Exudative age-related macular degeneration leads to severe central vision impairment due to choroidal neovascularization (CNV) resulting from increased vascular endothelial growth factor (VEGF) expression in the lesion area.1,2 Abnormal enhancement of VEGF expression in the Bruch's membrane and retinal pigment epithelium occurs as a consequence of cellular damages induced by intense light exposure and anoxia. VEGF is thought to play a pivotal role in pathological angiogenesis. Currently multiple factors, including adhesion molecules, matrix metalloproteinase (MMP), lipid deposition, inflammatory cytokines, degradation of the vascular basement membrane, migration and proliferation of the endothelial cells, and the formation of new capillaries, have been recognized to participate in the process of pathological angiogenesis.
The biological functions of VEGF are mediated by its specific receptors, namely, the fms-like tyrosine kinase (Flt-1) and the kinase insert domain-containing receptor (KDR). KDR is related to the proliferation and migration of endothelial cells, whereas Flt-1 only mediates cell migration, and it functions through different pathways. VEGF induces pathological angiogenesis mainly through KDR, which activates protein kinase C (PKC) and extracellular signal-regulated kinase (ERK), promotes the migration and proliferation of the vascular endothelial cells, and regulates the formation of new blood vessels. KDR is widely expressed in almost all the vascular endothelial cells.3,4
Annexin A2 (ANXA2) is a member of the calciumdependent phospholipid-binding protein superfamily and exists in the forms of monomers, heterodimers and heterotetramers. The N-terminal domain of the ANXA2 molecule comprises 30 amino acid residues and contains the binding site for calcium-binding protein S100A10 (P11) and phosphorylation and acetylation sites. The C-terminal of ANXA2 can bind to Ca2+, phospholipid and filamentous actin.5 The ANXA2 monomer is present mostly on the cell membrane, in the cytoplasm and cell nuclei, and can be expressed in vascular endothelial cells, mononuclear cells, macrophages, neural cells and some tumor cells. ANXA2 mediates a wide variety of biological effects, including signal transduction, cell migration, DNA synthesis and cell apoptosis.
Recent evidences suggest that ANXA2 overexpression is closely associated with a wide spectrum of neovascularization-related diseases,6,7 but the role of ANXA2 in retinal and choroidal neovascularization is poorly documented. ANXA2 is thought to play important roles in retinal neovascularization by promoting the degradation of extracellular matrix (ECM), vascular endothelial cell migration and vascular remodeling. As a plasminogen and an active receptor of tissue plasminogen activator (tPA), the membrane-bound ANXA2 promotes the synthesis of plasmin, which converts the pro-matrix metalloproteinases into MMPs, and in the presence of plasmin, the activated MMPs induce ECM degradation, vascular endothelial cell migration and neovascularization.8,9 Hypoxia can induce the overexpression of phospholipase A2 (PLA2),10 and after binding to its receptors, the membrane-bound or extracellular ANXA2, PLA2 can stimulate the synthesis of prostaglandins (PGs), increasing the expression of hypoxia-inducible factor-1α (HIF-1a) and inducing the expression of VEGF. In addition, PGs also participates in the angiogenesis process by inducing MMP expression and ECM degradation.11 The intracellular ANXA2 may serve as the substrate for PKC to have its serine residue at the position 25 phosphorylated, thereby enabling the entry of ANXA2 into the cell nuclei and promoting DNA synthesis and cell proliferation.12
In spite of the numerous studies examining the role of ANXA2 in angiogenesis, the relation between intracellular ANXA2 and VEGF in the course of angiogenesis remains poorly examined. Based on the current understanding of the regulatory role of ANXA2 in neovascularization, as either receptors or ligands for tPA, PLA, PKC, PLA2, and angiostatin,13-15 we presume that ANXA2 is in some way implicated in the signaling pathways of VEGF-induced neovascularization. In this study, we examined the expression of the membranebound annexin proteins and the effect of ANXA2 on VEGF expression in an animal model of CNV and a retinal pigment epithelial cell line RPE-J, aiming to further understand the roles of these annexin proteins and the mechanisms of their actions in neovascularization, which may shed light on the exploration of new treatment pathways of CNV.
