Regulation of Vascular Endothelial Growth Factor (VEGF) Splicing from Pro-angiogenic to Anti-angiogenic Isoforms
Vascular endothelial growth factor (VEGF) is produced either as a pro-angiogenic or anti-angiogenic protein depending upon splice site choice in the terminal, eighth exon. Proximal splice site selection (PSS) in exon 8 generates pro-angiogenic isoforms such as VEGF165, and distal splice site selection (DSS) results in anti-angiogenic isoforms such as VEGF165b. Cellular decisions on splice site selection depend upon the activity of RNA-binding splice factors, such as ASF/SF2, which have previously been shown to regulate VEGF splice site choice. To determine the mechanism by which the pro-angiogenic splice site choice is mediated, we investigated the effect of inhibition of ASF/SF2 phosphorylation by SR protein kinases (SRPK1/2) on splice site choice in epithelial cells and in in vivo angiogenesis models. Epithelial cells treated with insulin-like growth factor-1 (IGF-1) increased PSS and produced more VEGF165 and less VEGF165b. This down-regulation of DSS and increased PSS was blocked by protein kinase C inhibition and SRPK1/2 inhibition. IGF-1 treatment resulted in nuclear localization of ASF/SF2, which was blocked by SPRK1/2 inhibition. Pull-down assay and RNA immunoprecipitation using VEGF mRNA sequences identified an 11-nucleotide sequence required for ASF/SF2 binding. Injection of an SRPK1/2 inhibitor reduced angiogenesis in a mouse model of retinal neovascularization, suggesting that regulation of alternative splicing could be a potential therapeutic strategy in angiogenic pathologies. Vascular endothelial growth factor (VEGF) is produced either as a pro-angiogenic or anti-angiogenic protein depending upon splice site choice in the terminal, eighth exon. Proximal splice site selection (PSS) in exon 8 generates pro-angiogenic isoforms such as VEGF165, and distal splice site selection (DSS) results in anti-angiogenic isoforms such as VEGF165b. Cellular decisions on splice site selection depend upon the activity of RNA-binding splice factors, such as ASF/SF2, which have previously been shown to regulate VEGF splice site choice. To determine the mechanism by which the pro-angiogenic splice site choice is mediated, we investigated the effect of inhibition of ASF/SF2 phosphorylation by SR protein kinases (SRPK1/2) on splice site choice in epithelial cells and in in vivo angiogenesis models. Epithelial cells treated with insulin-like growth factor-1 (IGF-1) increased PSS and produced more VEGF165 and less VEGF165b. This down-regulation of DSS and increased PSS was blocked by protein kinase C inhibition and SRPK1/2 inhibition. IGF-1 treatment resulted in nuclear localization of ASF/SF2, which was blocked by SPRK1/2 inhibition. Pull-down assay and RNA immunoprecipitation using VEGF mRNA sequences identified an 11-nucleotide sequence required for ASF/SF2 binding. Injection of an SRPK1/2 inhibitor reduced angiogenesis in a mouse model of retinal neovascularization, suggesting that regulation of alternative splicing could be a potential therapeutic strategy in angiogenic pathologies. IntroductionVascular endothelial growth factor (VEGF-A, hereafter referred to as VEGF) 5The abbreviations used are: VEGFvascular endothelial growth factorIGF-1insulin-like growth factorntnucleotidePBSphosphate-buffered salinePKCprotein kinase CELISAenzyme-linked immunosorbent assayMBPmaltose-binding proteinPSSproximal splice site selectionOIRoxygen-induced retinopathyUTRuntranslated regionITSinsulin transferrin seleniumPMAphorbol myristate acetateHEKhuman embryonic kidney. is a key regulatory component in physiological and pathological angiogenesis. Inhibition of VEGF has shown to be effective in cancer (1.Kerbel R.S. N. Engl. J. Med. 2008; 358: 2039-2049Crossref PubMed Scopus (1867) Google Scholar) and ocular angiogenesis (2.Duh E. Aiello L.P. Diabetes. 