Introduction

Neovascular diseases of the retina are major causes of blindness that often affect young people in their productive ages.¹ Pathologic retinal angiogenesis is a final common pathway leading to vision loss in diabetic retinopathy, retinopathy of prematurity, ischemic retinal vein occlusion, and age-related macular degeneration. This pathologic angiogenesis is characterized by extensive proliferation of new blood vessels in the retina. Current therapies aimed at controlling this aberrant angiogenesis, such as pan retinal photocoagulation and cryotherapy, are only partially effective and are destructive to the retina. These therapies have side effects that include macular edema, decreased visual fields, and decreased visual acuity.^2,3 Moreover, it is estimated that as many as 50% of patients are not diagnosed early enough for these treatments to be effective. Alternative nondestructive therapies could benefit patients with neovascular diseases of the retina. Viral vector-mediated long-term expression of anti-angiogenic factors may promise alternative therapy.

Angiostatin is one of the most potent endogenous anti-angiogenic factors. Angiostatin is derived from plasminogen and was originally purified from the serum and urine of mice bearing primary Lewis lung carcinoma tumors.⁴ Angiostatin, which inhibits endothelial cell proliferation, has significantly greater tumor growth inhibition properties than most anti-angiogenic drugs described previously. Both purified recombinant angiostatin and vectors carrying the angiostatin expression unit have been successfully used for inhibition of tumor growth and metastasis in various cancer models.^5,6,7 Recombinant angiostatin generated by the yeast expression system is now being used for clinical trials. Potent inhibitory effects of angiostatin on endothelial cell proliferation are promising not only for the treatment of cancer, but also for the treatment of pathologic neovascularization in various noncancer disorders such as diabetic retinopathy and rheumatoid arthritis.

Gene therapy approaches have been applied to inhibit ocular angiogenesis. Choroidal neovascularizaiton has been inhibited by adeno-associated virus (AAV) vector-mediated expression of pigment epithelium-derived factor (PEDF),⁸ angiostatin,⁹ soluble VEGF receptor sFlt-1,^10,11 and adenovirus-mediated expression of endostatin.¹² In this experiment, using a mouse model of ischemia-induced retinal neovascularization,¹³ we demonstrated that HIV-mediated expression of angiostatin resulted in efficient inhibition of retinal neovascularization. Angiostatin gene therapy is a nondestructive treatment that may be effective in patients who do not respond to current therapies.

Results

HIV vector-mediated expression of angiostatin

The coding sequence of angiostatin (consisting of the first four kringle domains of plasminogen) was cloned by PCR and inserted downstream of the CAG promoter in the HIV vector construct. The VSV-G pseudotyped HIV vector containing the angiostatin expression unit (HIV-angiostatin) was generated and concentrated by the combination of ultrafiltration and ultracentrifugation. HIV vector-mediated gene transfer and expression was confirmed by RNA and protein analysis. RNA was extracted from HeLa cells transduced with HIV-angiostatin and subjected to Northern analysis. Blot hybridization with the mouse angiostatin cDNA probe showed the 2.4 Kb band (Figure 1a). Western analysis of the conditioned medium of transduced HeLa cells demonstrated the 67 kb band (Figure 1b).

Figure 1.

Characterization of HIV-angiostatin vector. (a) Northern blot analysis of angiostatin in HIV-angiostatin-transduced HeLa cells. RNA from HeLa cells was collected 3 days after vector transduction and analyzed with a mouse angiostatin cDNA probe. Lane 1: Nontransduced cells. Lane 2: HIV-EGFP-transduced cells. Lane 3: HIV-angiostatin-transduced cells. (b) Western blot analysis of angiostatin in conditioned medium from HIV-angiostatin-transduced HeLa cells. Conditioned medium was collected 3 days after vector transduction and analyzed with mouse anti-angiostatin antibody. Lane 1: Nontransduced cells. Lane 2: HIV-EGFP-transduced cells. Lane 3: HIV-angiostatin-transduced cells. (c) RT-PCR analysis of angiostatin mRNA and -actin mRNA in retinal tissue. Eye cups were harvested 5 days after injection of HIV-angiostatin in the left eye (lanes 4 and 5) and PBS in the right eye (lanes 1 and 2). The size marker (lane 3). Expression of -actin was observed in both the right and the left eyes (lanes 1 and 4). But expression of the angiostatin gene was only detected in the left eye (lane 4).