Animal model of CNV
A total of 132 adult brown Norwegian rats (10-12 weeks of age, weighing 200-250 g) meeting the Association for Research in Vision and Ophthalmology criteria for experimental animals were provided by the Experimental Animal Center of the Second Military Medical University. Animal models of laser-induced CNV were established according to the methods described in previous reports.16-18 Ten spots around the optic papilla were selected on the retina between the retinal vessels for laser coagulation. An argon laser slit lamp system (Coherent Radiation Systems, Novus 2000, USA) was used to induce CNV using a laser coagulation spot with a diameter of 50 μm, an exposure time of 0.05 seconds, and a laser power of 360 mW. Laser coagulation of the retina resulted in disruption of the Bruch's membrane and formation of bubbles. After the coagulation, the fundus oculi was photographed and the rats were sacrificed following fundus fluorescein angiography (FFA) at different time points. Laser coagulation was performed only in the left eyes (exposure group) of the rats, with the right eyes serving as the control.19,20
Tissue sample preparation
At days 0, 7, 14, 21, 28 and 56 after the laser coagulation, the eyes of the rats (n=6) were removed following FFA and immersed in RNA stabilizing buffer (RNAlater; Ambion, USA). Two hours later, the eyes were transferred to a refrigerator for preservation at -80°C. At each of the specified time points, the eyes of the rats (n=6) exposed to laser coagulation and other control eyes were for immunohistochemical study of the retina and choroid.21,22
Cell culture and laser ablation
RPE-J cells were provided by the Cell Source Center of Shanghai Institute of Life Sciences, Chinese Academy of Science. The argon laser generator with stable and controllable laser energy and the endolaser optical fiber were used for cell photocoagulation. At the preset laser energy (25 mW) and exposure time, cell photocoagulation was performed with the laser probe positioned at 1.5 mm from the cells.23 Each independent experiment was repeated for 3 times.
RNA extraction and quantitative real-time polymerase chain reaction (PCR)
Totally 6 rats were selected for quantitative real-time PCR. The total RNA was extracted using a RNAeasy Mini kit (Qiagen, USA) and quantified using spectrophotometry (A260/A280 ratio >1.8).24,25 The mRNA expressions of ANXA2, ANXA4, ANXA5, ANXA7 ANXA11, and VEGF (n=6) were quantified with real-time PCR following the method of SYBR® Green PCR Master Mix (Applied Biosystems, USA), and the primers were designed based on the GenBank data using Primer Express version 1.5 software (Applied Biosystems). The primer sequences, annealing temperature and the expected product length were list in Table.26 All the quantitative real-time PCR data were analyzed with an ABI Prism® 7700 Sequence Detector (Applied Biosystems). The mRNA expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the internal control for quantitative real-time PCR of the mRNA samples.
Immunohistochemistry for ANXA2 and VEGF
Also at the specified time points, 36 eyes were collected from the rats for immunohistochemistry examination of ANXA2 and VEGF protein. The eyeballs were fixed for 24 hours in 4% paraformaldehyde, and after careful removal of the crystalline lens and the vitreous body, the posterior wall of the eyeball containing the optic disc and the coagulated spots were prepared into paraffin sections. After routine deparaffinization and dehydration,27 the 5-μm-thick paraffin sections of the tissue samples were treated with H2O2 (Dako, Australia) for 10 minutes to inactivate the endogenous nucleases, and washed with TBS (pH 7.6) for 3 times prior to incubation with the rabbit anti-rat monoclonal antibodies (BD Transduction Labs, USA) of ANXA2 or VEGF (1:200) for 1 hour at room temperature. After washing with TBS again for 3 times, the sections were incubated for 30 minutes with biotinylated goat anti-rabbit antibody and washed with TBS for 3 times followed by staining with 3-amino-9-ethylcarbazole chromogen (AEC; Dako, Denmark) for 3 minutes, hematoxylin counterstaining and mounting.28,29 For the negative control, TBS was used instead of the first antibody. The IPP 4.5 software was used for analysis of the results of immunohistochemistry.30
Western blotting for ANXA2 and VEGF
Western blotting was performed according to the method of Yoon et al.31 Briefly, 3 μg of the total protein fractions extracted from each tissue sample (the retina containing the pigment epithelium but not the choroid) underwent electrophoresis and was transferred onto nitrocellulose filter (1.5 hours, 120 V). Rabbit anti-rat ANXA2 monoclonal antibody (mAb) or rabbit anti-rat VEGF mAb was applied on the blots followed by incubation with goat anti-rabbit IgG-horseradish peroxidase conjugate for 1 hour. The filter was then developed with 3,3'-diaminobenzidine (DAB) and analyzed after washing and drying.
