescein angiography
2.1. Animals
To confirm the inhibitory effect of anti-TF 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 intraperitoneal injection of 0.3 mL of 2% fluorescein sodium (Alcon Laboratories, Irvine, CA) was injected into the intraperitoneal cavity of the mice as previously described (Gu et al., 2010). The laser lesions were studied using fluorescein angiography to evaluate CNV development and its activity. 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 the CNV lesion was measured three times and averaged using Image J (USA National Institutes of Health, Bethesda, MD) (Xie et al., 2011).
All experimental procedures were held 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). 2.2. Laser-induced CNV and drug treatment 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 the 3, 6, 9, and 12 o'clock positions between the retinal vessels in a per papillary area of both eyes. Only burns that produced a bubble, indicating the rupture of the Bruch's membrane, were counted in the study. The intravitreal injection of 1 ml of anti-TF monoclonal antibody (200 ng/ml; sc-376361; Santa Cruz Biotechnology, Santa Cruz, CA) (anti-TF group) or vehicle (5% glucose solution, GS) was carried out at day 1, 2 or 3 after laser injury, and the control group means the laser-induced lesion without any injection. 2.3. Western blot analysis The protein extract of RPE-choroid complexes and retina from 3 mice at 1, 2, 3, 4, 6, 9 weeks after laser injury were prepared for western blot analysis. Protein and a molecular weight marker were separated by 5% SDS-PAGE, and transferred to a PVDF membrane. The membrane was then incubated with dilution of the monoclonal mouse anti-TF (1:120; Santa Cruz Biotechnology, Santa Cruz, CA) and anti-VEGF (1:200; Santa Cruz Biotechnology, Santa Cruz, CA) antibodies in blocking (5% skim milk) solution over-night at 4 C. and reaction with second antibodies HRP-conjugated goat antimouse IgG (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA) at 37 C for 2 h. Extensive washes in 0.05% Tween-20 in TBS and followed by incubation with anti-b-actin (1:10000 SigmaeAldrich, Saint Louis, MO). The signals were developed using a chemiluminescence solution (ECL western blotting detection reagents; Amersham Pharmacia Biotech, Piscataway, NJ). The intensity of the b-actin signal was used as an endogenous control and quantified the band optical density using Image J (National Institutes of Health, Bethesda, MD).
2.6. Histopathology and immunohistochemistry Eight eyes, heart, liver, brain and kidney were enucleated in each group, and 8 mm cryosections were prepared for either HE staining (on day 14 after laser photocoagulation) or immunohistochemistry of TF (on day 7 after laser photocoagulation). The cryosections were blocked with 1% BSA for 4 h at room temperature, and were incubated over-night at 4 C with monoclonal mouse anti-TF (1:50; Santa Cruz Biotechnology, Santa Cruz, CA), the slides then incubated with FITC-conjugated secondary antibody donkey antimouse IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) and hochest (1:2000 SigmaeAldrich, Saint Louis, MO). 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 that had been stained with hematoxylin and eosin were examined using a light microscope. IPP 6.0 was used to calculate the maximum thicknesses and lengths of each CNV lesion. 2.7. qRT-PCR We have randomly selected 3 mice in each group, The RPEchoroid complex and retina mRNA was isolated (TRIzol extraction protocol; Invitrogen) at 7 days after laser coagulation, and reverse transcription was performed with mRNA according to the manufacturer's protocol. Real-time PCR was performed with SYBR Green PCR Master Mix on the ABI 7500 Real Time System (Applied Biosystems, Foster City, CA), Reaction conditions were as follows: 30 s at 95 C, followed by 40 cycles of 5 s at 95 C, 34 s at 60 C. The sequences of primers used for mouse VEGF: 5-TTACTGCT
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3. Results 3.1. Expression of TF in various time points after laser photocoagulation To investigate the role of TF in choroidal neovascularization, we extracted the protein of RPE-choroid complexes and retinas from each group were prepared for western blotting analysis several weeks after laser photocoagulation and normal group. After laser injury, TF protein expression showed a significant up-regulation, reached the peak at 3rd week, and then gradually declined to the baseline from 4th to 9th week. We detected a weak expression of TF in the normal mice (Fig. 1). 3.2. Expression of TF in the CNV lesions
Fig. 1. Changes in TF protein expression in the retina and RPE-choroid complexes at 1, 2, 3, 4, 6, 9 weeks after laser photocoagulation by western blotting analysis. (A) TF protein level was increased gradually after injury, peaked at 3rd week, and reduced thereafter. TF was low in the normal mice, b-actin protein level was used as the internal control. (B)TF protein level represented a ratio between the amount of target protein and that of b-actin protein control, *P < 0.05; significantly different from the normal mice.
GTACCTCCACC-3 and 5-ACAGGACG GCTTGAAGATG-3; TF: 5CACCGAGCAATGGAAGAGTT-3 and 5-TTGCACAGTTCCCATCAGAG3; F4/80: 5-TCGCTGCTGGTTGAA TACAG-3 and 5-GCAACCTCGTG TCCTTGAGT-3; b-actin: 5eGAT GACCCAGATCATGTTTGA-3 and 5GGAGAGCATAGCCCTCGTA G-3. 2.8. Statistical analysis All values were presented as means ± standard error of mean (SEM). One-way ANOVA was used for statistical comparisons of multiple groups. Descriptive statistics were performed using Stata statistical software version 11.0. P values <0.05 were considered statistically significant. Each experiment consisted of at least three replicates.