Full figure and legend (50K)

To investigate the biological effects of HIV-mediated expression of angiostatin, the tube formation assay was performed using human umbilical vein endothelial cells (HUVECs) co-cultured with fibroblasts. When HUVECs were cultured in the presence of VEGF, efficient tube formation was observed (Figure 2a). No significant change in tube formation was observed after transduction with the HIV vector containing the enhanced green fluorescent protein (EGFP) marker gene (HIV-EGFP) (Figure 2b). However, when the cells were transduced with HIV-angiostatin, significant inhibition of tubular formation was detected (Figure 2c). Quantitative analyses of both tube area and length also confirmed that HIV-angiostatin is capable of efficient inhibition of in vitro angiogenesis (Figure 2d and e). These results indicate that HIV-angiostatin is useful for expression and secretion of functional angiostatin.

Figure 2.

Effects of HIV vector transduction on tube formation. HUVECs co-cultured with fibroblasts were incubated without vector (a), with HIV-EGFP (b), and with HIV-angiostatin (c) and cultured for 10 days. Their area (d) and tube length (e) of tube-like structures were measured quantitatively using an image analyzer and were analyzed statistically. (1) Without vector, (2) HIV-EGFP, (3) HIV-angiostatin.

Full figure and legend (235K)

To study the utility of HIV vectors for gene transfer into the retinal tissue, HIV-EGFP was directly injected into the vitreous on day P12. EGFP-positive cells were detected in ganglion, bipolar, and photoreceptor cells (Figure 3).

Figure 3.

Expression of EGFP by the vitreous injection of HIV vector. Cell nuclei were counterstained with DAPI to localize EGFP-positive cells. EGFP-positive neural retina (a) and photoreceptor cells (b) were detected in 3-m sections of the retina, 19 days postnatal.

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Finally, the retinal tissues injected with HIV-angiostatin were analyzed by RT-PCR. Angiostatin-specific RNA was detected only in the injected side of the eye (Figure 1c). We also performed RT-PCT analyses of brain, lung, heart, liver, and bone marrow, but no signal was detected in any tissues (data not shown). These results indicate that intravitreal injection of HIV-angiostatin can achieve local expression of angiostatin in the retinal tissue.

Reduced proliferative retinopathy in the murine model

We examined the effect of HIV vector-mediated angiostatin expression in a murine model of proliferative retinopathy. In this model, exposure of neonatal mice to 75% oxygen for 5 days (P7–12) results in cessation of normal retinal blood vessel development and obliteration of the posterior retinal vasculature. Return of animals to room air is believed to result in relative hypoxia of the nonperfused areas of the retina. Retinal neovascularization then occurs in 100% of the animals.¹² On day P12, 18 animals were injected with 0.5 l of HIV-EGFP or HIV-angiostatin in one eye and phosphate-buffered saline (PBS) in the other eye. Retinal neovascularization in the eye injected with HIV-angiostatin was reduced in 90% (9/10; P=0.025) of animals, compared to the control eye (Figures 4 and 5b). Average retinal neovascular nuclei in the eyes injected with HIV-EGFP were not reduced (P=0.76) (Figure 5a). Reduction of histologically evident neovascular nuclei per 6-m section averaged 68% for HIV-angiostatin, with maximal inhibitory effects of 87%. No significant cell infiltration and structural destruction was observed by light microscopy.

Figure 4.

Effects of HIV-angiostatin on the neovascularization of ischemic mouse retina in vivo. Model mice of proliferative retinopathy were generated as described in Materials and methods. HIV-EGFP or HIV-angiostatin was injected intravitreally into the left eyes, while PBS was injected into the right eyes as a control. (a) The right eye injected with PBS. Vascular cell nuclei internal to the inner limiting membrane represented areas of retinal neovascularization are indicated by arrows. (b) Magnified picture of (a). (c) The left eye injected with HIV-EGFP. (d) The left eye injected with HIV-angiostatin. Compared with (c), retinal neovascularization was inhibited.

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Figure 5.

Inhibition of neovascularization by HIV-mediated expression of angiostatin. Average neovascular cell nuclei per 6-m histological section per eye were determined as described. (a) HIV-EGFP, (b) HIV-angiostatin.

