WO2011008641A1 - Methids of modulating angiogenesis and treatment of angiogenesis-related diseases - Google Patents

Abstract

The present invention relates to a novel function for RGC-32 as an inhibitor of angiogenesis and a mediator between VEGF and FGF2 pathways. The present invention provides that expression of RGC-32 is induced in endothelial cells under hypoxia and that overexpression of RGC-32 in endothelial cells inhibited endothelial cell proliferation and migration via downregulation of another major angiogenic protein, FGF2, to further effect cyclin E. Also, RGC-32 promoted unstable vascular structure by increasing the numbers of apoptotic cells. The present invention provides methods of inhibiting proliferation of endothelial cells, angiogenesis, tumor growth by increasing expression of RGC-32 in the endothelial cells. The present invention relates to methods of increasing proliferation of endothelial cells as well as methods of treating ischemia by inhibiting expression of RGC-32 in the endothelial cells.

Description

METHODS OF MODULATING ANGIOGENESIS AND TREATMENT OF ANGIOGENESIS-RELATED DISEASES

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.

61/270,748, filed on July 13, 2009. The entire teachings of the above application are incorporated herein by reference.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by a grant HLROl 082837 from National Institutes of Health. The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Hypoxia/ischemia leading to cellular dysfunction is a complex process that involves numerous factors. In endothelial cells, it has been suggested that hypoxia induces angiogenesis via upregulation of hypoxia-inducible factor (HIF)-I alpha protein that in turn activates the transcription of several angiogenic genes, including VEGF, VEGF receptors flt-1 and neuropilin-1, and angiopointin-2 (Simons, M., Circulation 1 11 :1556-66, 2005). In contrast, hypoxia also directs endothelial cells toward apoptosis, which is caused by changes in p53 protein levels (Stempien-Otero, A., et al., J. Biol. Chem. 274:8039-45, 1999). Although functional genomic analyses have revealed specific genes that are involved in hypoxic signaling (Li, J. et al., Endothelial Biomedicine, Chapter 65, 602-608, 2007), gene regulation for maintaining endothelial cell homeostasis between angiogenesis and apoptosis under hypoxic conditions is still not fully understood.

The RGC-32 protein is localized in the cytoplasm and physically associates with cyclin-dependent kinase p34CDC2, which increases the kinase activity to induce quiescent aortic smooth muscle cells to enter S-phase (Badea, T. et al., J. Biol Chem. 277:502-8, 2002) and plays an important role in cell proliferation by downregulating cell cycle inhibitors (Oram, S. W., et al., BMC Cancer 6:154, 2006). However, studies of RGC-32 in tumor cell growth have yielded different results. It has been shown that RGC-32 showed p53-dependent transcriptional activity that suppressed tumor cell line growth via the arrest of mitotic progression (Saigusa K., el al., Oncogene 26: 1 110-21 2007). The disparities between these reports may be due to different RGC-32 functions in different cell types (Vlaicu, S. L, et al, Arch Immunol. Ther. Exp. (Warsz), 56: 115-22, 2008). However, the role of RGC-32 activity in hypoxia and angiogenesis has not been elucidated.

SUMMARY OF THE INVENTION

The present invention is based on the discovery of a novel function for RGC-

32 as a potential hypoxia-inducible inhibitor of angiogenesis and a mediator between VEGF and FGF2 pathways. It is determined that HIF- lα and VEGF significantly increased RGC-32 expression in hypoxia and ischemia and that HIF-I α/VEGF- induced RGC-32 expression does not follow the canonical VEGF pathway to promote angiogenesis. Rather, overexpression of RGC-32 in endothelial cells inhibited cell proliferation and migration via downregulation of another major angiogenic protein, FGF2, to further effect cyclin E. Also, RGC-32 promoted unstable vascular structure by increasing the numbers of apoptotic cells.

The present invention relates to methods for decreasing proliferation of endothelial cells by introducing an agent into said endothelial cells, whereby said agent increases expression of Response Gene to Component 32 (RGC-32) protein in said endothelial cells, and wherein RGC-32 protein is provided in an amount sufficient to increase apoptosis of the endothelial cells. In one embodiment, the agent is a nucleic acid comprising all, or a portion of, RGC-32 cDNA.

The present invention relates to methods of decreasing angiogenesis in a mammalian tissue comprising endothelial cells, comprising introducing an agent into said endothelial cells of said mammalian tissue, whereby said agent increases expression of Response Gene to Component 32 (RGC-32) protein in said endothelial cells, and wherein said RGC-32 protein is provided an amount sufficient to increase apoptosis of the endothelial cells. The present invention relates to methods of inhibiting tumor growth in a mammal by reducing angiogenesis, comprising administering to the mammal an agent, whereby said agent increases expression of Response Gene to Component 32 (RGC-32) protein in one or more endothelial cells associated with said tumor, and wherein said RGC-32 protein is expressed in an amount sufficient to increase apoptosis of said endothelial cells. In one embodiment, the tumor is associated with colon cancer.

The present invention relates to methods for increasing proliferation of endothelial cells, comprising introducing into said endothelial cells with an effective amount of an agent that reduces expression of Response Gene to Component 32 (RGC-32) protein, whereby reduced expression of RGC-32 results in increased proliferation of said endothelial cells. In one embodiment, the agent can be a highly specific antisense oligonucleotide against RGC-32 expression, for example, siRNA, peptide nucleic acid (PNA) and morpholino.

The present invention relates to methods for increasing angiogenesis in a tissue comprising endothelial cells, comprising introducing into said endothelial cells with an effective amount of an agent that reduces expression of Response Gene to Component 32 (RGC-32) protein, whereby reduced expression of RGC-32 results in increased angiogenesis in said tissue. In one embodiment, the agent is a highly specific antisense oligonucleotide against RGC-32 expression as described herein.

The present invention relates to methods of treating ischemia in mammalian tissue by preventing apoptosis of endothelial cells in said ischemic tissue, comprising contacting said mammalian tissue with an effective amount of an agent that reduces expression of Response Gene to Component 32 (RGC-32) protein in one or more endothelial cells of said ischemic mammalian tissue. In one embodiment, the agent can be a highly specific antisense oligonucleotide against RGC-32 expression, for example, siRNA, peptide nucleic acid (PNA) and morpholino.