To research whether TF expression transforms during CNV structure, we examined the expression of TF in the CNV lesions by immunofluorescence staining. We contrasted TF expression was detected in laser-induced CNV lesion and we also saw trace amount of TF expression in normal retina. Compared with the vehicle or control group, TF expression was weakened in the anti-TF group (Fig. 2). 3.3. Anti-TF monoclonal antibody suppresses the expression of TF and VEGF We research suppression of TF and VEGF by anti-TF monoclonal antibody treatment after laser injury. The TF levels of the RPEchoroid complexes and retina were decreased significantly at 7 days in the anti-TF group compared with the control or vehicle group. Surprisingly, the VEGF expression revealed a similar change (Fig. 3). 3.4. Time dependence of the effect of anti-TF monoclonal antibody on CNV formation To indicate the time dependence of the effect of anti-TF monoclonal antibody treatment on CNV formation in laser-treated mice, anti-TF monoclonal antibody was intravitreal injected at 1 day, 2
Fig. 2. Anti-TF monoclonal antibody significantly suppressed TF level in the RPE-choroid complexes at 7 days after photocoagulation. TF expression transforms during CNV structure and anti-TF monoclonal antibody significantly suppressed RPEechoroid protein levels of TF (AeC) vehicle treatment, (DeF) anti-TF monoclonal antibody treatment (GeI)the normal mice. Phase contrast image (left column), TF expression (center column, green channel) TF and hochest (right column, blue channel; merged images) Scale bars ¼ 100 mm.
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Fig. 3. Suppression of TF and VEGF by anti-TF monoclonal antibody treatment was shown in the RPE-choroid complex and retinal tissues at 7 days after laser coagulation (A, C) Western blot analysis revealed that TF and VEGF expression was suppressed by anti-TF monoclonal antibody treatment (B, D) Quantification graphs for TF and VEGF. Data of relative TF protein level to b-actin are presented as the mean ± SD of three independent experiments, *P < 0.01, significantly different from the vehicle group.
days, or 3 days after laser treatment. At day 7 after laser injury, the area of CNV was quantified by analysis of choroidal flat mounts. The RPE-choroid flat mounts distinctly decreased in a CNV lesion after anti-TF monoclonal antibody treatment (Fig. 4A). At 7 days, the average CNV area was 17,629.5 ± 1218 mm2 in the control mice (n ¼ 23e25 spots), 16,700.4 ± 1528 mm2 in the vehicle group (n ¼ 23e24 spots). The average CNV area were (4725 ± 1418 mm2,n ¼ 24 spots), (5169 ± 1334 mm2, n ¼ 24 spots) and (4359 ± 1423 mm2, n ¼ 23 spots) at 1 day, 2 days and 3 days injection respectively in the anti-TF group. Compared with the control group, there were decline rate of 72%, 69% and 74% in CNV area by anti-TF monoclonal antibody treatment. But there was no significant difference among various time points (Fig. 4B).
3.5. Anti-TF monoclonal antibody reduced the leakage of CNV lesions Comparisons of fluorescein angiograms between vehicle and anti-TF monoclonal antibody treatment group proved that the leakage area of CNV was less severe in the anti-TF group than in the vehicle group on day 7 after photocoagulation (Fig. 5A). AntiTF monoclonal antibody decreased the number of spots showing strong dye staining (score 2 or more) and increased the number showing weak staining (score 0 or 1) (Fig. 5B). The incidence of CNV formation was 92% in the control group, 96% in the vehicle group, 57% in the anti-TF group (Table 1). The incidence of fluorescence leakage in the eyes of the CNV mice was significantly
Fig. 4. Time dependence of the effect of anti-TF monoclonal antibody on CNV formation. (A) At 7 days after laser treatment, the CNV area in the anti-TF group was significantly attenuated compared to the vehicle or control group, and the experiments were repeated three times with similar results. Scale bar ¼ 100 mm (B) Quantitative analysis of CNV size in RPEechoroid flat mounts. Values are mean ± SEM, *P < 0.01.
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Fig. 5. Fluorescein angiograms, staining grade, the incidence and leakage area of CNV were obtained at 7 days after laser photocoagulation. (A) Hyper fluorescent leakage surrounding the laser spots were relative weak leakage (grade 1) in the anti-TF mice retina. (B) The intensity of FFA leakage grade was weaker in the anti-TF group than in the control or vehicle group. (C) Comparison of semi-quantitative leakage score between vehicle- and anti-TF -treated mice. The mean leakage score of CNV in the control group was set at 100. *P < 0.01.