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VEGF concentration in the retinal tissue

Recently, it was demonstrated that the anti-angiogenic effects of kringle 5 (K5) of plasminogen depends on a downregulation of VEGF.¹⁴ To study a possible mechanism of HIV-angiostatin-mediated inhibition of neovascularization in a murine retinopathy model, the concentration of VEGF in the eye was measured by the enzyme-linked immunosorbent assay (ELISA). Consistent with retinal neovascularization, the concentration of VEGF increased approximately twice after treatment with oxygen (Figure 6). However, HIV vector-mediated expression of angiostatin suppressed the oxygen-induced increase of the VEGF concentration. These results suggest that downregulation of VEGF may be involved in anti-angiogenic activities of angiostatin composed of kringles 1–4.

Figure 6.

VEGF concentration in the retinal tissues. VEGF in the vitreous fluid from control (1), ischemia induced (2), ischemia induced and treated with HIV-EGFP (3), ischemia induced and treated with HIV-angiostatin (4) were measured by ELISA. Values were the average of two experiments.

Full figure and legend (10K)

Discussion

Our results demonstrate that HIV-angiostatin can reduce retinal neovascularization in vivo without discernible short-term toxicity. Current therapies for retinal neovascularization, such as panretinal photocoagulation and cryotherapy, are only partially effective and are destructive to the retina. Furthermore, some patients do not effectively respond to these treatments. If patients do not respond to treatment, retinal neovascularization can lead to retinal detachment or secondary glaucoma, and can eventually cause phthisis bulbi and enucleation. In the case of diabetic retinopathy, more than 40% of patients do not effectively respond to treatment. For example, 43% of patients receiving panretinal photocoagulation develop macular edema after 6–10 weeks.¹⁵ In patients with severe retinal neovascularization that requires operations such as vitrectomy, the success rate is low and the visual prognosis is not good. The rate of treatment success in terminal patients may be higher if they receive gene therapy along with operations. Because of the nondestructive nature of gene therapy, we believe that patients may be able to maintain better vision acuity and visual fields than is possible with current therapy.

Angiostatin has been described as an endogenous inhibitor of angiogenesis.⁴ In mice, angiostatin reportedly inhibits primary tumor growth and metastasis. In vitro, angiostatin inhibits endothelial cell migration and induces endothelial cell-specific apoptosis.¹⁶ The mechanisms by which angiostatin inhibits angiogenesis are still unclear. Recently, angiomotin was discovered using a yeast two-hybrid screen for proteins that bind to angiostatin.¹⁷ Angiomotin was localized to the leading edge of migrating endothelial cells. Expression of angiomotin in endothelial cells resulted in increased cell migration. However, treatment with angiostatin inhibited migration and tube formation in angiomotin-expressing cells, indicating that angiostatin inhibits cell migration by interfering with angiomotin activity in endothelial cells.

Downregulation of endogenous angiogenic stimulators may be another possible mechanism of angiostatin-mediated inhibition of neovascularization.¹⁴ Angiogenesis is thought to be regulated by the balance between angiogenic stimulators and inhibitors.¹⁸ In the field of ophthalmology, it was demonstrated that VEGF levels are elevated in the vitreous of patients with retinal neovascularization,¹⁹ choroidal neovascular membrane²⁰ and the mouse model of retinal neovascularization.²¹ Furthermore, decreased levels of PEDF, a strong anti-angiogenic factor²² originally isolated from the conditioned media of human fetal retinal pigment epithelium cells,²³ have been associated with ischemia-induced retinal neovascularization.²⁴ In contrast, therapeutic effects of retinal photocoagulation in proliferative diabetic retinopathy are thought to be due to downregulation of VEGF mediated by locally induced angiostatin.²⁵ Recently, kringle 5, another angiogenic inhibitor derived from proteolytic products of plaminogene, was shown to be able to induce PEDF expression and decrease VEGF levels in the rat model of ischemic retinopathy.¹⁴ Our data also suggest that angiostatin composed of kringles 1–4 may inhibit neovascularization through downregulation of VEGF.

Although angiostatin has therapeutic potential, a serious limitation is short half-life of protein molecules. Previous studies in a variety of animal tumor models have demonstrated that if angiostatin treatment is discontinued, tumor growth resumes.²⁶ Retinal neovascularization should also be treated chronically, and therefore repeated injections of angiostatin are required. However, repeated intravitreal injections may cause serious complications, such as retinal detachment, cataract formation, cystoid macular edema, and endophthalmitis due to infection. Several biodegradable polymers have been investigated for possible use in sustained drug delivery. Using codrug pellets, triamcinolone acetonide and 5-FU are successfully released in the rabbit vitreous²⁷ and inhibit the progression of proliferative vitreous retinopathy.²⁸ But, the effect lasted only a month. Moreover, when the device is placed in the vitreous, surgical complications such as vitreous hemorrhage and astigmatism may occur.