The present invention relates to methods for treating a vascular disorder by reducing angiogenesis in a subject in need thereof, comprising administering an agent to said subject in need, whereby said agent increases expression of Response Gene to Component 32 (RGC-32) protein in one or more endothelial cells, wherein said RGC- 32 protein is expressed in an amount sufficient to increase apoptosis of said endothelial cells. In one embodiment, the vascular disorder is a disease in the eye characterized by abnormal blood vessel growth, for example, retinopathy of prematurity, ischemic retinopathy, retinal vein or artery occlusion, diabetic retinopathy, choroidal neovascularization, age related macular degeneration, corneal neovascularization, neovascular glaucoma and corneal transplantation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 A-ID depict the effect of hypoxia on RGC-32 expression. RGC-32 expression in HUVECs was assessed after varying durations of hypoxia, from 1 hour to 48 hours using Northern (FIG. IA) and Western (FIG. IB) blotting and confirmed in the Quantitative-PCR assay (FIG. 1 C). RGC-32 half-life was detected by Northern blot after HUVECs were exposed to either normoxia or hypoxia for 5 h before administering actinomyocin D (ACD; 5 ug/ml) (FIG. ID top). Total RNA was extracted from HUVECs exposed to nomoxia or hypoxia at 1 hour intervals for 5 hours after ACD administration and subjected to Northern blot with a RGC-32 RNA probe. Signals were quantified and plotted as a percentage of the 0-Hour value against time (FIG. ID bottom). Hypoxia extended the half-life of RGC-32 mRNA from 1.96 to 6.05 h. Both mRNA and protein were upregulated by hypoxia. Results are means ± SEM, based on 3 experiments quantified by Image J; *P < 0.05.

FIGS. 2A-2G depict the effect of HIF-I α and VEGF on RGC-32 expression. RGC-32 mRNA was significantly induced by CoCl₂, DMOG, and 3,4-DHB in

HUVECs under normoxic conditions with HIF-I α accumulated (FIG. 2A). RGC-32 promoter activity was increased 1.62±0.22 fold in luciferase assay in response to HIF-lα. Results are means ± SD (FIG. 2B). *P<0.05. ChIP assay showing the RGC-32 promoter co-immunoprecipitating with HIFl α antibody in hypoxic conditions (FIG. 2C). HUVECs were stimulated by VEGF (25ng/ml), FGF2 (25ng/ml), TGF-β (lOng/ml), IL- lβ (10ng/ml), TNF-α (10ng/ml) or PDGF

(20ng/ml) for 12 hours, and RGC-32 expression was analyzed by Northern blot (FIG. 2D). VEGF was upregulated by hypoxia in HUVECs (FIG. 2E). RGC-32 was upregulated by VEGF in a time (FIG. 2F left) and dose dependent manner (FIG. 2F right). RGC-32 induced by VEGF was partially inhibited by SU4312 under hypoxia (FIG. 2G left) and by anti-VEGF-neutralizing antibody (FIG. 2G right). FIGS. 3A-3E depict the effect of RGC-32 on angiogenesis. RGC-32 overexpression in HUVECs was determined by Western blot (FIG. 3A). Growth curve analysis indicates that RGC-32 (o/e) attenuated 51% of endothelial cell proliferation (FIG. 3B); Migration assay for RGC-32 (o/e) shows about 45% lower migrating rates compared to controls (FIG. 3C); Two siRNA sequences of RGC-32 showed knockdown efficiency (FIG. 3D left): RGC-32 knockdown increased HUVECs proliferation (FIG. 3D middle) and migration (FIG. 3D right) compared to negative siRNA controls; Macroscopic (FIG. 3E top) and CD-31 stained

microscopic (FIG. 3E bottom) images of the angiogenic response induced 3 days after implantation of Matrigels with VEGF-Al 65-secreting SKMEL/VEGF cells and PT67 cells packaging pBMN-GFP or pBMN-RGC32 (Note: VEGF stimulated blood vessels (c/g arrows) can be blocked by RGC-32 treatment (d/h)).

FIGS. 4A-4C depict the effect of RGC-32 endothelial cell apoptosis:

Vascular structure formation in Matrigel showed no significant change at 20 hours between RGC-32 (o/e) and control HUVECs (FIG. 4A). At 40 hours, the vascular structure of RGC-32 (o/e) cells was unstable from enhanced apoptosis shown by Annexin V staining (FIG. 4A lower panel). Flow cytometry analysis indicated that the number of apoptotic cells increased in RGC-32 (o/e) HUVECs in hypoxia (FIG. 4B). Western Blot shows increase of cleaved caspase 3 in RGC-32 (o/e) HUVECs (FIG. 4C).

FIGS. 5A-5E depict the effect of RGC-32 on expression of FGF2 and FGF2- stimulated cyclin E: Three major angiogenesis pathway proteins including

VEGFR2, FGF/FGFR1 and Angiopointin I/Tie 2 were examined (FIG. 5A). Note the decreased expression of FGF2 in RGC-32 (o/e) HUVECs and the lack of significant changes for other tested genes (FIG. 5A). Decreased FGF2 expressions in RGC-32 (o/e) HUVECs show in normoxia and hypoxia at 6, 24 and 48 hours (FIG. 5B). Western Blotting analysis indicated that FGF2-stimulated cyclin E, but not cyclin B and D, was attenuated in RGC-32 o/e cells (FIG. 5C). Western blotting indicates that there were no significant differences for expression levels of these VEGF downstream signaling pathway genes between RGC-32 o/e and control cells (FIG. 5D). Western blotting also indicates that RGC-32 did not effect FGF2 stimulated MAPK pathway (FIG. 5E) FIGS. 6A-6E depict the effect of RGC-32 on angiogenesis and blood flow recovery after ischemia in vivo. RGC-32 protein expression increased after femoral artery ligation (FIG. 6A). Infrared imaging from mice subjected to femoral artery ligation and retroviral injection of either pBMN or pBMN-RGC-32 on quadriceps (FIG. 6B). Quantification of blood-flow recovery after treating either control or RGC-32 retrovirus (FIG. 6C). Data are means ± SEM n=8. Sections of quadriceps from pBMN or RGC-32 o/e mice stained for CD31 (FIG. 6D left) and quantification of capillaries (FIG. 6D right). GFP expression showed the successful RGC-32 gene delivery from quadriceps sections (FIG. 6E).