1999; 48: 1899-1906Crossref PubMed Scopus (280) Google Scholar), and it is up-regulated by a number of growth factors also implicated in these conditions, including insulin-like growth factor-1 (IGF-1) (3.Slomiany M.G. Rosenzweig S.A. Invest. Ophthalmol. Vis. Sci. 2004; 45: 2838-2847Crossref PubMed Scopus (90) Google Scholar). VEGF is generated as multiple isoforms by alternative splicing (4.Harper S.J. Bates D.O. Nat. Rev. Cancer. 2008; 8: 880-887Crossref PubMed Scopus (385) Google Scholar). There are two principal families of VEGF isoforms, the pro-angiogenic VEGFxxx isoforms, generated by proximal splice site selection in the terminal exon, exon 8a (5.Houck K.A. Ferrara N. Winer J. Cachianes G. Li B. Leung D.W. Mol. Endocrinol. 1991; 5: 1806-1814Crossref PubMed Scopus (1230) Google Scholar), and the anti-angiogenic VEGFxxxb isoforms (6.Woolard J. Wang W.Y. Bevan H.S. Qiu Y. Morbidelli L. Pritchard-Jones R.O. Cui T.G. Sugiono M. Waine E. Perrin R. Foster R. Digby-Bell J. Shields J.D. Whittles C.E. Mushens R.E. Gillatt D.A. Ziche M. Harper S.J. Bates D.O. Cancer Res. 2004; 64: 7822-7835Crossref PubMed Scopus (382) Google Scholar), generated by use of a distal splice site 66 bp further into exon 8, generating mRNA isoforms that contain exon 8b. As the stop codon for the protein is encoded in exon 8, these two isoforms contain alternate six amino acids at the C terminus (Fig. 1A). The pro-angiogenic isoforms such as VEGF165 encode a terminal six amino acid sequence of CDKPRR, and the anti-angiogenic isoforms such as VEGF165b encode SLTRKD (7.Bates D.O. Cui T.G. Doughty J.M. Winkler M. Sugiono M. Shields J.D. Peat D. Gillatt D. Harper S.J. Cancer Res. 2002; 62: 4123-4131PubMed Google Scholar). Many normal tissues, including the eye generate both isoforms (8.Perrin R.M. Konopatskaya O. Qiu Y. Harper S. Bates D.O. Churchill A.J. Diabetologia. 2005; 48: 2422-2427Crossref PubMed Scopus (201) Google Scholar), and previous studies have shown that the anti-angiogenic isoforms dominate in non-angiogenic tissues such as the normal colon (9.Varey A.H. Rennel E.S. Qiu Y. Bevan H.S. Perrin R.M. Raffy S. Dixon A.R. Paraskeva C. Zaccheo O. Hassan A.B. Harper S.J. Bates D.O. Br. J. Cancer. 2008; 98: 1366-1379Crossref PubMed Scopus (177) Google Scholar) and the vitreous (8.Perrin R.M. Konopatskaya O. Qiu Y. Harper S. Bates D.O. Churchill A.J. Diabetologia. 2005; 48: 2422-2427Crossref PubMed Scopus (201) Google Scholar). However, there is a splicing switch in angiogenic conditions such as proliferative diabetic retinopathy (8.Perrin R.M. Konopatskaya O. Qiu Y. Harper S. Bates D.O. Churchill A.J. Diabetologia. 2005; 48: 2422-2427Crossref PubMed Scopus (201) Google Scholar), colon (9.Varey A.H. Rennel E.S. Qiu Y. Bevan H.S. Perrin R.M. Raffy S. Dixon A.R. Paraskeva C. Zaccheo O. Hassan A.B. Harper S.J. Bates D.O. Br. J. Cancer. 2008; 98: 1366-1379Crossref PubMed Scopus (177) Google Scholar), prostate (10.Rennel E. Waine E. Guan H. Schüler Y. Leenders W. Woolard J. Sugiono M. Gillatt D. Kleinerman E. Bates D. Harper S. Br. J. Cancer. 2008; 98: 1250-1257Crossref PubMed Scopus (117) Google Scholar), renal (7.Bates D.O. Cui T.G. Doughty J.M. Winkler M. Sugiono M. Shields J.D. Peat D. Gillatt D. Harper S.J. Cancer Res. 2002; 62: 4123-4131PubMed Google Scholar), and skin cancers (11.Pritchard-Jones R.O. Dunn D.B. Qiu Y. Varey A.H. Orlando A. Rigby H. Harper S.J. Bates D.O. Br. J. Cancer. 