In vitroandin vivoRNA interference of ANXA2, VEGF and KDR
The double-stranded small interfering RNAs (siRNAs) targeting ANXA2 (siANXA2, Sense: 5′-CAATGCACA-GAGGCAGGACAT-3′ and antisense: 5′-TTGGTTC- TTGAGCAGATGATC-3′), VEGF (siVEGF, Sense: 5′-AGCCCTCCCGGCTCTACGACG-3′ and antisense: 5′-CAGGAGTAAGTTCTCCAAGTT-3′) and KDR (siKDR, Sense: 5′-AAGAGCACACCUCUGACGGCUGAUGGA-3′ and antisense: 5′-UCCAUCAGCCGUCAGAGGUCUGCUCUU-3′) were synthesized using a commercially available kit (Ambion, Austin, TX, USA). Irrelevant oligonucleotides were used as the control for siANXA2, siVEGF and siKDR (SiANXA2_M, SiVEGF_M, and SiKDR_M, respectively). The RPE-J cells were plated in a 6-well plate and transfected with the siRNAs or their corresponding controls using siPORT (Ambion), and analysis was carried out 1 day after the transfection.32
The recombinant adenoviral vector containing ANXA2 gene (Ad_ANXA2) was constructed using the method described by Ishii et al.33 Homologous recombination of the ANXA2 gene and the adenoviral genome was accomplished using a commercial kit for adenoviral vector construction (TaKaRa, Ohtsu, Japan). The ANXA2 gene fragment was inserted into a shuttle plasmid containing the homologous sequence of the adenoviral genome, and the shuttle plasmid was co-transfected with a plasmid harboring the adenoviral genome into 293 cells. By homologous recombination between the homologous sequences in the shuttle plasmid and the adenoviral genome, ANXA2 gene was integrated into the adenoviral genome to generate the recombinant adenovirus with ANX2 gene insertion at the E1 or E3 depletion site. The recombinant adenovirus was then replicated and packaged into viral particles in 293 cells. The adenoviral vector containing no target gene fragment served as the control (AdNull).
The rats were anesthetized and when pupil dilation occurred, 1 μg of the siRNAs or 1 μl Ad_ANXA2 (containing 1×109 /μl viral particles) was injected into the vitreous cavity (n=6) using a Harvard microinjection pump and a glass microinjection syringe prior to laser coagulation of the retina. Fourteen days after laser coagulation, quantitative real-time PCR, Western blotting of the retina containing the pigment epithelium but not the choroid and pathological examination of the retina and choroid were performed. For pathological examination, the eyeballs of the rats were removed and the cup-shaped posterior portion of the eyeball was fixed in buffered 4% paraformaldehyde overnight. The tissues were then cut into rectangular blocks containing the 10 spots of laser coagulation and the optic disc, and the blocks were prepared routinely into serial paraffin sections (5 μm) for HE staining. The sections were observed microscopically (×400) and the field of interest was digitized using software.