FIGS 7A-7E depict the effect of RGC-32 on angiogenesis in SW480 melanoma tumor. Nude mice bearing established SW480 xenograft were treated with control or RGC-32 retrovirus (FIG. 7A). Note: tumor size was significantly reduced as shown on day 13 after treatment. Data are means ± SEM, * P<0.05 n=4 (FIG. 7A). Q-PCR analysis shows increased RGC-32 mRNA expression in the RGC-32 virus treated tumor compared to the control (* n=4, P<0.0005) (FIG. 7B). Representative view of tumor section with CD31 staining (FIG. 7C) and statistic analysis of vessel number with 12 HMF counts n=12; P<0.01 (FIG. 7D) on day 13 after treatment. Western blotting indicated that FGF2, cyclin E and CD31 were downregulated in RGC-32 treated mice (FIG. 7E). DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on Applicants' discovery that RGC-32 expression is increased under hypoxia in endothelial cell and changes in RGC-32 expression modulate proliferation of endothelial cells. The present invention provides that RGC-32 is a hypoxia-inducible gene and anti-angiogenic factor in endothelial cells.

RGC-32 plays an important homeostatic role, as it contributes to

differentiating the pathways for VEGF and FGF2 in angiogenesis of endothelial cells, and provides a new target for ischemic disorder and tumor therapies.

Overexpression of RGC-32 reduces the proliferation and migration and destabilized vascular structure formation in vitro, and inhibited angiogenesis in Matrigel assays in vivo. Expression of RGC-32 also stimulates apoptosis as shown by the increased apoptotic cells and caspase-3 cleavage. RGC-32's effect on the anti-angiogenic response is via attenuating FGF2 expression and further inhibiting expression of cyclin E without impacting VEGF and FGF2 signaling in endothelial cells.

The present invention also provides that silencing RGC-32 by highly specific antisense oligonucleotide has an opposing, stimulatory effect on proliferation of endothelial cells. For example, in the mouse hindlimb ischemia model, RGC-32 inhibits capillary density with a significant attenuation in blood flow.

The present invention also provides that treatment with RGC-32 in the xenograft tumor model resulted in reduced growth of blood vessels which negatively affected tumor growth.

The present invention also includes methods for treating a vascular disorder by reducing angiogenesis in a subject. The method of the invention is useful for treating a vascular disorder related to abnormal blood vessel growth or angiogenesis by administering an agent capable of increasing expression of RGC-32 protein in the endothelial cells. In a preferred embodiment, the method of the invention is useful for treating the eye disorders characterized by abnormal blood vessel growth. The vascular disorders in the eye include, but are not limited to, retinopathy of prematurity, ischemic retinopathy, retinal vein or artery occlusion, diabetic retinopathy, choroidal neovascularization, age related macular degeneration, corneal neovascularization, neovascular glaucoma and corneal transplantation. Methods of evaluating candidate agents for efficacy in treating angiogenesis-associated vascular disorders, including those of the eye, are described herein and also known to those of skill in the art, for example, in U.S. Patent No. 7,527,936 or in U.S. Patent Application Publication 2008/0181893, the entire teachings of which are

incorporated herein by reference.

A description of example embodiments of the invention follows. The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety. EXAMPLE

Cell Culture and Hypoxia

Human Umbilical Vein Endothelial Cells (HUVECs) (LONZA^®) were cultured in EBM2 containing 2% FBS with growth supplements. Hypoxia (<1%O2) was induced using a Modular Incubator Chamber (Billumps-Rothenberg). CoCl₂, dimethyl-oxalylglycine (DMOG), and 3,4-dihydroxybenzoate(3,4-DHB) (SIGMA^®) were used to mimic hypoxia. Retroviral Construction

The RGC-32 (NMJ) 14059.1) coding sequence was cloned into pBMN-GFP vector (OBIGEN^®) for retrovirus packaging. pBMN-GFP or pBMN-GFP-RGC-32 were transfected to 293T using Polyethylenimine (PEI) with pVSV-G, pJK3, and pCMVtat. The medium with retrovirus/RGC-32 was collected and filtered before being used to infect HUVECs.

RNA Analysis

Total RNA from HUVECs was extracted using TRIzol Reagent

(INVITROGEN^®) and cDNA was synthesized by reverse transcription. A 420bp RGC-32 cDNA was cloned with primers (forward: 5'-

GCGCTGTGCGAGTTTGAC-3' (SEQ ID NO: 1), reverse: 5'- CCCCTCTGGCAGCAGATT-3' (SEQ ID NO:2)) for probing. Northern blots were performed using a DIG Northern Starter Kit (ROCHE^®). RNA levels were also measured by Quantitative PCR (Q-PCR) using the primers

5'-CAAAGACGTGC ACTC AACCTTC-3 ' (SEQ ID NO: 3), and

5 '-CTGTCTAAATTGCCCAGAAATGG -3 ' (SEQ ID NO:4). A TaqMan probe, 5'-FAM (ό-carboxyfluorescein)-ACCAGGCCACTCTC AGGCTC ACCTT AA-3 ' TAMRA (6-carboxy-tetramethylrhodamine) (SEQ ID NO:5) was included during Q- PCR.

RGC-32 Transcription Studies

A nucleotide fragment (-1205 to +379 of human sequence at GeneBank: 28984) encompassing basal elements of the human RGC-32 promoter was cloned into a PGL-3 vector (PROMEGA^®). HEK293 were transfected with the RGC-32 promoter construct co-transfected with HIF- lα cDNA using Polyethylenimine (PEI). The luciferase activity was determined using a Dual-luciferase assay system

(PROMEGA^®).

Chromatin Immunoprecipitation

Chromatin immunoprecipitation (ChIP) was performed with ChIP-IT

Express Kit (Active Motif) in accordance with the manufacturer's instructions. The RGC-32 and actin promoters were amplified with the primer pairs

5 '-ATTTAATTAGCCGTCTGTGGTGAG-S ' (SEQ ID NO: 6) and

5 '-AGCCTGACTTTATCTAGAAGGGGT-S ' (SEQ ID NO:7), and

5 '-TGCACTGTGCGGCGAAGC-S ' (SEQ ID NO:8) and

5 '-TCGAGCCATAAAAGGCAA-S ' (SEQ ID NO: 9), respectively. RGC-32 primers were designed to the putative hypoxia response elements (HREs) binding site for HIF- lα. siRNA transfection

siRNA targeting human RGC-32 were synthesized by GENPHARMA^® . (ZhangJiang, Shanghai). Two duplexes of siRNA of RGC-32

(siRNA 1 : 5'-GAUUCACUUUAUAGGAACATT-S ' (SEQ ID NO: 10),

5'-UGUUCCUAUAAAGUGAAUCTG-S ' (SEQ ID NO: 1 1);

siRN A2: 5'-CAUUGCUGAUCUUGACAAATT-S ' (SEQ ID NO: 12),

5'-UUUGUCAAGAUCAGCAAUGTT-S ' (SEQ ID NO: 13))

were confirmed to have knockdown efficiency by Northern Blotting. Another duplex of siRNA that is not targeted to any human genes was used as negative control. HUVECs were transfected with siRNA at a final concentration of 50 nM using Lipofectamine 2000 (INVITROGEN^®).