2007; 97: 223-230Crossref PubMed Scopus (114) Google Scholar), and in Denys Drash Syndrome (12.Schumacher V.A. Jeruschke S. Eitner F. Becker J.U. Pitschke G. Ince Y. Miner J.H. Leuschner I. Engers R. Everding A.S. Bulla M. Royer-Pokora B. J. Am. Soc. Nephrol. 2007; 18: 719-729Crossref PubMed Scopus (61) Google Scholar). In contrast, in non-angiogenic conditions where VEGF is up-regulated, such as glaucoma and rhegmatogenous retinal detachment associated with proliferative vitreoretinopathy (13.Dieudonné S.C. La Heij E.C. Diederen R.M. Kessels A.G. Liem A.T. Kijlstra A. Hendrikse F. Ophthalmic. Res. 2007; 39: 148-154Crossref PubMed Scopus (31) Google Scholar) or glaucoma (14.Ergorul C. Ray A. Huang W. Darland D. Luo Z.K. Grosskreutz C.L. Mol. Vis. 2008; 14: 1517-1524PubMed Google Scholar), the anti-angiogenic isoforms are up-regulated. We have previously shown that IGF-1 can switch splicing in cultured epithelial cells from anti-angiogenic to pro-angiogenic isoforms (15.Nowak D.G. Woolard J. Amin E.M. Konopatskaya O. Saleem M.A. Churchill A.J. Ladomery M.R. Harper S.J. Bates D.O. J. Cell Sci. 2008; 121: 3487-3495Crossref PubMed Scopus (263) Google Scholar). As IGF-1 has been implicated in a number of angiogenic conditions including diabetic retinopathy and colon cancer, we hypothesized that the mechanism through which IGF-1 mediates this change in splicing may be a potential therapeutic target to prevent angiogenesis. To this end, we have investigated the signaling pathways, the splicing factors involved, and the possibility of therapeutic intervention in the pathway in an animal model of diabetic retinopathy.DISCUSSIONVEGF induction by IGF-1 occurs via different signaling pathways including PKC (22.Beckert S. Farrahi F. Perveen Ghani Q. Aslam R. Scheuenstuhl H. Coerper S. Königsrainer A. Hunt T.K. Hussain M.Z. Biochem. Biophys. Res. Commun. 2006; 341: 67-72Crossref PubMed Scopus (45) Google Scholar) and PI3-K (23.Miele C. Rochford J.J. Filippa N. Giorgetti-Peraldi S. Van Obberghen E. J. Biol. Chem. 2000; 275: 21695-21702Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 24.Poulaki V. Mitsiades C.S. McMullan C. Sykoutri D. Fanourakis G. Kotoula V. Tseleni-Balafouta S. Koutras D.A. Mitsiades N. J. Clin. Endocrinol. Metab. 2003; 88: 5392-5398Crossref PubMed Scopus (92) Google Scholar, 25.Slomiany M.G. Black L.A. Kibbey M.M. Day T.A. Rosenzweig S.A. Biochem. Biophys. Res. Commun. 2006; 342: 851-858Crossref PubMed Scopus (48) Google Scholar). There is increasing evidence that transducing components that link the cell surface with the nuclear splicing machinery implicate signaling pathways such as PKC (26.Lynch K.W. Weiss A. Mol. Cell. Biol. 2000; 20: 70-80Crossref PubMed Scopus (113) Google Scholar), PI3-K (27.Patel N.A. Chalfant C.E. Watson J.E. Wyatt J.R. Dean N.M. Eichler D.C. Cooper D.R. J. Biol. Chem. 2001; 276: 22648-22654Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 28.Blaustein M. Pelisch F. Coso O.A. Bissell M.J. Kornblihtt A.R. Srebrow A. J. Biol. Chem. 2004; 279: 21029-21037Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), or PKB/Akt (29.Blaustein M. Pelisch F. Tanos T. Muñoz M.J. Wengier D. Quadrana L. Sanford J.R. Muschietti J.P. Kornblihtt A.R. Cáceres J.F. Coso O.A. Srebrow A. Nat. Struct. Mol. Biol. 2005; 12: 1037-1044Crossref PubMed Scopus (187) Google Scholar, 30.Patel N.A. Kaneko S. Apostolatos H.S. Bae S.S. Watson J.E. Davidowitz K. Chappell D.S. Birnbaum M.J. Cheng J.Q. Cooper D.R. J. Biol. Chem. 2005; 280: 14302-14309Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). IGF-1 modulates splicing of VEGF isoforms by preferential use of the PSS to increase expression