elevated relative to that in the vehicle mice at 7 days after photocoagulation. 3.6. Anti-TF monoclonal antibody decreases neovascularization area On day 14 after photocoagulation, Histopathology analysis showed that anti-TF monoclonal antibody treatments significantly decreased the thickness and length of retinal lesions caused by choroidal neovascularization, we also found that the retina detachment occur more often in the eye treated with laser (Fig. 6). And there were no pathological changes on liver, heart, brain or kidney tissue. 3.7. Anti-TF monoclonal antibody down-regulated TF, VEGF, F4/80 mRNA Quantitative RT-PCR detected a very low-level F4/80, TF and VEGF mRNA expression in RPE-choroid complexes in normal mice (without laser treatment). On day 7 after laser photocoagulation, the expression of F4/80, TF and VEGF mRNA increased. But anti-TF monoclonal antibody treatment significantly suppressed their expression (Fig. 7). 4. Discussion Our study demonstrated at first that treatment with anti-TF monoclonal antibody significantly decreases the dimensions of laser-induced CNV in mice model. The results further suggest that Table 1 Incidence of CNV after various treatments on 7th day after photocoagulation, the fewer incidence of CNV lesions in the anti-TF group was exhibited. A Chi-square test showed *P < 0.001. Groups
No. of mice
Laser burns
The incidence of CNV (%)
Control 5%GS Anti-TF
3 3 3
24 24 24
22/24 (92) 23/24 (96) 14/24 (57)*
TF may be an important molecular target for anti-angiogenesis therapies (Belting et al., 2005, 2004; Abe et al., 1999; Darmoul et al., 2003). CNV is a complex process that develops in several stages. Different growth factors (such as TF, VEGF) and cellular types are important in each stage of the pathogenesis of CNV (Grossniklaus et al., 2002; Green, 1991; Schlingemann, 2004). A break or defect in Bruchs' membrane is necessary for CNV to develop. In our experiments, we used anti-TF monoclonal antibody act on laserinduced mouse model of CNV, which is well established for investigating ocular angiogenesis (Ryan, 1979), Many studies have reported that krypton laser in C57BL6 mice with modified settings including a smaller spot size, higher intensity, and shorter duration resulted in a higher frequency of histologic evidence of CNV(Seo et al., 1999; Tobe et al., 1998). The mechanism of accelerating angiogenesis by TF could be that the TF/FVIIa complex promotes inflammation and angiogenesis through a protease-activated receptor that regulates intracellular signal transduction is safe. It has been reported that murine anti-TF antibodies have been successfully used in experimental melanoma metastasis models (Mueller et al., 1992). The mechanism underlying the tumor suppression by anti-TF monoclonal antibody might contribute to the apoptosis triggered by TF inhibition, which was due to inhibition of TF: FVIIa signaling (Hembrough et al., 2003). Some studies showed that overexpression of TF in gastric cancer cells (Zhang et al., 2005), enhances neovascularization growth by increasing the transcription of pro-angiogenic VEGF. And downregulated TF expression can contribute to decrease pathologic angiogenesis (Zhang et al., 2005). Therefore we consider that the reduction of TF can also reduce the expression of VEGF, thus reduce CNV. Our results suggest anti-TF monoclonal antibody can reduce the TF expression as well VEGF expression at the same time. Evidences suggest that the cytokines released from inflammatory responses predisposes retina to CNV (Sakurai et al., 2003; Hollyfield et al., 2008). TF can also increase the expression of cytokines such as interleukin (IL)-1b, IL-6, IL-8 and other factors, which are associated with inflammation (Chu, 2006). Our results first showed that anti-TF monoclonal antibody reduced F4/80 levels in the laser-induced CNV
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Fig. 6. Anti-TF monoclonal antibody cans specific inhibition the development of CNV 2 weeks after laser photocoagulation. HE stains images of the effect of anti-TF monoclonal antibody on CNV formation. (A)Normal mice retinal and choroid structure, (BeD)Each photograph showed the central area of CNV lesions in the control, vehicle or anti-TF group mice retinal and choroid, Scale bar ¼ 100 mm (S: sclera C: choroid, RPE: retinal pigment epithelium; OS: outer segment; IS: inner segment; ONL: outer nuclear layer; OPL: outer plexi form layer; INL: inner nuclear layer; IPL: inner plexiform layer; NFL: nerve fiber layer) (E)Statistical analysis of the data presented from B to D, n ¼ 12e16 spots *P < 0.001. Black dash lines represented the edge of area of CNV lesions.
Fig. 7. Anti-TF monoclonal antibody significantly suppressed RPEechoroid protein levels of TF, VEGF, and F4/80 on day 7. In anti-TF monoclonal antibody groups, TF (A) VEGF (B) F4/ 80 (C) mRNA expression significantly increased compared with no laser photocoagulation and reduced in anti-TF group. **P < 0.001 *P < 0.005.