Growth factor and inhibitor studies

Selective growth factors and cytokines including VEGF, FGF2, TGF-β, IL- lβ, TNF-α, and PDGF-BB (SIGMA^®) were added to HUVECs. To specifically block secreted VEGF, the monoclonal anti-human VEGF-neutralizing antibody, murine isotype control IgG (R&D Systems^®), or SU4312 (SIGMA^®), a suppressor of VEGFR2 activation that inhibits VEGFR2 phosphorylation, were pre-incubated with HUVECs at 37°C for 1 hour before hypoxia. Western Blot Analysis

HUVECs were lysed in RIPA buffer and blot with antibodies as described in Wu et al. (Wu et al, Am. J. Physiol. Heart Circ. Physiol. 285:2420-9, 2003).

Western Blot Analysis HUVECs were lysed in RIPA buffer (BIOPRODUCTS^®, MA) containing cocktailed protease inhibitors (ROCHE^®, NJ). Samples were subjected to 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes (MILLIPORE^®, MA). Membranes were incubated with the indicated antibodies including anti-RGC32 (GENEMED^®); anti-FGF2, anti-cyclin Bl₅ Dl, E, E2F, Tie- 2, angiopointin, FGFRl, actin (SANTA CRUZ BIOTECHNOLOGY^®); anti-Akt, pAkt, eNOS, p-eNOS, ERK, pERK, p38, p-p38, VEGF receptor 2, and caspase 3 (CELL SIGNALING^®); anti-HIF-lα (BD TRANSDUCTION LABORATORIES^®) and anti-vinculin (SIGMA^®) followed by incubation with HRP anti-mouse or anti- rabbit IgG (CALBIOCHEM^®). The blots were developed using the ECL detection system. Proliferation and Migration Assays

HUVECs were seeded in 12-well plates (3x10⁴ cells/well) and incubated for

5 hours counting from day 1. At the indicated time point, culture was stopped by fixing with 4% paraformaldehyde for 15 minutes and subsequently stained with 0.1% crystal violet dissolved in 10% ethanol for 20 minutes. After washing, 10% acetic acid was added to the cells and absorbance was measured at 590 nm wavelength. HUVECs migration was measured by "wounding" assays. Cells were grown to subconfluence in 12-well plates (10⁵ cells/well) then starved for 24 hours in 0.5% serum. The cell layer was scratched with a pipette tip, producing a gap ~2- mm wide. The gap width was measured at marked locations from images taken by inverted microscope (TMS-F; NIKON^®) immediately after the scratching and again

6 hours later at the same marked locations as described (Horowitz, A., et al., Biochemistry 38:15871-7, 1999, the entire teachings which are incorporated herein by reference).

Apoptosis Assay

Apoptotic cells were analyzed using Vybrant Apoptosis Assay Kit 13 for

FACS and Annexin V kit for Matrigel (INVITROGEN^®). The cells were incubated with PO-PRO^™-1 and 7-AAD on ice for 30 minutes. Stained HUVECs were analyzed by FACScan flow cytometry using BD FACSDiva software (BD

PHARMINGEN^®). Cells that were PO-PRO™-1 positive and 7-AAD negative were counted for apoptosis to exclude necrotic cells. Caspase Inhibitor Z-VAD- FMK was from CALBIOCHEM^®.

Matrigel Analysis

BD MatrigelTM Matrix Growth Factor Reduced (BD BIOSCIENCE^®) was coated on a pre-chilled 24-well culture plate on ice. After Matrigel solidification for 30 minutes, HUVECs were plated 10⁵ cells/well with EBM2. Twenty-four to 48 hours later, the extent of network formation was observed and photographed. In vivo Matrigel angiogenesis assays were carried out as described (Zeng H., et al., J. Exp. Med. 203:719-29, 2006). IxIO⁷ PT67 cells were infected with retroviruses expressing full-length RGC-32 or GFP cDNAs. The infected PT67 alone or mixed with SKMEL/VEGF cells (IxIO⁷) were suspended in 0.5ml of growth factor- reduced Matrigel (BD BIOSCIENCE^®) and injected subcutaneously (sc) into nu/nu mice. Tissues were photographed and fixed with 4% paraformaldehyde for immunohistochemistry. Each experiment was replicated with 4 mice.

Hindlimb ischemia model

Hindlimb ischemia in 12-week-old male FVB mice was induced by ligation of the femoral artery as described (Couffinhal, T.. et al, Am. J. Pathol. 152: 1667-79, 1998). Immediately after surgery, animals received in the right hindlimb an i.m. injection consisting of 50 μl of either Retrovirus pBMN-GFP (IXlO⁸ pfu/ml) or pBMN-GFP-RGC-32 (IXlO⁸ pfu/ml) at the quadriceps. Tissue section was performed to assess the expression of retro viral-encoded GFP from injected ischemic muscle specimens 7 and 28 days after surgery. Blood flow was measured with a MoorLDI2 infrared scanner.

Tumor analysis

1x10⁸ SW480 cells were implanted s.c. into BALB/c nude mice. Animals were inspected daily for tumor development. Tumors were measured using a digital caliper, and volume was calculated by length x width² x 0.52, which approximates the volume of an elliptical solid. After two weeks, mice harboring subcutaneous tumors (approximately 100 mm³) were randomly divided into two groups (n=4) and were treated with an intraneoplastic inoculation of 1 x 10⁷ PFU retrovirus of either pBMN or RGC-32 every 3 days. Mice were sacrificed on day 13 post-injection.

Immunostaining

Sections of in vivo Matrigel blocks, hindlimb ischemia quadriceps and colon tumor tissues were fixed in 4% Paraformaldehyde for capillary density studies. Immunohistochemistry and immunofluorescence were performed with the CD31 antibody (BD PHARMINGEN^®) as described previously (Zeng H., et al, J. Exp. Med. 203:719-29, 2006, the entire teaching of which are incorporated herein by reference).