of pro-angiogenic isoforms (15.Nowak D.G. Woolard J. Amin E.M. Konopatskaya O. Saleem M.A. Churchill A.J. Ladomery M.R. Harper S.J. Bates D.O. J. Cell Sci. 2008; 121: 3487-3495Crossref PubMed Scopus (263) Google Scholar). Moreover, previously we have shown that ASF/SF2 overexpression preferentially increases usage of the proximal splice site (15.Nowak D.G. Woolard J. Amin E.M. Konopatskaya O. Saleem M.A. Churchill A.J. Ladomery M.R. Harper S.J. Bates D.O. J. Cell Sci. 2008; 121: 3487-3495Crossref PubMed Scopus (263) Google Scholar) and gives the same effect as IGF-1. SRPK1 has been shown specifically to phosphorylate 12 serines of the RS domain in ASF/SF2 (31.Ma C.T. Velazquez-Dones A. Hagopian J.C. Ghosh G. Fu X.D. Adams J.A. J. Mol. Biol. 2008; 376: 55-68Crossref PubMed Scopus (43) Google Scholar), and SRPK2 has been involved in the localization of ASF/SF2 within the nucleus. Thus, in this report, we have investigated the link between the splicing machinery and IGF-1 signaling.We have shown that the IGF-1-mediated increase in VEGF isoforms using the proximal splice site is inhibited by blocking PKC and SRPK1/2, and that this can be overcome by the use of a PKC inhibitor or mimicked by a PKC agonist or overexpression of SRPK1. This firmly suggests that this kinase cascade is involved in splice site selection in the VEGF gene. We have used RT-PCR, ELISA, and Western blotting to investigate VEGF splicing. VEGF isoform mRNA expression depends on transcription, splicing, and degradation of mRNA, and protein expression additionally depends upon translational rate and degradation rate. The finding that the mRNA and protein isoforms are both altered in a similar way by each intervention suggests that this is an mRNA switch, at least in part. As the mRNAs are generated by alternative splicing it is unlikely that differential isoform production is due to differential transcription, as both isoforms are transcribed from the same promoter region. However, VEGF has been shown to have two alternate transcription start sites. Although there is no evidence to date to show that these are differentially used for the different exon 8 isoforms, they do confer different exon 7 inclusion (32.Bastide A. Karaa Z. Bornes S. Hieblot C. Lacazette E. Prats H. Touriol C. Nucleic Acids Res. 2008; 36: 2434-2445Crossref PubMed Scopus (60) Google Scholar). If alternate transcription start sites are used, the splicing machinery would still need to be different in order for the transcription complex to recognize the different exon 8 splice sites. It is possible that these two different isoform families are differentially degraded, and that IGF mediates a decrease in degradation of mRNA encoding the proximal splice site. This is a possibility that we have not as yet excluded. However, the finding that ASF/SF2, a known splicing factor, requires the presence of a specific short sequence in the polypyrimidine tract upstream of the proximal splice site and that ASF/SF2 is induced to nuclear localization by SRPK1 activation and IGF-1 activation strongly suggest that this is a splicing mechanism rather than a degradation mechanism. We have shown that ASF/SF2 requires this sequence, which contains both a U2AF65 and consensus ASF/SF2 sequence, for binding to the VEGF pre-mRNA, but does not demonstrate that this is the sequence it binds to. ASF/SF2 binding to a region upstream of a splice site is generally considered a splicing repressor. There is evidence that SR proteins can interact with sequences upstream of the splice site that act as intronic splicing enhancer or silencer regions (reviewed in Ref. 33.Voelker R.B. J.A. Res. 2007; PubMed