mice, suggesting the anti-TF monoclonal antibody maybe acts as an effective tool for decreasing macrophages leakage and inflammatory reaction. Our results coincide with these data and indicate that the suppression of macrophages infiltration acts as an important cellular mechanism for anti-TF monoclonal antibody treatment in the current model (Xie et al., 2012). Our findings further unveiled the involvement of TF in angiogenesis. Anti-TF may act as an attractive therapeutic approach for the treatment of CNV in human AMD, and have the potential to be extended to the management of other angiogenesis related diseases. The additional experiments remain necessary to test the optimum efficiency of anti-TF monoclonal antibody administration for CNV. Acknowledgments The study was supported by National Natural Science Foundation of China (81200680). References Abe, K., Shoji, M., Chen, J., Bierhaus, A., Danave, I., Micko, C., Casper, K., Dillehay, D.L., Nawroth, P.P., Rickles, F.R., 1999. Regulation of vascular endothelial growth
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hypoxia-inducible factor-1-independent mechanisms. Cancer Res. 66 (14), 7067e7074. Ruf, W., Disse, J., Carneiro-Lobo, T.C., Yokota, N., Schaffner, F., 2011. Tissue factor and cell signalling in cancer progression and thrombosis. J. Thromb. Haemost. 9 (Suppl. 1), 306e315. Ryan, S.J., 1979. The development of an experimental model of subretinal neovascularization in disciform macular degeneration. Trans. Am. Ophthalmol. Soc. 77, 707e745. Sakurai, E., Anand, A., Ambati, B.K., van Rooijen, N., Ambati, J., 2003. Macrophage depletion inhibits experimental choroidal neovascularization. Investig. Ophthalmol. Vis. Sci. 44 (8), 3578e3585. Schlingemann, R.O., 2004. Role of growth factors and the wound healing response in age-related macular degeneration. Graefes Arch. Clin. Exp. Ophthalmol. 242 (1), 91e101. Seo, M.S., Kwak, N., Ozaki, H., Yamada, H., Okamoto, N., Yamada, E., Fabbro, D., Hofmann, F., Wood, J.M., Campochiaro, P.A., 1999. Dramatic inhibition of retinal and choroidal neovascularization by oral administration of a kinase inhibitor. Am. J. Pathol. 154 (6), 1743e1753. Smith, W., Assink, J., Klein, R., Mitchell, P., Klaver, C.C., Klein, B.E., Hofman, A., Jensen, S., Wang, J.J., de Jong, P.T., 2001. Risk factors for age-related macular degeneration: pooled findings from three continents. Ophthalmology 108 (4), 697e704. Spaide, R.F., Chang, L.K., Klancnik, J.M., Yannuzzi, L.A., Sorenson, J., Slakter, J.S., Freund, K.B., Klein, R., 2009. Prospective study of intravitreal ranibizumab as a treatment for decreased visual acuity secondary to central retinal vein occlusion. Am. J. Ophthalmol. 147 (2), 298e306. Swaroop, A., Chew, E.Y., Rickman, C.B., Abecasis, G.R., 2009. Unraveling a multifactorial late-onset disease: from genetic susceptibility to disease mechanisms for age-related macular degeneration. Annu. Rev. Genomics Hum. Genet. 10, 19e43. Takehana, Y., Kurokawa, T., Kitamura, T., Tsukahara, Y., Akahane, S., Kitazawa, M., Yoshimura, N., 1999. Suppression of laser-induced choroidal neovascularization by oral tranilast in the rat. Investig. Ophthalmol. Vis. Sci. 40 (2), 459e466. Tezel, T.H., Bodek, E., Sonmez, K., Kaliappan, S., Kaplan, H.J., Hu, Z., Garen, A., 2007. Targeting tissue factor for immunotherapy of choroidal neovascularization by intravitreal delivery of factor VII-Fc chimeric antibody. Ocul. Immunol. Inflamm. 15 (1), 3e10. Tobe, T., Ortega, S., Luna, J.D., Ozaki, H., Okamoto, N., Derevjanik, N.L., Vinores, S.A., Basilico, C., Campochiaro, P.A., 1998. Targeted disruption of the FGF2 gene does not prevent choroidal neovascularization in a murine model. Am. J. Pathol. 153 (5), 1641e1646. Uusitalo-Jarvinen, H., Kurokawa, T., Mueller, B.M., Andrade-Gordon, P., Friedlander, M., Ruf, W., 2007. Role of protease activated receptor 1 and 2 signaling in hypoxia-induced angiogenesis. Arterioscler. Thromb. Vasc. Biol. 27 (6), 1456e1462. Valmaggia, C., Niederberger, H., Lang, C., Kloos, P., Haueter, I., 2008. The treatment of choroidal neovascularisation with intravitreal injections of bevacizumab (Avastin). Klin. Monbl Augenheilkd. 225 (5), 380e384. Wang, G.F., Zou, X.L., 2012. Tissue factor with age-related macular degeneration. Int. J. Ophthalmol. 5 (5), 609e613. Wickremasinghe, S.S., Xie, J., Lim, J., Chauhan, D.S., Robman, L., Richardson, A.J., Hageman, G., Baird, P.N., Guymer, R., 2011. Variants in the APOE gene are associated with improved outcome after anti-VEGF treatment for neovascular AMD. Investig. Ophthalmol. Vis. Sci. 52 (7), 4072e4079. Xie, P., Kamei, M., Suzuki, M., Matsumura, N., Nishida, K., Sakimoto, S., Sakaguchi, H., 2011. Suppression and regression of choroidal neovascularization in mice by a novel CCR2 antagonist, INCB3344. PLoS One 6 (12), e28933. Xie, P., Zhang, W., Yuan, S., Chen, Z., Yang, Q., Yuan, D., Wang, F., Liu, Q., 2012. Suppression of experimental choroidal neovascularization by curcumin in mice. PLoS One 7 (12), e53329. Yoshida, T., Ohno-Matsui, K., Yasuzumi, K., Kojima, A., Shimada, N., Futagami, S., Tokoro, T., Mochizuki, M., 2003. Myopic choroidal neovascularization: a 10-year follow-up. Ophthalmology 110 (7), 1297e1305. Zhang, J., Ding, J., Zhang, X., Shao, X., Hao, Z., 2005. Regulation of vascular endothelial growth factor (VEGF) production and angiogenesis by tissue factor (TF) in SGC-7901 gastric cancer cells. Cancer Biol. Ther. 4 (7), 769e772.