Enzyme-linked immunosorbent assay (ELISA) for NAXA2 and VEGF contents in RPE-J cell culture
After laser coagulation of the RPE-J cells, 200 μl of the supernatant of the cell culture was collected and centrifuged at 750 ×g for 5 minutes. The protein concentrations of ANXA2 and VEGF were determined using the ELISA kits for ANXA2 and VEGF (R&D Systems, USA) according to the manufacturer's instructions. Each sample was examined in duplicate. Colorimetric analysis was performed on a BioRad DC microplate reader (Bio-Rad Laboratories, USA) and the optical density of the samples was measured at 450 nm and 570 nm to calculate the protein concentrations according to the standard curve generated.34
Immunofluorescent assay for VEGF protein expressionin vitro
The RPE-J cells were transfected with siANXA2 and siANXA2_M, and treated at temperature for 5 minutes with PBS containing 2% paraformaldehyde and 0.25% Triton X-100, or alternatively with 2% paraformaldehyde only. The cells were blocked with 20% normal goat serum (NGS) and incubated at 37°C for 60 minutes with FITC-labeled rabbit anti-rat VEGF mAb diluted at 1:100 with PBS containing 1% NGS. The protein expression of VEGF in the transfected cells was detected using a fluorescence microscope (Nikon, Japan).35
SPSS 12.0 software was used for statistical analysis of the data. Data were presented as mean ± standard error (SE). Paired t test was performed for comparison of the data from the laser-exposed and control eyes, and the correlation between VEGF and the annexin genes in the choroid tissues was assessed using Spearman rank correlation test. A P value less than 0.05 was considered to indicate statistically significant difference.35
Animal models of laser coagulation-induced CNV
Laser coagulation resulted in pale spots of burn injuries at all the selected sites, often with vacuolization in the center (Figure 1A-1F). The fluorescein angiograms displayed a bright central area in the area of CNV due to fluorescein leakage with gradually dimmed peripherals illustrating the formation of irregular neovessels. This fluorescence pattern associated with CNV occurred 1 week after laser coagulation and persisted till 8 weeks.
Annexins and VEGF mRNA expressions in rat models of CNV
Three weeks after laser coagulation, ANXA2 and VEGF mRNA expressions showed significant changes in the choroid and retina of the rats, whereas the mRNAs of the membrane bound ANXA4, ANXA5, ANXA7, or ANXA11 underwent no obvious changes. ANXA2 and VEGF mRNA expressions increased obviously 7 days to 21 days after the coagulation, reaching the highest level on day 14 (Figure 1G). A significant positive correlation was noted between ANXA2 and VEGF mRNA expressions, with the Spearman rank correlation coefficient being 0.609 (P=0.0127).
ANXA2 and VEGF protein expressions in the retina
Immunohistochemistry identified the presence of ANXA2 and VEGF proteins in the vascular endothelial cells, ganglion cells, inner nuclear layer and retinal pigment epithelial cells (Figure 2A-2F). Maximal ANXA2 and VEGF protein expressions occurred on day 14 following the coagulation (Figure 2C), which was 5.1 and 4.3 folds those in the control eyes ((17.20±1.78) IAU vs. (4.84±1.58) IAU (image arbitrary unit), P <0.0001; (15.86±2.12) IAU vs. (3.56±1.22) IAU, P <0.0001, Figure 2G and 2H), respectively. On day 3 after the coagulation, ANXA2 and VEGF protein expressions in the coagulated eyes showed no significant differences from those in the control eyes ((4.01±1.238) IAU vs. (4.32±1.25) IAU, P=0.9480; (4.77±2.003) IAU vs. (3.90±2.15) IAU, P=0.9020). Western blotting also yielded similar results with regard to ANXA2 and VEGF expressions in the choroid and retina, which increased to the maximum on day 14 and began to decrease on day 28 till recovery of the control level on day 56 (Figure 2I and 2J).
VEGF expression after RNA interferencein vitroandin vivo
Quantitative PCR showed that, in the RPE-J cells, transfection with siANXA2 significantly inhibited the expression of VEGF mRNA, resulting in reduction of the ANXA2 and VEGF mRNA expressions by 90% and 77%, respectively. But the transfection did not produce obvious changes in the expressions of VEGF receptor 2 (VGFR2, or KDR) or VEGFR1 (Flt-1) (Figure 3A). Three days after transfection of the cells with siKDR, quantitative PCR showed markedly increased expression of ANXA2 mRNA, whereas siVEGF or siFlt-1 transfection did not cause significant changes in ANXA2 mRNA expression (Figure 3B).