Scopus Google for in the in of the sequences in the intronic region upstream of the splice site resulted in altered splicing Nucleic Acids Res. 2006; PubMed Scopus Google Scholar). However, an alternative is that ASF/SF2 requires the U2AF65 consensus sequence to exon 8a to be in order for it to to consensus sequences and distal splice site and or DSS is in preferential proximal splice site is required to the mechanism of splicing regulation by is still not the IGF-1 a in the growth of However, there are increasing that IGF-1 increases angiogenesis by of VEGF (22.Beckert S. Farrahi F. Perveen Ghani Q. Aslam R. Scheuenstuhl H. Coerper S. Königsrainer A. Hunt T.K. Hussain M.Z. Biochem. Biophys. Res. Commun. 2006; 341: 67-72Crossref PubMed Scopus (45) Google Scholar, 25.Slomiany M.G. Black L.A. Kibbey M.M. Day T.A. Rosenzweig S.A. Biochem. Biophys. Res. Commun. 2006; 342: 851-858Crossref PubMed Scopus (48) Google Scholar, PubMed Scopus Google Scholar). Moreover, IGF-1 angiogenesis in the C. M. J. Diabetologia. 36: PubMed Scopus Google Scholar) and S. K. K. S. T. K. J. 1999; PubMed Google Scholar), and of endothelial cells J. H. T. I. S. Full Text PDF PubMed Scopus Google Scholar). IGF-1 is also implicated in pathological angiogenesis. of IGF-1 was increased in the vitreous of with diabetic retinopathy M. B. C. Diabetes. PubMed Google Scholar). There is a between and IGF-1 in different of cancers such as cancer R.S. H. M.R. Ferrara N. D.B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), cancer J.E. K. L. J. J.C. Dunn 2002; PubMed Google Scholar), and and cell M.G. Black L.A. Kibbey M.M. Day T.A. Rosenzweig S.A. Biochem. Biophys. Res. Commun. 2006; 342: 851-858Crossref PubMed Scopus (48) Google the encoding ASF/SF2, the of a D. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). of ASF/SF2 in resulted in of into R. E. R. D. A.R. Nat. Struct. Mol. Biol. 2007; 14: PubMed Scopus Google Scholar). of ASF/SF2 in which has expression of that inhibited in R. E. R. D. A.R. Nat. Struct. Mol. Biol. 2007; 14: PubMed Scopus Google are increasing that of the splicing machinery can be used as therapeutic M. Biophys. 2005; PubMed Scopus Google Scholar). that can target splicing factors and kinases involved in splicing are for T. T. S. K. T. Y. M. N. L.A. L.A. M. Sci. 2006; PubMed Scopus Google Scholar, B. E. M. C. J.F. J. J. Cancer Res. 2001; Google Scholar, M. B. T. H. J. T. K. J. H. K. H. A.R. M. M. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar, N. S. L. F. C. D. B. C. J. 2007; PubMed Scopus Google Scholar). the use of such as which can to the specific splice sites to splicing has been investigated W. Mol. 2004; PubMed Scopus Google Scholar, J.D. J. Clin. Invest. 2007; Google Scholar). SRPK1 to be a target for cancer but is more as expression of SRPK1 has been in and and increased expression is associated with the of a Cancer Res. 2007; PubMed Scopus Google Scholar). Moreover, it has been shown that known such as and increase cell with a effect phosphorylation of SR proteins was inhibited by using SRPK1 Cancer Res. 2006; PubMed Scopus Google Scholar). SRPK2 is to and phosphorylate an SR and it from nuclear to the in the activation of S.J. A. S. H. J. K. Cancer Res. 2008; PubMed Scopus Google Scholar). Moreover, overexpression of or SRPK2 increased cell SRPK2 and also in and with expression S.J. A. S. H. J. K. Cancer Res. 2008; PubMed Scopus Google Scholar). two kinases are to phosphorylate the splicing factor, ASF/SF2 and these components are involved in the choice of the PSS in The inhibitor of SRPK1/2 the down-regulation of the VEGFxxxb Moreover, SRPK1 and SRPK2 are known to phosphorylate the