Statistics

The results are expressed as mean ± S. E. M or S. D based on triplet experiments. Statistical analysis used analysis of variance and Student's t-test (two- tailed). A p-value <0.05 was considered statistically significant.

RESULTS

RGC-32 expression is induced by hypoxia

To investigate the role of RGC-32 in angiogenesis, RGC-32 expression under hypoxia compared with normoxia in endothelial cells was first examined. A significant increase in RGC-32 gene expression occurred from 3 hours (2.21±0.12 fold) to 36 hours (3.47±0.57 fold), and returned to baseline after 48 hours in hypoxia (FIG. IA). Immunoblot analysis shows that RGC-32 protein response to hypoxia began at 6 hours and remained at a high level until 48 hours (FIG. IB). The increase in RGC-32 expression from 3 hours to 24 hours was also confirmed in the

Quantitative-PCR assay (FIG. 1C). mRNA half-life assay indicated that RGC-32 mRNA half-life was prolonged under hypoxia (6.05 vs 1.96 hours) (FIG. ID).

These results demonstrate that the increased expression of RGC-32 in response to hypoxia is mediated, at least partially, at the posttranscriptional level, which could reasonably account for its steady elevated protein level.

HIF- lα and VEGF mediate the induction of RGC-32 expression in hypoxia To determine whether hypoxia-induced RGC-32 mRNA was mediated by

HIF-I, HUVECs were exposed to CoCl₂, 3,4-DHB, or DMOG, which stabilize HIF-I α expression via hydroxylase inhibition (Epstein, A. C, et ah, Cell 107:43-54, 2001). RGC-32 mRNA was significantly increased when the HIF-I protein was protected from degradation (FIG. 2A). In addition, co-transfection with the RGC-32 promoter and HIF- lα cDNA led to a significant increase in RGC-32 promoter activity (FIG. 2B). ChIP assay revealed direct interaction between HIF-lα and HRE sites on the RGC-32 promoter (FIG. 2C). Thus, hypoxia-induced RGC-32 expression was regulated at both the transcriptional and posttranscriptional levels via HIF-I or HIF-I target genes. Compared with other reported hypoxia responsive genes, RGC-32 showed a steady, prolonged induction, suggesting that some other indirect induction may also be involved in this response. A panel of growth factors and cytokines, VEGF, FGF-2, TGF-β, ILl -β, TNF-α and PDGF, were incubated with HUVECs for 12 hours, and RGC-32 mRNA levels were examined. As FIG. 2D shows, VEGF, but not TGF-β or PDGF, significantly induced RGC-32 mRNA expression, whereas TNF-α, FGF2 and IL- lβ decreased the expression of RGC-32 mRNA. VEGF was markedly induced by hypoxia in HUVECs (FIG. 2E), and VEGF-induced RGC-32 expression showed a time- and dose-dependence (FIG. 2F), indicating that VEGF might play an important role in hypoxia-related RGC-32 stimulation. To test this hypothesis, SU4312, a VEGF receptor 2 activation suppressor, was incubated with HUVECs for 1 hour prior to hypoxia. Hypoxia- induced RGC-32 expression was significantly blocked (FIG. 2G left). In addition, treating HUVECs with anti-VEGF-neutralizing antibody prior to hypoxia decreased the hypoxia-induced RGC-32 mRNA (FIG. 2G right). Thus, VEGF appears to be the predominant factor for hypoxia induction of RGC-32 in endothelial cells.

RGC-32 attenuates angiogenesis in vitro and in vivo

To test the hypothesis that RGC-32, as a downstream gene of VEGF, might enhance angiogenesis, RGC-32 cDNA carried by a retrovirus was generated in endothelial cells. RGC-32 proteins were successfully expressed in HUVECs (FIG. 3A) and angiogenic activities of RGC-32 (o/e) were examined. Surprisingly, a growth curve assay showed that the RGC-32 (o/e) cells grew significantly slower compared with those infected by retrovirus vector only (FIG. 3B). The migration rate of the RGC-32 (o/e) cells was dramatically decreased compared to control cells shown in FIG. 3C.

To further validate that RGC-32 does not act as an angiogenic enhancer within the VEGF pathways, three sequences of siRNA that targeted RGC-32 were synthesized, and two of them showed superior knockdown of RGC-32 expression (FIG. 3D left). FIG. 3D (middle) demonstrates that the HUVECs decreased the velocity of growth under hypoxic conditions. However, knockdown of RGC-32 by siRNA indicated that the HUVECs growth rate was accelerated in normoxia and significantly enhanced by hypoxia in comparison with the control siRNA. The migration rate was also accelerated in RGC-32 knockdown HUVECs compared with the control cells (FIG. 3D right).

To further verify RGC-32's mechanism of action, the Matrigel assay was used to introduce genes of interest into the vascular endothelium in vivo (Hoang M.V., et al, Methods MoI. Biol. 294:269-85, 2005). On day 3 after implantation of Matrigel plugs containing various cell mixtures, angiogenic responses were evaluated macroscopically (FIG. 3E, top panels) and by histology and

immunohistochemistry for the endothelial cell marker CD31 (FIG. 3E, bottom panels). Plugs containing only PT67 cells did not significantly induce angiogenesis (FIG. 3E-a/e). However, strong angiogenesis was induced in plugs containing SK- MEL/VEGF cells (FIG. 3E-c/g). When PT67/RGC-32 cells were included in the Matrigel, the angiogenic response induced by SKMEL/VEGF cells was strikingly inhibited (FIG. 3E-d/h). Taken together, these results suggest that RGC-32 does not have pro- angiogenesis capability, instead it can inhibit endothelial cell proliferation and migration and VEGF-induced angiogenesis (Folkman J., Exp. Cell. Res. 312:594- 607, 2006).