To overcome these problems, gene-mediated expression of angiostatin should be much superior to direct injection of purified angiostatin. Various viral vectors have been used for gene transfer into the eye. Adenoviral vectors are able to efficiently transduce all eye tissues, but expression of the transgene is short-lived.²⁹ Conventional Moloney murine leukemic virus-based retroviral vectors (MLV) are not suited for gene transfer in the retina, because MLV infects only dividing cells.³⁰ AAV vector is known to infect nondividing cells, but no efficient packaging system is available and the transduction efficiency is very low without adenoviral helper function.³¹ On the contrary, HIV-based lentiviral vectors have been shown to achieve stable gene transfer and expression in retinal cells and corneal cells.³² The present study demonstrated that the HIV vector is highly useful for angiostatin gene therapy in the retina.

HIV-based lentiviral vectors were originally designed for gene therapy in AIDS because of their specificity for CD4⁺ T cells.³³ Subsequently, it was demonstrated that lentiviral vectors can efficiently infect nondividing cells and integrate into their chromosomes, which is unusual for an oncoretrovirus. To expand their tropism, lentiviral vector particles have been pseudotyped with the vesicular stomatitis virus G glycoprotein (VSV-G).³⁴ Such VSV-pseudotyped lentiviral vectors have proven useful for stable gene transfer into human hematopoietic stem cells,³⁵ terminal differentiated macrophages,³⁶ and neuronal cells.^37,38

A potential problem of viral vector-mediated protein therapy is viral shedding. Because HIV vectors stably integrate into the chromosome, long-term expression of angiostatin in nontarget organs may cause serious biological effects. However, the eye is a highly closed organ, because of the tight retina–blood barrier. No expression was detected in any organs examined except the eye where the vector was injected. Intravitreal injection of HIV vectors may appear to be the safe local therapeutic strategy for ocular disorders.

A practical problem for the clinical implementation of dormancy therapy may be the requirement for long-term, systemic drug delivery, a particularly vexing problem for a protein drug like angiostatin.³⁹ HIV vector-mediated targeted anti-angiogenesis therapy may offer a solution to this problem for the treatment of localized areas of pathological angiogenesis, such as seen in diabetic retinopathy and rheumatoid arthritis.

Materials and methods

Plasmid construction

The coding sequence of the mouse angiostatin gene was amplified by PCR from the mouse plasminogen gene (IMAGE clone no. 2225648) purchased at Incyte Genomics, inc (Palo Alto, CA, USA). The amino acid residues 1–6 and 461–466 of mouse plasminogen were amplified with a sense primer containing a SalI site (5'-TCGTCGACATGGACCATAAGGAAGTAATCC-3') and an antisense primer containing a XhoI site (5'-TCTCTCGAGTTATGTGGGCAATTCCACAACA-3').⁵ PCR was performed as previously described.⁴⁰ The 1.4 kb mouse angiostatin cDNA (pAngiostatin), which includes a signal peptide and the first four triple loop structures (kringle regions) of plasminogen, was cloned into the pGEM-T EASY vector (Promega, Madison, WI, USA). All sequences were confirmed. The HIV vector packaging plasmids, pMDG and pCMVR8.2, and the HIV vector plasmid pHR were kindly provided by Dr D Trono. The HIV vector plasmid pHIV-CS-PRE was kindly provided by Dr H Miyoshi.⁴¹ The CAG promoter (pCAG/72) was constructed by inserting the SalI-MspI fragment from pCAGGS⁴² into pSP72 vector (Promega, Madison, WI, USA). The pHIV-CS-CAG-PRE was constructed by inserting the BamI-PstI CAG promoter from pCAG/72 into pHIV-CS-PRE. pHIV-angiostatin was constructed by inserting a 1.4 kb XhoI-salI fragment from pAngiostatin into XhoI of pHIV-CS-CAG-PRE. The HIV vector plasmid expressing EGFP (pEGFP/HR) was constructed by inserting the EGFP gene driven by the CAG promoter into the pHR plasmid.⁴³