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Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer
Blockage of tissue factor ameliorates the lesion of laser-induced choroidal neovascularization in mice Li Wang a, Zhongkai Yang b, Ying Yu a, Chen Cui a, Huaijin Guan a, Hui Chen a, * a b
Eye Institute, Affiliated Hospital of Nantong University, 20 Xisi Road, Nantong 226001, Jiangsu, China Department of Ophthalmology, Xinhua Hospital, Yili, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 February 2014 Accepted in revised form 8 July 2014 Available online 22 July 2014
Choroidal neovascularization (CNV) occurs as a result of age-related macular degeneration (AMD) and causes severe vision loss among elderly patients. High expression of tissue factor (TF) was found in the retinas of AMD patients. In this study, we used anti-TF monoclonal antibody to test the effect of the TF blockage on experimental CNV induced by laser photocoagulation of retina in mice. Anti-TF monoclonal antibody or vehicle was administered intravitreally at day 1, 2 or 3 after laser application. We found that TF expression increased, and reached the peak at the 3rd week after the after laser application. Anti-TF monoclonal antibody can predominantly decrease the expression of TF, VEGF and F4/80, and reduced the area of CNV. Anti-TF monoclonal antibody also decreased incidence of CNV and leakage area. There were no pathological changes in the liver, heart, brain or kidney tissue. We conclude that TF plays an important role in CNV and anti-TF monoclonal antibody significantly decreases CNV in mouse model and anti-TF monoclonal antibody may have therapeutic potential in AMD. © 2014 Elsevier Ltd. All rights reserved.
Keywords: tissue factor choroidal neovascularization anti-TF monoclonal antibody mouse
1. Introduction Age-related macular degeneration (AMD) is the leading cause of blindness in most developed countries (Smith et al., 2001). Choroidal neovascularization (CNV) beneath the macula, which occurs at the end stage of the disease and is characterized as the ‘‘wet form’’ of AMD, causes severe central vision loss and it has serious effects on the quality of life in patients (Calabrese et al., 2011; Swaroop et al., 2009). CNV is pathological angiogenic process that arises from the choroid into the Sub-retinal pigment epithelium (RPE) or subretinal spaces (Yoshida et al., 2003; Campochiaro, 2000). Nevertheless, the pathogenic mechanisms underlying CNV are complex and still largely unknown, the current therapeutic options (VEGF trap (Bakall et al., 2013; Heier et al., 2012) and anti-VEGF antibodies (Wickremasinghe et al., 2011)) for CNV are effective but a portion of patients do not respond to the treatment well. In addition, current anti-VEGF drugs can increase the risks of RPE tears (Arias et al., 2007), cardiovascular disease (Valmaggia et al., 2008), nervous system disease (transient ischemic attack) (Spaide et al., 2009) and other systemic adverse events.
* Corresponding author. Tel.: þ86 13809082586; fax: þ86 513 85519820. E-mail address: [email protected] (H. Chen). http://dx.doi.org/10.1016/j.exer.2014.07.006 0014-4835/© 2014 Elsevier Ltd. All rights reserved.
Some studies showed that TF is one of the key mediators of physiological and pathologic angiogenesis (Cho et al., 2011; Belting et al., 2004; Rak et al., 2006; Ahamed and Ruf, 2004; Wang and Zou, 2012). TF is also known as coagulation factor III, which is a cellsurface glycoprotein receptor which initiates the blood coagulation cascade after vessel injury and implicated in various pathological processes (Harlos et al., 1994). TF can regulate the angiogenesis factor (Pendurthi et al., 2000), for instance, early growth response gene 1 (Egr-1) and vascular endothelial growth factor (VEGF) (Rong et al., 2006). Binding of FVIIa to TF directly causes protease-activated receptor (PAR)-2 and results in phosphorylation of the TF (Uusitalo-Jarvinen et al., 2007), subsequently promoting pathological angiogenesis. The lack of negative regulatory control by the TF cytoplasmic domain is also a pathway by which pro-angiogenic signaling of PAR-2 can be turned on (Bluff et al., 2008). A strong link exists between tumor VEGF and endothelial TF in vivo angiogenic conditions. VEGF and TF can upregulate each other (Croce and Libby, 2007). TF is over expressed in AMD tissue and it is triggered by inflammatory factors High expression of TF was found in RPE cells and macrophages by immunostaining of CNV biopsy (Grossniklaus et al., 2002), but not in the normal blood vessels (Belting et al., 2004). Our previous studies found that anti-TF siRNA can inhibit cell proliferation, cell migration, and tube formation in an in vitro model of neovascularization (Peng et al., 2013). Some studies
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2.4. Choroidal flat mount
showed that the frequency of leakage in CNV lesions was significantly reduced using fVII-verteporfin, a ligand that binds with exceptional affinity and specificity to its receptor TF (Lu et al., 2009). Earlier studies showed that the Icon (Tezel et al., 2007), which is composed of factor VII(fVII), conjugated to the Fc domain of an IgG1 Ig, the TFeIcon complex is strongly efficacious in preventing CNV formation, and the Icon functions as an anti-TF antibody (Bora et al., 2003). Anti-TF monoclonal antibody can significantly inhibit the growth of pulmonary metastases angiogenesis (Mueller et al., 1992) experimental lung metastasis of invasive breast cancer angiogenesis (Ngo et al., 2007) as well attenuated tumor growth and angiogenesis (Ruf et al., 2011). These results suggest that anti-TF monoclonal antibody may have beneficial and potential effects on CNV. In this paper, we tested first the efficacy of anti-TF monoclonal antibody on laser-induced mouse model of CNV.