Immunohistochemistry with FITC-labeled VEGF mAb demonstrated the absence of VEGF protein expression in cultured RPE-J cells following siANXA2 transfection (Figure 4C). In contrast, obvious VEGF expression was found in the non-transfected cells or cells transfected with siANXA2_M (Figure 4A and 4B). The results of Western blotting showed a substantial reduction in VEGF protein by about 85% in RPE-J cells transfected with siANXA2 (P=0.02025) (Figure 4D). ELISA performed on days 1, 3, 5, 7 and 9 following siANXA2 transfection of the cells revealed that the lowest VEGF protein level occurred on day 3 (Figure 4E).
As the number of neovessels varies in different models of CNV, comparison of the absolute number of the newly generated blood vessels may seem unconvincing. Instead, comparison of the relative number of the newly formed blood vessels in the same experiment is more scientifically valid, which is defined as the ratio of the average newly generated vessel numbers in a unit area between the eyes with siANXA2 injection and those with Ad_ANXA2 injection. We administered the siANXA2 and Ad_ANXA2 into the vitreous body of the rats 1 day after laser coagulation and detected the VEGF expressions at both mRNA and protein levels 14 days later. The results showed that in comparison with siANXA2_M, microinjection of siANXA2 significantly lowered the expression levels of VEGF mRNA and protein (Figure 5), and reduced the number of newly formed vessels in the choroid (Figure 6). In addition, significantly higher ANXA2 mRNA and protein expressions were found in the eyes with siKDR injection than those with siKDR_M injection (P <0.05, Figure 5C). Microinjection of siVEGF, however, did not significantly affect the expressions of ANXA2. Compared with the control group, the expression levels of VEGF mRNA and protein were significantly increased in Ad_ANXA2 group (P <0.05, Figure 6).
An animal model that mimics human choroid CNV using argon laser coagulation was established in this study.18,19 In this model, the choroidal neovessels penetrated the disrupted Brunch's membrane and grew beneath the retina; we also observed the retinal pigment epithelial cells and the proliferation and migration of the choroidal vascular endothelial cells. In both the animal model of choroid CNV and the retinal pigment epithelial RPE-J cells, we investigated the expression of the annexin proteins and their association with VEGF.
The expressions of ANXA2 and VEGF mRNA were both increased in rats with laser coagulation-induced choroid CNV, as may also occur in other pathologies involving neovascularization due to various reasons.13,14 Of much importance was our finding that siANXA2, which significantly inhibited the expression of ANXA2 mRNA, also substantially lowered the expression of VEGF mRNA in models of CNV, suggesting that the expression of ANXA2 may promote the transcription of VEGF gene. In both rat models of CNV and RPE-J cells, siANXA2 did not cause significant changes in the expressions of Flt-1 or KDR mRNAs, whereas the application of siKDR, but not siFlt-1 or siVEGF, resulted in increased expression of ANXA2 mRNA expression in the retina, suggesting that KDR regulates the mRNA expression of ANXA2 through a negative feedback mechanism both in vivo and in vitro. Flt-1 and VEGF, according to these results, do not affect the expression of ANXA2, indicating that ANXA2 regulates VEGF expression through a positive feedback mechanism.