ASF/SF2 splicing factor with W. J.A. J.M. Z. Fu X.D. J. Cell Biol. PubMed Scopus Google Scholar, C.T. Velazquez-Dones A. Hagopian J.C. Ghosh G. Fu X.D. Adams J.A. J. Mol. Biol. 2008; 376: 55-68Crossref PubMed Scopus (43) Google Scholar, K. J.M. Cáceres J.F. T. Fu X.D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). results that an of SRPK1 can phosphorylation of However, have including known to VEGFxxxb such as (15.Nowak D.G. Woolard J. Amin E.M. Konopatskaya O. Saleem M.A. Churchill A.J. Ladomery M.R. Harper S.J. Bates D.O. J. Cell Sci. 2008; 121: 3487-3495Crossref PubMed Scopus (263) Google Scholar), and the of phosphorylation to by binding to the distal splice enhancer be It that phosphorylation of ASF/SF2 could an activation of the PSS and increased production of pro-angiogenic overexpression suggest that are they are not for proximal splice site these that SRPK1 may be and to that we to investigate this in a model of angiogenesis in the of resulted in inhibition of angiogenesis and increased normal we have not VEGF165b in these there is a between and exon isoforms that is a of altered splicing. The angiogenesis is known to be by pro-angiogenic VEGF and can be inhibited by anti-angiogenic VEGF165b O. Churchill A.J. Harper S.J. Bates D.O. T.A. Mol. Vis. 2006; 12: Google to a between anti-angiogenic splice and inhibition of splice factors that pro-angiogenic splicing in the same retinal angiogenesis suggest that may be anti-angiogenic suggesting a use in cancer as as diabetic retinopathy or IntroductionVascular endothelial growth factor (VEGF-A, hereafter referred to as VEGF) 5The abbreviations used are: VEGFvascular endothelial growth factorIGF-1insulin-like growth factorntnucleotidePBSphosphate-buffered salinePKCprotein kinase CELISAenzyme-linked immunosorbent assayMBPmaltose-binding proteinPSSproximal splice site selectionOIRoxygen-induced retinopathyUTRuntranslated regionITSinsulin transferrin seleniumPMAphorbol myristate acetateHEKhuman embryonic kidney. is a key regulatory component in physiological and pathological angiogenesis. Inhibition of VEGF has shown to be effective in cancer (1.Kerbel R.S. N. Engl. J. Med. 2008; 358: 2039-2049Crossref PubMed Scopus (1867) Google Scholar) and ocular angiogenesis (2.Duh E. Aiello L.P. Diabetes. 1999; 48: 1899-1906Crossref PubMed Scopus (280) Google Scholar), and it is up-regulated by a number of growth factors also implicated in these conditions, including insulin-like growth factor-1 (IGF-1) (3.Slomiany M.G. Rosenzweig S.A. Invest. Ophthalmol. Vis. Sci. 2004; 45: 2838-2847Crossref PubMed Scopus (90) Google Scholar). VEGF is generated as multiple isoforms by alternative splicing (4.Harper S.J. Bates D.O. Nat. Rev. Cancer. 2008; 8: 880-887Crossref PubMed Scopus (385) Google Scholar). There are two principal families of VEGF isoforms, the pro-angiogenic VEGFxxx isoforms, generated by proximal splice site selection in the terminal exon, exon 8a (5.Houck K.A. Ferrara N. Winer J. Cachianes G. Li B. Leung D.W. Mol. Endocrinol. 1991; 5: 1806-1814Crossref PubMed Scopus (1230) Google Scholar), and the anti-angiogenic VEGFxxxb isoforms (6.Woolard J. Wang W.Y. Bevan H.S. Qiu Y. Morbidelli L. Pritchard-Jones R.O. Cui T.G. Sugiono M. Waine E. Perrin R. Foster R. Digby-Bell J. Shields J.D. Whittles C.E. Mushens R.E. Gillatt D.A. Ziche M. Harper S.J. Bates D.O. Cancer Res. 2004; 64: 7822-7835Crossref PubMed Scopus (382) Google Scholar), generated by use of a distal splice site 66 bp further into exon 8, generating mRNA isoforms that contain exon 8b. As the stop codon for the protein is encoded in exon 8, these two isoforms contain alternate six amino acids at the C terminus (Fig. 1A). The pro-angiogenic isoforms such as VEGF165 encode a terminal six amino acid sequence of CDKPRR, and the anti-angiogenic isoforms such as VEGF165b encode SLTRKD (7.Bates D.O. Cui T.G. Doughty J.M. Winkler M. Sugiono M. Shields J.D. Peat D. Gillatt D. Harper S.J. Cancer Res. 2002; 62: 4123-4131PubMed Google Scholar). Many normal tissues, including the eye generate both isoforms (8.Perrin R.M. Konopatskaya O. Qiu Y. Harper S. Bates D.O. Churchill A.J. Diabetologia. 