Three mice per group were killed at 7 days after laser injury, and the area of the CNV lesions was quantified using the choroidal flat mount technique (Nagai et al., 2006). Briefly, eyes were enucleated and immediately fixed with 4% paraformaldehyde for 30 min at 4 C or ice. The anterior segment and retina of each eye were removed to obtain the RPE-choroidal complex, and the complex were washed three times in PBS (136.9 mMNaCl, 8.1 mMNa2HPO412H2O, 1.46 mM Na2H2PO2.2H2O). The RPE-choroidal complex then incubated overnight at 4 C with fluorescein isothiocyanate-conjugated isolectin B4 (1:150; Vector Laboratories, Burlingame, CA). The fluorescence microscope (DM400 B, Leica, Germany) with wavelength (488 nm) laser was used to observe CNV. The CNV-related fluorescence area was measured with Image J (USA National Institutes of Health, Bethesda, MD).
2. Methods
2.5. Fundus fluorescein angiography
2.1. Animals
To confirm the inhibitory effect of anti-TF 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 intraperitoneal injection of 0.3 mL of 2% fluorescein sodium (Alcon Laboratories, Irvine, CA) was injected into the intraperitoneal cavity of the mice as previously described (Gu et al., 2010). The laser lesions were studied using fluorescein angiography to evaluate CNV development and its activity. 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 the CNV lesion was measured three times and averaged using Image J (USA National Institutes of Health, Bethesda, MD) (Xie et al., 2011).
All experimental procedures were held 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). 2.2. Laser-induced CNV and drug treatment 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 the 3, 6, 9, and 12 o'clock positions between the retinal vessels in a per papillary area of both eyes. Only burns that produced a bubble, indicating the rupture of the Bruch's membrane, were counted in the study. The intravitreal injection of 1 ml of anti-TF monoclonal antibody (200 ng/ml; sc-376361; Santa Cruz Biotechnology, Santa Cruz, CA) (anti-TF group) or vehicle (5% glucose solution, GS) was carried out at day 1, 2 or 3 after laser injury, and the control group means the laser-induced lesion without any injection. 2.3. Western blot analysis The protein extract of RPE-choroid complexes and retina from 3 mice at 1, 2, 3, 4, 6, 9 weeks after laser injury were prepared for western blot analysis. Protein and a molecular weight marker were separated by 5% SDS-PAGE, and transferred to a PVDF membrane. The membrane was then incubated with dilution of the monoclonal mouse anti-TF (1:120; Santa Cruz Biotechnology, Santa Cruz, CA) and anti-VEGF (1:200; Santa Cruz Biotechnology, Santa Cruz, CA) antibodies in blocking (5% skim milk) solution over-night at 4 C. and reaction with second antibodies HRP-conjugated goat antimouse IgG (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA) at 37 C for 2 h. Extensive washes in 0.05% Tween-20 in TBS and followed by incubation with anti-b-actin (1:10000 SigmaeAldrich, Saint Louis, MO). The signals were developed using a chemiluminescence solution (ECL western blotting detection reagents; Amersham Pharmacia Biotech, Piscataway, NJ). The intensity of the b-actin signal was used as an endogenous control and quantified the band optical density using Image J (National Institutes of Health, Bethesda, MD).
2.6. Histopathology and immunohistochemistry Eight eyes, heart, liver, brain and kidney were enucleated in each group, and 8 mm cryosections were prepared for either HE staining (on day 14 after laser photocoagulation) or immunohistochemistry of TF (on day 7 after laser photocoagulation). The cryosections were blocked with 1% BSA for 4 h at room temperature, and were incubated over-night at 4 C with monoclonal mouse anti-TF (1:50; Santa Cruz Biotechnology, Santa Cruz, CA), the slides then incubated with FITC-conjugated secondary antibody donkey antimouse IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) and hochest (1:2000 SigmaeAldrich, Saint Louis, MO). 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 that had been stained with hematoxylin and eosin were examined using a light microscope. IPP 6.0 was used to calculate the maximum thicknesses and lengths of each CNV lesion. 2.7. qRT-PCR We have randomly selected 3 mice in each group, The RPEchoroid complex and retina mRNA was isolated (TRIzol extraction protocol; Invitrogen) at 7 days after laser coagulation, and reverse transcription was performed with mRNA according to the manufacturer's protocol. Real-time PCR was performed with SYBR Green PCR Master Mix on the ABI 7500 Real Time System (Applied Biosystems, Foster City, CA), Reaction conditions were as follows: 30 s at 95 C, followed by 40 cycles of 5 s at 95 C, 34 s at 60 C. The sequences of primers used for mouse VEGF: 5-TTACTGCT
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3. Results 3.1. Expression of TF in various time points after laser photocoagulation To investigate the role of TF in choroidal neovascularization, we extracted the protein of RPE-choroid complexes and retinas from each group were prepared for western blotting analysis several weeks after laser photocoagulation and normal group. After laser injury, TF protein expression showed a significant up-regulation, reached the peak at 3rd week, and then gradually declined to the baseline from 4th to 9th week. We detected a weak expression of TF in the normal mice (Fig. 1). 3.2. Expression of TF in the CNV lesions
Fig. 1. Changes in TF protein expression in the retina and RPE-choroid complexes at 1, 2, 3, 4, 6, 9 weeks after laser photocoagulation by western blotting analysis. (A) TF protein level was increased gradually after injury, peaked at 3rd week, and reduced thereafter. TF was low in the normal mice, b-actin protein level was used as the internal control. (B)TF protein level represented a ratio between the amount of target protein and that of b-actin protein control, *P < 0.05; significantly different from the normal mice.