RGC-32 induces apoptosis

When the effect of RGC-32 in Matrigel capillary structure formation in vitro was examined, it did not show significant differences at an early stage (10-24 hours) between RGC-32 (o/e) and control cells. However, at a late stage (40 hours), the vascular structures formed by RGC-32 (o/e) cells were less stable, but no significant difference was seen when the cells were incubated with the caspase inhibitor Z- VAD-FMK compared with control cells. This indicated that endothelial cell apoptosis took place in RGC-32 (o/e) cells, which was confirmed by the increase of Annexin V positive cells (FIG. 4A). PO-PRO™-l/7-aminoactinomycin D staining measured the number of apoptotic cells (FIG. 4B). The obvious changes were observed after cells had undergone hypoxia for 48 hours. The number of apoptotic cells increased from 7.7% to 14.2% for controls, but increased from 8.5% to 22.1% for RGC-32 (o/e) HUVECs. In agreement with the increased apoptotic cell number, there was also a significant increase of cleaved caspase 3 in RGC-32 (o/e) HUVECs after 48 hour hypoxia (FIG. 4C). These results suggest that RGC-32 accelerates hypoxia-induced endothelial apoptosis.

RGC-32 attenuates FGF2-related cyclin E expression

One possible mechanism that might explain the non-canonical VEGF angiogenic pathway is that RGC-32 could be a VEGF negative feedback regulator (Lobov, LB., et al, Proc. Natl. Acad. Sc.i USA. 104:3219-24, 2007). However, there were no significant differences found between RGC-32 (o/e) cells and control cells in terms of phosphorylation of eNOS, Akt and MAP kinases (ERK and p38) by VEGF stimulation (FIG. 5D). Examination of three major angiogenic pathways (Oettgen, P., Circ. Res. 89:380-8 2001) in RGC-32 (o/e) cells showed marked decreases in the expressions of the FGF2 isoforms 23kd and 17kd (FIG. 5A), and RGC-32 down-regulated FGF2 expression enhanced by hypoxia (FIG. 5B). However, no effect on the FGF2-stimulated signaling pathway was observed (FIG. 5E). Meanwhile, the protein levels of anti-angiogenic genes, including angiostatin and endostatin was examined, but no significant differences were found between RGC-32 (o/e) and control cells. These results suggest that RGC-32 had no effect on the VEGF downstream pathway, but did influence expression of FGF2.

In light of RGC-32 perturbations of the cell cycle described for malignant transformation (Saigusa K., et al, Oncogene, 26:1110-21 2007; Fosbrink, M., et al, Exp. MoI. Pathol. 78:116-22, 2005), RGC-32 might act in conjunction with cyclin in response to the down-regulation of FGF2. FIG. 5C indicate that RGC-32 inhibited FGF2-stimulated cyclin E, but not cyclin B, D and E2F. VEGF-induced cycline E was also attenuated in RGC-32 o/e cells in comparison to controls. Collectively, these data suggested that RGC-32 impacts FGF2's pro-proliferating pathway via inhibiting FGF2's up-regulation of cyclin E in endothelial cells, which explains RGC-32's ability to reduce proliferation.

RGC-32 impaired perfusion recovery after hindlimb ischemia

In the mouse hindlimb ischemia, the expression of RGC-32 was markedly induced in all ischemic muscle beds (FIG. 6A). To assess whether RGC-32 can impact the normal vascular response to ischemia, RGC-32 and control retroviruses were delivered to quadriceps immediately after the procedure. The expression of the delivered gene was confirmed by GFP (FIG. 6E). Blood perfusion in ischemic hindlimbs was measured by Doppler analysis on days 0, 7, 14, 21 and 28. The blood flow in the pBMN vector-only treated quadriceps area was improved 88% of the non-ischemic control at day 14. However, treatment with RGC-32 decreased the blood flow recovery, showing about 64% of the non-ischemic control (FIGS. 6B and 6C). The angiographic analysis at day 7 and 28 after ligation of the femoral artery revealed a decreased number of vessels in the RGC-32-treated group (FIG. 6D).

RGC-32 reduced tumor angiogenesis

To determine whether RGC-32 has the ability to regulate tumor

angiogenesis, an intraneoplastic inoculation of either pBMN or RGC-32 retrovirus started when tumors were around 100mm³. Growth of RGC-32 expressed tumors was greatly inhibited. Compared to the control, the average tumor size in the RGC- 32 group recessed by 35% on day 9 and 45% on day 13 (FIG. 7A). Additionally, it was found that suppression of tumor growth by RGC-32 (FIG. 7B) was associated with reduced angiogenesis, particularly with a reduction in number of vessels, which was assessed by CD31 staining (FIGS. 7C and 7D). RGC-32 in regulation of FGF2 and cyclin E was also found in RGC-32 treated tumor tissues. FIG. 7E indicates that RGC-32-treated tumor tissue significantly reduced expression of FGF2, cyclin E, and endothelial cell marker CD31.

DISCUSSION

The principal finding of this study is that RGC-32 is a hypoxia-induced anti- angiogenesis factor in endothelial cells. It is demonstrated that the mechanism of HIF-lα/VEGF-induced RGC-32 expression inhibits angiogenesis via RGC-32- dependent attenuation of the FGF2 pathway for cyclin E expression. Several lines of evidence support this finding. First, RGC-32 expression was induced by hypoxia in both mRNA and protein levels. Second, inhibitory studies indicated that hypoxia- induced RGC-32 expression is dependent on HIF- lα stabilization, and HIF- lα is able to stimulate RGC-32 promoter activity. Third, RGC-32 was induced by VEGF but did not effect VEGF signaling. Instead, RGC-32 significantly downregulated FGF2 and attenuated FGF2-dependent cyclin E expression. Finally, the ability of RGC-32 to inhibit endothelial cell proliferation and migration could be blocked by inhibition of RGC-32. Injection of RGC-32 in the mouse hindlimb ischemia and the tumor xenograph models can reduce the number of blood vessels in association with downregulation of FGF2 and cyclin E.

The experimental data above provide evidence that hypoxia induced RGC-32 expression via the HIFlα/VEGF pathway. The results for RGC-32 as an inducible gene were consistent with previous reports indicating that RGC-32 mRNA expression is induced by C5b-9 complement activation (Badea, T. et al., J. Biol. Chem. 277:502-8, 2002), steroid hormones 18 and TGF-β (Li, F., et al, J, Biol,

Chem, 282:10133-7, 2007). However, it was not observed TGF-β induced RGC-32 expression in endothelial cells, although this might be due to TGF-β activating RGC-32 through specific Smad and RhoA signaling to initiate cell differentiation in smooth muscle cells (Li, F., et al, J. Biol. Chem. 282:10133-7, 2007).