Lentiviral vector production

For transfection with lentiviral vectors, Cos (monkey kidney) cells (110⁶) were plated on 10-cm plates of 200 pieces for 16–24 h. On the following day, the cells were co-transfected with 7 g of pCMVR8.2, 7 g of pMDG, and 7 g of pHIV-angiostatin or pHIV-EGFP/HR by calcium phosphate precipitation. After 4–6 h at 37°C, the medium was removed and fresh 0.1 DMEM (D0.1) medium was added. Cells were cultured for an additional 3–4 days. The viral supernatant was passed through a 45-m filter and concentrated to a volume of 5 ml at room temperature using a CENTRIPREP (Millipore Corporation, Bedford, MA, USA; 3000 rpm, CH3.8 rotor, Beckman Coulter, Boulevard, PO, USA). 5 ml of the viral supernatant was concentrated to a pellet by ultracentrifugation. The pellet was diluted with 50 l PBS. To determine the titer of the HIV vector encoding EGFP (HIV-EGFP), HeLa cells (2–310⁵ cells/well in six-well plates) were incubated for 3 days with vector stock (up to 1 ml) in 3 ml of medium also containing 8 g/ml of polybrane (Sigma-Aldrich, Milwaukee, WI, USA). EGFP-positive cells were then counted by fluorescence activated cell-sorting analysis. The titer of the final vector stock was approximately 1.010¹¹ transduction unit (TU)/ml.

Northern blotting

Total RNA (10 g) was isolated from HeLa cells transduced by HIV-EGFP or HIV-angiostatin. Northern blotting was performed according to the standard method reported previously.⁴⁴ A uniformly labeled DNA probe was synthesized from the 712 bp pstI fragment of pAngiostatin.

Western immunoblotting

HeLa cells (210⁵), which were plated in six-well plates and transduced by HIV-angiostatin, were added to 1 ml of D0.1. Three days later, the conditioned medium which was concentrated to one-tenth using a CENTRIPREP was added to 50 l of SDS sample buffer and samples were heated at 95°C for 2 min. Angiostatin or 10 l of media were resolved by 10% SDS-PAGE. To determine the purity of the protein preparations, gels were stained with Coomassie Brilliant Blue. Western blotting was performed essentially as previously described.⁴⁵ The transfer membrane was incubated with antimouse angiostatin antibody (Yanaihara, Fukuoka, Japan) diluted 1:500 for 1 h and with secondary antibody (antirabbit IgG HRP conjugate, Promega, Madison, WI, USA) diluted 1:1000 for 1 h. The blot was then immersed in an enhanced chemiluminescence solution (ECL plus; Amersham Pharmacia Biotech, Buckinghamshire, UK) and exposed to Kodak KAR5R film.

In vitro tube formation asssay

Experiments on tube formation were conducted in triplicate in 24-multiwell dishes using Angiogenesis kit (Kurabo, Okayama, Japan), according to the manufacturer's instruction. Briefly, HUVECs co-cultured with human fibroblasts were transduced with HIV-angiostatin or HIV-EGFP (110⁸ TU/ml) and cultured in the medium containing 10 ng/ml of VEGF. The medium was changed every 3 days. After 10 days, dishes were washed with PBS and fixed with 70% ethanol at 4°C. After the fixed cells were rinsed three times with PBS, cells were incubated with mouse antihuman CD31 (Kurabo, Okayama, Japan) in PBS containing 1% BSA for 60 min. After washing with 1% BSA–PBS three times, cells were incubated with goat anti-mouse IgG AlkP conjugate (Kurabo, Okayama, Japan). Metal-enhanced 3,3'-diamino-benzidine-tetrahydrochloride (DAB) was the substrate, the reaction yielding a dark reddish-brown insoluble end-product. Finally, the cells were washed with PBS five times, and viewed in an Olympus microscope. The area and tube length were measured quantitatively with the Kurabo angiogenesis image analyzer (Kurabo, Okayama, Japan) in five different fields per each well, and analyzed statically.

Mouse model of ischemia-induced retinal neovascularization

All investigations proceeded in accordance with the regulations of the Ethical Committee of Nippon Medical School. This animal model has been previously described.^13,21 C57BL/6J mice were exposed to 75% O₂ from postnatal day 7 (P7) to P12 along with nursing mothers. At day P12, the mice were returned to room air. Maximal retinal neovascularization was observed 5 days after return to room air. Intraocular injections were performed at day P12 as described below. On days P17–19 the mice were killed by cardiac perfusion of 4% paraformaldehyde in PBS. The eyes were enucleated and fixed in 4% paraformaldehyde overnight at 4°C before paraffin embedding.