Immunohistochemistry revealed obviously increased expression of ANXA2 and VEGF proteins in the choroid after laser coagulation in comparison with normal choroid. This endogenous ANXA2 protein may not be the most important factor responsible for occurrence of CNV, but can be involved in regulating the internal environment and promoting angiogenesis. This phenomenon was also confirmed in studies of tumor angiogenesis. These results support the hypothesis that ANXA2 plays the role of an activator in the process of neovascularization.36 Immunohistochemistry and pathological examination showed obviously lowered ANXA2 protein expression and fewer newly generated blood vessels in rat choroid and retina with siANXA2 transfection following laser exposure as compared with those in the control group. This suggests inhibition of ANXA2 expression produces inhibitory effects on choroidal and retinal neovascularization, and ANXA2 may serve as a potential biomarker for neovascularization.37
VEGF and its receptors play critical roles in neovascularization.38 Three types of VEGF receptors have been identified, namely Flt-1, KDR and Flt-4, among which KDR is thought to play the major role in the angiogenesis process of neovascularization.39 The expressions of VEGF and its receptors are regulated by a complex network to maintain their relative balance in promoting angiogenesis,40 and ANXA2 is probably a part of this network. In the event of laser exposure to induce choroidal or retinal injuries, both VEGF and ANXA2 expressions are up-regulated. Increased VEGF induces enhancement of Flt-1 and KDR expression,41 and increased KDR results in inhibition of ANXA2 expression and VEGF expression. Lowered VEGF expression lowers the expression levels of KDR accordingly, which leads to relief of ANXA2 inhibition. In the process of neovascularization, therefore, all the factors involved maintain a relative balance.
Choroidal and retinal neovascularization is one of the important causes of blindness, and remains the focus of current molecular therapy. Previous studies identified VEGF as the major stimulator of choroidal and retinal neovascularization,42 and therefore VEGF has been the important target in researches to inhibit neovascularization. Although currently we do not fully understand the role ANXA2 in the mechanism of neovascularization, its capacity to enhance VEGF activity is confirmed.43 Further studies of the exact role of ANXA2 in the process of neovascularization are needed, which may facilitate the development of the therapeutic approaches targeting ANXA2.
1. Iranmanesh R, Eandi CM, Peiretti E, Klais CM, Garuti S, Goldberg DE, et al. The nature and frequency of neovascular age-related macular degeneration. Eur J Ophthalmol 2007; 17: 75-83.
2. Lin RC, Rosenfeld PJ. Antiangiogenic therapy in neovascular age-related macular degeneration. Int Ophthalmol Clin 2007; 47: 117-137.
3. Wong C, Jin ZG. Protein kinase C-dependent protein kinase D activation modulates ERK signal pathway and endothelial cell proliferation by vascular endothelial growth factor. J Biol Chem 2005; 280: 33262-33269.
4. Lukiw WJ, Ottlecz A, Lambrou G, Grueninger M, Finley J, Thompson HW, et al. Coordinate activation of HIF-1 and NF-kappaB DNA binding and COX-2 and VEGF expression in retinal cells by hypoxia. Invest Ophthalmol Vis Sci 2003; 44: 4163-4170.
5. Yoshiji H, Kuriyama S, Ways DK, Yoshii J, Miyamoto Y, Kawata M, et al. Protein kinase C lies on the signaling pathway for vascular endothelial growth factor-mediated tumor development and angiogenesis. Cancer Res 1999; 59: 4413-4418.
6. Sharma MC, Sharma M. The role of annexin II in angiogenesis and tumor progression: a potential therapeutic target. Curr Pharm Des 2007; 13: 3568-3575.
7. Rescher U, Gerke V. Annexins — unique membrane binding proteins with diverse functions. J Cell Sci 2004; 117: 2631-2639.
8. Ling Q, Jacovina AT, Deora A, Febbraio M, Simantov R, Silverstein RL, et al. Annexin II regulates fibrin homeostasis and neoangiogenesis in vivo.
J Clin Invest 2004; 113: 38-48.
9. Lobov S, Croucher DR, Saunders DN, Ranson M. Plasminogen activator inhibitor type 2 inhibits cell surface associated tissue plasminogen activator in vitro:
potential receptor interactions. Thromb Haemost 2008; 1002: 319-329.
10. Michiels C, Renard P, Bouaziz N, Heck N, Eliaers F, Ninane N, et al. Identification of the phospholipase A(2) isoforms that contribute to arachidonic acid release in hypoxic endothelial cells: limits of phospholipase A(2) inhibitors. Biochem Pharmacol 2002; 63: 321-332.