Unlike other VEGF-induced genes, such as COX-2 (Wu, G., et al, Am. J. Physiol. Heart Circ. Physiol. 285:H2420-9, 2003), RGC-32 did not follow the canonical VEGF-induced angiogenic pathway. One possible mechanism that might explain the role of RGC32 during proliferation and vascular growth is that RGC-32 could be a VEGF negative feedback regulator. Similarly, Delta-like ligand 4 (D114) was dynamically induced by VEGF, but D114 blockade enhanced angiogenic sprouting while suppressing ectopic pathological neo-vascularization in the retinal vasculature (Lobov, LB. , et al, Proc. Natl. Acad. Sc.i USA. 104:3219-24, 2007). In the present study, RGC-32 did not influence VEGF-mediated signaling pathways indicated by VEGF-stimulated phosphorylation of eNOS, Akt and MAPK (ERK, P38) that showed no significant differences in RGC-32 overexpressed endothelial cells compared with control cells. As shown here, RGC-32 significantly attenuated expression of FGF2, but it did not interrupt the expressions of the FGF receptor 1 and angiopointin-l/Tie2 pathways. The anti-angiogenic proteins angiostatin and endostatin were also not regulated by RGC-32.

Although FGF2 is involved in angiogenesis, its expression regulation during hypoxia is poorly documented. In HUVECs, it was observed that FGF2 protein was downregulated in 23 kd and 17 kd isoforms under hypoxic conditions. This result contradicted another report showing that FGF2 was induced at a protein level concomitant with a decrease in FGF2 mRNA caused by internal ribosome entry site (IRES) during hypoxia (Conte, C, et al, PLoS ONE 3:e3078, 2008). However, the present data is in agreement with previous reports suggesting that hypoxia induced expression of VEGF, but not FGF2 (Aparicio, S., et al, Biochem. Biophys. Res. Commun. 326:387-94, 2005 and Brogi, E., et al, Circulation 90:649-52, 1994). Despite the fact that expression of FGF2 was reduced by RGC-32, it had no effect on the FGF2-stimulated signaling pathways.

In contrast to these active constitutive forms, it was found that, in RGC-32 overexpressing cells, cyclin E expression was attenuated in response to FGF2.

Cyclin E is one of the major cyclins that is involved in the Gl to S phase transition. This result is consistent with a previous report showing that RGC-32 suppressed the growth of glioma cells via p53 regulation (Saigusa K., et al, Oncogene, 26: 1110-21 2007). However, RGC-32 did not directly alter activation of cyclin E in endothelial cells. These results suggest that RGC-32 inhibits endothelial cell proliferation not by directly interrupting the cell cycle, but via influencing FGF2 pathways, in which cyclin E and cyclin-dependent kinase (cdks) 2 and 4 were expressed after FGF2 infusion (Olson, N.E. et al, J. Biol. Chem. 275:11270-7, 2000). Furthermore, FGF2 decreased levels of the cdk inhibitor p27 (Kip 1) to enhance association of cyclin E- cdk2 (Frederick, TJ., et al, MoI. Cell Neurosci. 25:480-92, 2004).

RGC-32 had no impact on vascular structure formation, but it did decrease its stability. This suggests that RGC-32 not only inhibits angiogenesis, but also stimulates apoptosis. RGC-32 has been shown to be a direct transcriptional target of p53 in human cells of various tissue origins (Saigusa K., et al., Oncogene, 26:1110- 21 2007). Cell cycle regulation and apoptosis are the most important features of p53-dependent tumor suppression. The regulator of FGF2 transcription (RFT) is a transcriptional repressor and induces glioma cell death by its overexpression, suggesting that RFT regulates the Gl-S transition and apoptosis via the

p53/p21Wafl pathway (Kano, H., et al, Biochem. Biophys. Res. Commun. 317:902- 8, 2004). Thus, down-regulation of FGF2 expression by RGC-32 may trigger suppression of cell growth, especially mitotic progression through the p53 pathway in endothelial cells. Moreover, the FGF2-treated group in the hindlimb ischemia increased angiogenesis and number of collateral vessels after ligation of the femoral artery (Cao R., et al., Nat Med. 9:604-13, . 2003), therefore, downregulation of FGF2 expression by RGC-32 could impact the return of blood flow to the ischemic limb and inhibit progressive neovascularization.

As a cell cycle regulator, RGC-32 has shown two opposite results for tumor growth. In addition to targeting the G/M phase transition and suppressing tumor growth (Saigusa K., et al, Oncogene, 26:1110-21 2007), RGC-32 has also reportedly activated cdc2 kinases to induces cell cycle activation (Fosbrink, M., et al, Exp. MoI. Pathol. 78:116-22, 2005). These contradictory results have been explained by the possibility that RGC-32 may play dual roles by enhancing cell proliferation and acting as a tumor suppressor gene in certain types of cancers (Vlaicu, S.I., et ah, Arch Immunol. Ther. Exp. (Warsz), 56:115-22, 2008). The model in the present study demonstrated that RGC-32 reduced colon cancer tumor size with reduced numbers of vessels. In the present study, a significant decrease of VEGF-induced blood vessel growth by RGC-32 in Matrigels was observed. Many stimuli including hypoxia can increase a major angiogenic factor (e.g. VEGF) expression in tumor cells, which is correlated with increased microvessel counts and poor prognosis in many human cancers (Lyden et al., Nat Med, 7: 1194-201, 2001). However, hypoxia/ischemia-induced gene regulation in tumor growth is not fully understood. Here, it is reported that RGC-32 has the ability to promote both anti- angiogenesis and pro-apoptosis in endothelial cells, which results in decreased blood vessel growth during colon cancer proliferation and, in turn, reduced tumor size.

Overexpression of FGF2 mRNA was associated with significantly increased risk for tumor recurrence (Barclay, C, et ah, Clin. Cancer Res. 11 :7683-91, 2005).

Targeting FGF receptors and/or FGF signaling can affect both the tumor cells directly and tumor angiogenesis (Kwabi-Addo, B., et ah, Endocr. Relat. Cancer 11 :709-24, 2004). Decreased FGF2 expression in the RGC-32 treated xenograph model may not be limited to vessels due to the FGF signaling involved in both the cancer cells and surrounding vasculature to enhance proliferation and resistance to cell death, thereby, enhancing tumor progression (Kwabi-Addo, B., et al., Endocr. Relat. Cancer 11 :709-24, 2004). The results in the present study add an additional mechanism, anti-angiogensis, for RGC-32 as a tumor suppressor.