GTACCTCCACC-3 and 5-ACAGGACG GCTTGAAGATG-3; TF: 5CACCGAGCAATGGAAGAGTT-3 and 5-TTGCACAGTTCCCATCAGAG3; F4/80: 5-TCGCTGCTGGTTGAA TACAG-3 and 5-GCAACCTCGTG TCCTTGAGT-3; b-actin: 5eGAT GACCCAGATCATGTTTGA-3 and 5GGAGAGCATAGCCCTCGTA G-3. 2.8. Statistical analysis All values were presented as means ± standard error of mean (SEM). One-way ANOVA was used for statistical comparisons of multiple groups. Descriptive statistics were performed using Stata statistical software version 11.0. P values <0.05 were considered statistically significant. Each experiment consisted of at least three replicates.
To research whether TF expression transforms during CNV structure, we examined the expression of TF in the CNV lesions by immunofluorescence staining. We contrasted TF expression was detected in laser-induced CNV lesion and we also saw trace amount of TF expression in normal retina. Compared with the vehicle or control group, TF expression was weakened in the anti-TF group (Fig. 2). 3.3. Anti-TF monoclonal antibody suppresses the expression of TF and VEGF We research suppression of TF and VEGF by anti-TF monoclonal antibody treatment after laser injury. The TF levels of the RPEchoroid complexes and retina were decreased significantly at 7 days in the anti-TF group compared with the control or vehicle group. Surprisingly, the VEGF expression revealed a similar change (Fig. 3). 3.4. Time dependence of the effect of anti-TF monoclonal antibody on CNV formation To indicate the time dependence of the effect of anti-TF monoclonal antibody treatment on CNV formation in laser-treated mice, anti-TF monoclonal antibody was intravitreal injected at 1 day, 2
Fig. 2. Anti-TF monoclonal antibody significantly suppressed TF level in the RPE-choroid complexes at 7 days after photocoagulation. TF expression transforms during CNV structure and anti-TF monoclonal antibody significantly suppressed RPEechoroid protein levels of TF (AeC) vehicle treatment, (DeF) anti-TF monoclonal antibody treatment (GeI)the normal mice. Phase contrast image (left column), TF expression (center column, green channel) TF and hochest (right column, blue channel; merged images) Scale bars ¼ 100 mm.
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Fig. 3. Suppression of TF and VEGF by anti-TF monoclonal antibody treatment was shown in the RPE-choroid complex and retinal tissues at 7 days after laser coagulation (A, C) Western blot analysis revealed that TF and VEGF expression was suppressed by anti-TF monoclonal antibody treatment (B, D) Quantification graphs for TF and VEGF. Data of relative TF protein level to b-actin are presented as the mean ± SD of three independent experiments, *P < 0.01, significantly different from the vehicle group.
days, or 3 days after laser treatment. At day 7 after laser injury, the area of CNV was quantified by analysis of choroidal flat mounts. The RPE-choroid flat mounts distinctly decreased in a CNV lesion after anti-TF monoclonal antibody treatment (Fig. 4A). At 7 days, the average CNV area was 17,629.5 ± 1218 mm2 in the control mice (n ¼ 23e25 spots), 16,700.4 ± 1528 mm2 in the vehicle group (n ¼ 23e24 spots). The average CNV area were (4725 ± 1418 mm2,n ¼ 24 spots), (5169 ± 1334 mm2, n ¼ 24 spots) and (4359 ± 1423 mm2, n ¼ 23 spots) at 1 day, 2 days and 3 days injection respectively in the anti-TF group. Compared with the control group, there were decline rate of 72%, 69% and 74% in CNV area by anti-TF monoclonal antibody treatment. But there was no significant difference among various time points (Fig. 4B).
3.5. Anti-TF monoclonal antibody reduced the leakage of CNV lesions Comparisons of fluorescein angiograms between vehicle and anti-TF monoclonal antibody treatment group proved that the leakage area of CNV was less severe in the anti-TF group than in the vehicle group on day 7 after photocoagulation (Fig. 5A). AntiTF monoclonal antibody decreased the number of spots showing strong dye staining (score 2 or more) and increased the number showing weak staining (score 0 or 1) (Fig. 5B). The incidence of CNV formation was 92% in the control group, 96% in the vehicle group, 57% in the anti-TF group (Table 1). The incidence of fluorescence leakage in the eyes of the CNV mice was significantly
Fig. 4. Time dependence of the effect of anti-TF monoclonal antibody on CNV formation. (A) At 7 days after laser treatment, the CNV area in the anti-TF group was significantly attenuated compared to the vehicle or control group, and the experiments were repeated three times with similar results. Scale bar ¼ 100 mm (B) Quantitative analysis of CNV size in RPEechoroid flat mounts. Values are mean ± SEM, *P < 0.01.
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Fig. 5. Fluorescein angiograms, staining grade, the incidence and leakage area of CNV were obtained at 7 days after laser photocoagulation. (A) Hyper fluorescent leakage surrounding the laser spots were relative weak leakage (grade 1) in the anti-TF mice retina. (B) The intensity of FFA leakage grade was weaker in the anti-TF group than in the control or vehicle group. (C) Comparison of semi-quantitative leakage score between vehicle- and anti-TF -treated mice. The mean leakage score of CNV in the control group was set at 100. *P < 0.01.