2005; 48: 2422-2427Crossref PubMed Scopus (201) Google Scholar), and previous studies have shown that the anti-angiogenic isoforms dominate in non-angiogenic tissues such as the normal colon (9.Varey A.H. Rennel E.S. Qiu Y. Bevan H.S. Perrin R.M. Raffy S. Dixon A.R. Paraskeva C. Zaccheo O. Hassan A.B. Harper S.J. Bates D.O. Br. J. Cancer. 2008; 98: 1366-1379Crossref PubMed Scopus (177) Google Scholar) and the vitreous (8.Perrin R.M. Konopatskaya O. Qiu Y. Harper S. Bates D.O. Churchill A.J. Diabetologia. 2005; 48: 2422-2427Crossref PubMed Scopus (201) Google Scholar). However, there is a splicing switch in angiogenic conditions such as proliferative diabetic retinopathy (8.Perrin R.M. Konopatskaya O. Qiu Y. Harper S. Bates D.O. Churchill A.J. Diabetologia. 2005; 48: 2422-2427Crossref PubMed Scopus (201) Google Scholar), colon (9.Varey A.H. Rennel E.S. Qiu Y. Bevan H.S. Perrin R.M. Raffy S. Dixon A.R. Paraskeva C. Zaccheo O. Hassan A.B. Harper S.J. Bates D.O. Br. J. Cancer. 2008; 98: 1366-1379Crossref PubMed Scopus (177) Google Scholar), prostate (10.Rennel E. Waine E. Guan H. Schüler Y. Leenders W. Woolard J. Sugiono M. Gillatt D. Kleinerman E. Bates D. Harper S. Br. J. Cancer. 2008; 98: 1250-1257Crossref PubMed Scopus (117) Google Scholar), renal (7.Bates D.O. Cui T.G. Doughty J.M. Winkler M. Sugiono M. Shields J.D. Peat D. Gillatt D. Harper S.J. Cancer Res. 2002; 62: 4123-4131PubMed Google Scholar), and skin cancers (11.Pritchard-Jones R.O. Dunn D.B. Qiu Y. Varey A.H. Orlando A. Rigby H. Harper S.J. Bates D.O. Br. J. Cancer. 2007; 97: 223-230Crossref PubMed Scopus (114) Google Scholar), and in Denys Drash Syndrome (12.Schumacher V.A. Jeruschke S. Eitner F. Becker J.U. Pitschke G. Ince Y. Miner J.H. Leuschner I. Engers R. Everding A.S. Bulla M. Royer-Pokora B. J. Am. Soc. Nephrol. 2007; 18: 719-729Crossref PubMed Scopus (61) Google Scholar). In contrast, in non-angiogenic conditions where VEGF is up-regulated, such as glaucoma and rhegmatogenous retinal detachment associated with proliferative vitreoretinopathy (13.Dieudonné S.C. La Heij E.C. Diederen R.M. Kessels A.G. Liem A.T. Kijlstra A. Hendrikse F. Ophthalmic. Res. 2007; 39: 148-154Crossref PubMed Scopus (31) Google Scholar) or glaucoma (14.Ergorul C. Ray A. Huang W. Darland D. Luo Z.K. Grosskreutz C.L. Mol. Vis. 2008; 14: 1517-1524PubMed Google Scholar), the anti-angiogenic isoforms are up-regulated. We have previously shown that IGF-1 can switch splicing in cultured epithelial cells from anti-angiogenic to pro-angiogenic isoforms (15.Nowak D.G. Woolard J. Amin E.M. Konopatskaya O. Saleem M.A. Churchill A.J. Ladomery M.R. Harper S.J. Bates D.O. J. Cell Sci. 2008; 121: 3487-3495Crossref PubMed Scopus (263) Google Scholar). As IGF-1 has been implicated in a number of angiogenic conditions including diabetic retinopathy and colon cancer, we hypothesized that the mechanism through which IGF-1 mediates this change in splicing may be a potential therapeutic target to prevent angiogenesis. To this end, we have investigated the signaling pathways, the splicing factors involved, and the possibility of therapeutic intervention in the pathway in an animal model of diabetic