11. Ottino P, Bazan HE. Corneal stimulation of MMP-1, -9 and uPA by platelet-activating factor is mediated by cyclooxygenase-2 metabolites. Curr Eye Res 2001; 23: 77-85.
12. Luo W, Yan G, Li L, Wang Z, Liu H, Zhou S, et al. Epstein-Barr virus latent membrane protein 1 mediates serine 25 phosphorylation and nuclear entry of annexin A2 via PI-PLC-PKCalpha/PKCbeta pathway. Mol Carcinog 2008; 47: 934-946.
13. Katanasaka Y, Asai T, Naitou H, Ohashi N, Oku N. Proteomic characterization of angiogenic endothelial cells stimulated with cancer cell-conditioned medium. Biol Pharm Bull 2007; 30: 2300-2307.
14. Syed SP, Martin AM, Haupt HM, Arenas-Elliot CP, Brooks JJ. Angiostatin receptor annexin II in vascular tumors including angiosarcoma. Hum Pathol 2007; 38: 508-513.
15. Sharma MR, Rothman V, Tuszynski GP, Sharma MC. Antibody-directed targeting of angiostatin's receptor annexin II inhibits Lewis lung carcinoma tumor growth via blocking of plasminogen activation: possible biochemical mechanism of angiostatin's action. Exp Mol Pathol 2006; 81: 136-145.
16. Bora NS, Kaliappan S, Jha P, Xu Q, Sivasankar B, Harris CL, et al. CD59, a complement regulatory protein, controls choroidal neovascularization in a mouse model of wet-type age-related macular degeneration. J Immunol 2007; 178: 1783-1790.
17. Apte RS, Richter J, Herndon J, Ferguson T. Macrophages inhibit neovascularization in a murine model of age-related macular degeneration. PloS Med 2006; 3: 310-319.
18. Tobe T, Takahashi K, Ohkuma H, Uyama M. Experimental choroidal neovascularization in the rat. J Jpn Ophthalmol Soc 1994; 98: 837-845.
19. Imamura Y, Noda S, Hashizume K, Shinoda K, Yamaguchi M, Uchiyama S, et al. Drusen, choroidal neovascularization, and retinal pigment epithelium dysfunction in SOD1-deficient mice: a model of age-related macular degeneration. Proc Natl Acad Sci USA 2006; 103: 11282-11287.
20. Kiilgaard JF, Andersen MV, Wiencke AK, Scherfig E, la Cour M, Tezel TH, et al. A new animal model of choroidal neovascularization. Acta Ophthalmol Scand 2005; 83: 697-704.
21. Dejneka NS, Kuroki AM, Fosnot J, Tang W, Tolentino MJ, Bennett J. Systemic rapamycin inhibits retinal and choroidal neovascularization in mice. Mol Vis 2004; 22: 964-972.
22. Shen WY, Yu MJ, Barry CJ, Constable IJ, Rakoczy PE. Expression of cell adhesion molecules and vascular endothelial growth factor in experimental choroidal neovascularisation in the rat. Br J Ophthalmol 1998; 82: 1063-1071.
23. Reich SJ, Fosnot J, Kuroki A, Tang W, Yang X, Maguire AM, et al. Small interfering RNA (siRNA) targeting VEGF effectively inhibits ocular neovascularization in a mouse model. Mol Vis 2003; 30: 210-216.
24. Piri N, Kwong JM, Song M, Elashoff D, Caprioli J. Gene expression changes in the retina following optic nerve transection. Mol Vis 2006; 22: 1660-1673.
25. Chan CK, Pham LN, Zhou J, Spee C, Ryan SJ, Hinton DR. Differential expression of pro-and antiangiogenic factors in mouse strain-dependent hypoxia-induced retinal neovascularization. Lab Invest 2005; 85: 721-733.
26. Morrison TB, Weis JJ, Wittwer CT. Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification. Biotechniques 1998; 24: 954-958.
27. Murata T, Ishibashi T, Inomata H. Immunohistochemical detection of blood-retinal barrier breakdown in steptozotocindiabetic rats. Graefes Arch Clin Exp Ophthalmo 1993; 231: 175-177.