elevated relative to that in the vehicle mice at 7 days after photocoagulation. 3.6. Anti-TF monoclonal antibody decreases neovascularization area On day 14 after photocoagulation, Histopathology analysis showed that anti-TF monoclonal antibody treatments significantly decreased the thickness and length of retinal lesions caused by choroidal neovascularization, we also found that the retina detachment occur more often in the eye treated with laser (Fig. 6). And there were no pathological changes on liver, heart, brain or kidney tissue. 3.7. Anti-TF monoclonal antibody down-regulated TF, VEGF, F4/80 mRNA Quantitative RT-PCR detected a very low-level F4/80, TF and VEGF mRNA expression in RPE-choroid complexes in normal mice (without laser treatment). On day 7 after laser photocoagulation, the expression of F4/80, TF and VEGF mRNA increased. But anti-TF monoclonal antibody treatment significantly suppressed their expression (Fig. 7). 4. Discussion Our study demonstrated at first that treatment with anti-TF monoclonal antibody significantly decreases the dimensions of laser-induced CNV in mice model. The results further suggest that Table 1 Incidence of CNV after various treatments on 7th day after photocoagulation, the fewer incidence of CNV lesions in the anti-TF group was exhibited. A Chi-square test showed *P < 0.001. Groups
No. of mice
Laser burns
The incidence of CNV (%)
Control 5%GS Anti-TF
3 3 3
24 24 24
22/24 (92) 23/24 (96) 14/24 (57)*
TF may be an important molecular target for anti-angiogenesis therapies (Belting et al., 2005, 2004; Abe et al., 1999; Darmoul et al., 2003). CNV is a complex process that develops in several stages. Different growth factors (such as TF, VEGF) and cellular types are important in each stage of the pathogenesis of CNV (Grossniklaus et al., 2002; Green, 1991; Schlingemann, 2004). A break or defect in Bruchs' membrane is necessary for CNV to develop. In our experiments, we used anti-TF monoclonal antibody act on laserinduced mouse model of CNV, which is well established for investigating ocular angiogenesis (Ryan, 1979), Many studies have reported that krypton laser in C57BL6 mice with modified settings including a smaller spot size, higher intensity, and shorter duration resulted in a higher frequency of histologic evidence of CNV(Seo et al., 1999; Tobe et al., 1998). The mechanism of accelerating angiogenesis by TF could be that the TF/FVIIa complex promotes inflammation and angiogenesis through a protease-activated receptor that regulates intracellular signal transduction is safe. It has been reported that murine anti-TF antibodies have been successfully used in experimental melanoma metastasis models (Mueller et al., 1992). The mechanism underlying the tumor suppression by anti-TF monoclonal antibody might contribute to the apoptosis triggered by TF inhibition, which was due to inhibition of TF: FVIIa signaling (Hembrough et al., 2003). Some studies showed that overexpression of TF in gastric cancer cells (Zhang et al., 2005), enhances neovascularization growth by increasing the transcription of pro-angiogenic VEGF. And downregulated TF expression can contribute to decrease pathologic angiogenesis (Zhang et al., 2005). Therefore we consider that the reduction of TF can also reduce the expression of VEGF, thus reduce CNV. Our results suggest anti-TF monoclonal antibody can reduce the TF expression as well VEGF expression at the same time. Evidences suggest that the cytokines released from inflammatory responses predisposes retina to CNV (Sakurai et al., 2003; Hollyfield et al., 2008). TF can also increase the expression of cytokines such as interleukin (IL)-1b, IL-6, IL-8 and other factors, which are associated with inflammation (Chu, 2006). Our results first showed that anti-TF monoclonal antibody reduced F4/80 levels in the laser-induced CNV
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Fig. 6. Anti-TF monoclonal antibody cans specific inhibition the development of CNV 2 weeks after laser photocoagulation. HE stains images of the effect of anti-TF monoclonal antibody on CNV formation. (A)Normal mice retinal and choroid structure, (BeD)Each photograph showed the central area of CNV lesions in the control, vehicle or anti-TF group mice retinal and choroid, Scale bar ¼ 100 mm (S: sclera C: choroid, RPE: retinal pigment epithelium; OS: outer segment; IS: inner segment; ONL: outer nuclear layer; OPL: outer plexi form layer; INL: inner nuclear layer; IPL: inner plexiform layer; NFL: nerve fiber layer) (E)Statistical analysis of the data presented from B to D, n ¼ 12e16 spots *P < 0.001. Black dash lines represented the edge of area of CNV lesions.
Fig. 7. Anti-TF monoclonal antibody significantly suppressed RPEechoroid protein levels of TF, VEGF, and F4/80 on day 7. In anti-TF monoclonal antibody groups, TF (A) VEGF (B) F4/ 80 (C) mRNA expression significantly increased compared with no laser photocoagulation and reduced in anti-TF group. **P < 0.001 *P < 0.005.
mice, suggesting the anti-TF monoclonal antibody maybe acts as an effective tool for decreasing macrophages leakage and inflammatory reaction. Our results coincide with these data and indicate that the suppression of macrophages infiltration acts as an important cellular mechanism for anti-TF monoclonal antibody treatment in the current model (Xie et al., 2012). Our findings further unveiled the involvement of TF in angiogenesis. Anti-TF may act as an attractive therapeutic approach for the treatment of CNV in human AMD, and have the potential to be extended to the management of other angiogenesis related diseases. The additional experiments remain necessary to test the optimum efficiency of anti-TF monoclonal antibody administration for CNV. Acknowledgments The study was supported by National Natural Science Foundation of China (81200680). References Abe, K., Shoji, M., Chen, J., Bierhaus, A., Danave, I., Micko, C., Casper, K., Dillehay, D.L., Nawroth, P.P., Rickles, F.R., 1999. Regulation of vascular endothelial growth
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