In summary, it has been demonstrated that RGC-32 is a hypoxia inducible gene dependent on HIF- lα and VEGF. Induced RGC-32 performed anti-angiogenic activity through downregulating endothelial cyclin E via the FGF2 pathway. This study reveals the important role that RGC-32 plays in homeostasis of hypoxic endothelial cells. This may contribute to understanding the crosstalk between different angiogenic gene pathways and also highlights the potential for tumor treatment by targeting RGC-32.

While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

CLAIMS What is claimed is:

1. A method for decreasing proliferation of endothelial cells by introducing an agent into said endothelial cells, whereby said agent increases expression of Response Gene to Component 32 (RGC-32) protein in said endothelial cells, and wherein RGC-32 protein is provided in an amount sufficient to increase apoptosis of the endothelial cells, thereby decreasing proliferation of the endothelial cells.

2. The method of Claim 1, wherein the agent is a nucleic acid comprising all, or a portion of, RGC-32 cDNA.

3. The method of Claim 2, wherein said RGC-32 cDNA is operably linked to a promoter and transfected into the endothelial cells using a viral vector.

4. The method of Claim 3, wherein said viral vector is pBMN-RGC32.

5. A method of decreasing angiogenesis in a mammalian tissue comprising

endothelial cells, comprising introducing an agent into said endothelial cells of said mammalian tissue, whereby said agent increases expression of Response Gene to Component 32 (RGC-32) protein in said endothelial cells, and wherein said RGC-32 protein is provided an amount sufficient to increase apoptosis of the endothelial cells, thereby decreasing angiogenesis in the mammalian tissue.

6. The method of Claim 5, wherein the agent is a nucleic acid comprising all, or a portion of, RGC-32 cDNA.

7. The method of Claim 6, wherein said RGC-32 cDNA is operably linked to a promoter and transfected into the endothelial cells using a viral vector.

8. The method of Claim 7, wherein said viral vector is pBMN-RGC32.

9. A method of inhibiting tumor growth in a mammal by reducing angiogenesis, comprising administering to the mammal an agent, whereby said agent increases expression of Response Gene to Component 32 (RGC-32) protein in one or more endothelial cells associated with said tumor, and wherein said RGC-32 protein is expressed in an amount sufficient to increase apoptosis of said endothelial cells, thereby inhibiting tumor growth in the mammal.

10. The method of Claim 9, wherein said tumor is associated with colon cancer.

11. The method of Claim 9, wherein the agent is a nucleic acid comprising all, or a portion of, RGC-32 cDNA.

12. The method of Claim 11, wherein said RGC-32 cDNA is operably linked to a promoter and transfected into the endothelial cells associated with the tumor using a viral vector.

13. The method of Claim 12, wherein said viral vector is pBMN-RGC32.

14. A method for increasing proliferation of endothelial cells, comprising

introducing into said endothelial cells with an effective amount of an agent that reduces expression of Response Gene to Component 32 (RGC-32) protein, whereby reduced expression of RGC-32 results in increased proliferation of said endothelial cells.

15. The method of Claim 14, wherein said agent is a highly specific antisense oligonucleotide against RGC-32 expression and selected from the group consisting of siRNA, peptide nucleic acid (PNA) and morpholino.

16. The method of Claim 15, wherein said agent is siRNA comprising a sequence selected from the group consisting of:

5'-GAUUCACUUUAUAGGAACATT-S' (SEQ ID NO:10), 5'-UGUUCCUAUAAAGUGAAUCTG-S' (SEQ ID NO:11), 5'-CAUUGCUGAUCUUGACAAATT-S' (SEQ ID NO:12), and 5'-UUUGUCAAGAUCAGCAAUGTT-S' (SEQ ID NO:13).

17. A method for increasing angiogenesis in a tissue comprising endothelial cells, comprising introducing into said endothelial cells with an effective amount of an agent that reduces expression of Response Gene to Component 32 (RGC-32) protein, whereby reduced expression of RGC-32 results in increased

angiogenesis in said tissue.

18. The method of Claim 17, wherein said agent is a highly specific antisense

oligonucleotide against RGC-32 expression and selected from the group consisting of siRNA, peptide nucleic acid (PNA) and morpholino.

19. The method of Claim 18, wherein said agent is siRNA comprising a sequence selected from the group consisting of:

5'-GAUUCACUUUAUAGGAACATT-S ' (SEQ ID NO: 10), 5 '-UGUUCCUAUAAAGUGAAUCTG-S ' (SEQ ID NO: 11), 5 '-CAUUGCUGAUCUUGACAAATT-S ' (SEQ ID NO: 12), and 5'-UUUGUCAAGAUCAGCAAUGTT-S ' (SEQ ID N0: 13).

20. A method of treating ischemia in mammalian tissue by preventing apoptosis of endothelial cells in said ischemic tissue, comprising contacting said mammalian tissue with an effective amount of an agent that reduces expression of Response Gene to Component 32 (RGC-32) protein in one or more endothelial cells of said ischemic mammalian tissue, thereby decreasing apoptosis of said endothelial cells.

21. The method of Claim 20, wherein said agent is a highly specific antisense

oligonucleotide against RGC-32 expression and selected from the group consisting of siRNA, peptide nucleic acid (PNA) and morpholino.

22. The method of Claim 21, wherein said agent is siRNA comprising a sequence selected from the group consisting of:

5'-GAUUCACUUUAUAGGAACATT-S ' (SEQ ID NO:10), S'-UGUUCCUAUAAAGUGAAUCTG-S' (SEQ ID NO:11),

S'-CAUUGCUGAUCUUGACAAATT-S' (SEQ ID NO: 12), and 5'-UUUGUCAAGAUCAGCAAUGTT-S ' (SEQ ID NO: 13).

23. A method for treating a vascular disorder by reducing angiogenesis in a subject in need thereof, comprising administering an agent to said subject in need, whereby said agent increases expression of Response Gene to Component 32 (RGC-32) protein in one or more endothelial cells, wherein said RGC-32 protein is expressed in an amount sufficient to increase apoptosis of said endothelial cells.

24. The method of Claim 23, wherein said vascular disorder is a disease in the eye and selected from the group consisting of: retinopathy of prematurity, ischemic retinopathy, retinal vein or artery occlusion, diabetic retinopathy, choroidal neovascularization, age related macular degeneration, corneal

neovascularization, neovascular glaucoma and corneal transplantation.