Intraocular injections

Mice were deeply anesthetized by intramuscular injection of ketamine hydrochloride and by intraperitoneal injection of pentobarbital sodium. Anesthetic eye drops (oxybuprocain hydrochloride, Santen, Osaka, Japan) were applied. Intravitreal injections were performed at the posterior limbus. A 32-gauge Hamilton needle and syringe were used to deliver 0.5 l of HIV-angiostatin or HIV-EGFP. The amount of solution actually remaining in the vitreous cavity was less than 0.5 l in some animals due to a small amount of leakage at the injection site.

Histological evaluation

To study the retinal location of EGFP-positive cells, mice injected with HIV-EGFP were killed. Eyes were enucleated, fixed overnight at 4°C, and then transferred to 0.01 M PBS containing 10%, 20%, and 30% sucrose every 2 h until equilibrated. Eyes were frozen in O.C.T. compound on dry ice and 2-m-thick cryostat sections were cut in parallel to the vertical meridian of the eye. Mounting medium with 4', 6-diamidino-2-phenylindole (DAPI; Vector, Burlingame, CA, USA) was used. The specimens were observed using an Olympus fluorescent microscope with NIBA and WO filters (Olympus, Tokyo, Japan).

Detection of angiostatin mRNA in tissues

The expression of transgene in retinal tissue was confirmed by reverse transcription-PCR (RT-PCR). Mice were killed by diethyl ether and the eyes were immediately enucleated. After the removal of the anterior segment and the vitreous, total RNA (10 g) was prepared from the homogenized eye cup. The total RNAs extracted from brain, lung, heart, liver, kidney, and bone marrow were also analyzed by RT-PCR to study the distribution of HIV vectors.

One microgram of RNA treated with DNase (Promega, Madison, WI, USA) was used for RT-PCR. RT-PCR was performed using an RT-PCR kit (Takara, Tokyo, Japan) according to the manufacturer's instructions. The primers were designed to detect angiostatin mRNA derived from the HIV vector. One primer encoding HIV vector cDNA and the other primer encoding angiostatin cDNA were designed using Oligo 6.0 and primer software. The HIV vector forward primer was 5'-ATTCATCGATTCTAGAGTTA-3' and the mouse angiostatin reverse primer was 5'-GCATTCCTCTTCACATTCA-3'. The PCR products were separated by 0.8% agarose gel electrophoresis, stained with ethidium bromide, and photographed. As a control for RNA integrity, expression of mouse -actin mRNA was assessed. The mouse -actin forward primer was 5'-TGTGATGGTGGGAATGGGTCAG-3' and the mouse -actin reverse primer was 5'-TTTGATGTCACGCACGATTTCC-3'.

Quantification of retinal neovascularization

Serial 6-m sections from whole eyes were cut sagittally parallel to the optic nerve and stained with hematoxylin and eosin according to a standard protocol.⁸ The extent of neovascularization was determined by counting neovascular cell nuclei extending through the internal limiting membrane into the vitreous. All counting was done using a masked protocol. For each eye, 10 intact sections of equal length, each 30 m apart, were evaluated. The mean number of neovascular nuclei per section per eye was then determined. Ten percent of the eyes exhibited retinal detachment or endophthalmitis and were excluded from the evaluation. The extent of neovascularization in angiostatin-treated eyes was then compared with that found in control eyes using paired Student's t-test. All statistical calculations were performed using SigmaStat for Windows (Hulinks, Berkeley, CA, USA).

Determination of VEGF concentration

The amounts of secreted VEGF in the vitreous and retina were determined using the commercial VEGF-ELISA kit (R&D systems, Minneapolis, MN, USA), according to the manufacturer's instructions. Briefly, the vitreous fluid was prepared from the eye injected with PBS, HIV-angiostatin or HIV-EGFP at P19. One microliter of the vitreous fluid collected from six mice was incubated with 50 l of buffered protein base for 2 h at RT in a 96-well plate coated with a monoclonal antibody against mouse VEGF. After three washing, a conjugate consisting of polyclonal anti-VEGF antibody and horseradish peroxidase was added and incubated for 2 h at RT. After the addition of tetramethylbenzidine, absorbance was measured at 450 nm in a BIO-RAD Model 3550 microplate reader (Bio-Rad Laboratories, Philadelphia, PA, USA). Using a serial dilution of the VEGF standard provided with the kit, we obtained a linear curve between 0 and 125 pg/ml, plotted against optical density at 450 nm.

Acknowledgements

We thank M Kato, N Suzuki, H Takahashi, S Hisayasu, and M Okabe for valuable technical assistance. This work was supported in part by grants from the Ministry of Health and Welfare of Japan and the Ministry of Education, Science and Culture of Japan.

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