28. Zambarakji HJ, Nakazawa T, Connolly E. Dose dependent effect of pitavastatin on VEGF and angiogenesis in a mouse model of choroidal neovascularization. Invest Ophthalmol Vis Sci 2006; 47: 2623-2631.
29. Itaya M, Sakurai E, Nozaki M, Yamada K, Yamasaki S, Asai K, et al. Upregulation of VEGF in murine retina via monocyte recruitment after retinal scatter laser photocoagulation. Invest Ophthalmol Vis Sci 2007; 48: 5677-5683.
30. Nicholls SJ, Cutri B, Worthley SG, Kee P, Rye KA, Bao S, et al. Impact of short-term administration of high-density lipoproteins and atorvastatin on atherosclerosis in rabbits. Arterioscler Thromb Vasc Bio 2005; 125: 2416-2421.
31. Yoon HS, Rho SH, Jeong JH, Yoon S, Yoo KS, Yoo YH. Genistein produces reduction in growth and induces apoptosis of rat RPE-J cells. Curr Eye Res 2000; 20: 215-224.
32. Tolentino MJ, Brucker AJ, Fosnot J, Ying GS, Wu IH, Malik G, et al. Intravitreal injection of vascular endothelial growth factor small interfering RNA inhibits growth and leakage in a nonhuman primate, laser-induced model of choroidal neovascularization. Retina 2004; 24: 660-672.
33. Ishii H, Yoshida M, Hajjar KA, Yasukochi Y, Numano F. Construction of recombinant adenoviral vector of annexin II. Ann NY Acad Sci 2000; 902: 311-314.
34. Ottino P, Finley J, Rojo E, Ottlecz A, Lambrou GN, Bazan HE, et al. Hypoxia activates matrix metalloproteinase expression and the VEGF system in monkey choroid-retinal endothelial cells: involvement of cytosolic phospholipase A2 activity. Molecular Vision 2004; 10: 341-350.
35. She H, Nakazawa T, Matsubara A, Connolly E, Hisatomi T, Noda K, et al. Photoreceptor protection after photodynamic therapy using dexamethasone in a rat model of choroidal neovascularization. Invest Ophthalmol Vis Sci 2008; 49: 5008-5014.
36. Katanasaka Y, Asai T, Naitou H, Ohashi N, Oku N. Proteomic characterization of angiogenic endothelial cells stimulated with cancer cell-conditioned medium. Biol Pharm Bull 2007; 30: 2300-2307.
37. Yu GR, Kim SH, Park SH, Cui XD, Xu DY, Yu HC, et al. Identification of molecular markers for the oncogenic differentiation of hepatocellular carcinoma. Exp Mol Med 2007; 31: 641-652.
38. Kroll J, Waltenberger J. The vascular endothelial growth factor receptor KDR activates multiple signal transduction pathways in porcine aortic endothelial cells. J Biol Chem 1997; 272: 32521-23527.
39. Bussolati B, Dunk C, Grohman M, Kontos CD, Mason J, Ahmed A. Vascular endothelial growth factor receptor-1 modulates vascular endothelial growth factor-mediated angiogenesis via nitric oxide. Am J Pathol 2001; 159: 993-1008.
40. Gille H, Kowalski J, Li B, LeCouter J, Moffat B, Zioncheck TF, et al. Analysis of biological effects and signaling properties of Flt-1 (VEGFR-1) and KDR (VEGFR-2). A reassessment using novel receptor-specific vascular endothelial growth factor mutants. J Biol Chem 2001; 276: 3222-3230.
41. Zachary I, Gliki G. Signaling transduction mechanisms mediating biological actions of the vascular endothelial growth factor family. Cardiovasc Res 2001; 49: 568-581.
42. Campochiaro PA, Hackett SF. Ocular neovascularization: a valuable model system. Oncogene 2003; 29: 6537-6548.
43. Hayes MJ, Moss SE. Annexins and diseases. Biochem Biophys Res Commun 2004; 322: 1166-1170.