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Anti-chemorepulsive Effects of Vascular Endothelial Growth Factor and Placental Growth Factor-2 in Dorsal Root Ganglion Neurons Are Mediated via Neuropilin-1 and Cyclooxygenase-derived Prostanoid Production

2004; Elsevier BV; Volume: 279; Issue: 29 Linguagem: Inglês

10.1074/jbc.m402488200

ISSN

1083-351X

Autores

Lili Cheng, Haiyan Jia, Marianne Löhr, Azadeh Bagherzadeh, David Holmes, David L. Selwood, Ian Zachary,

Tópico(s)

Lymphatic System and Diseases

Resumo

Vascular endothelial growth factor (VEGF) displays neurotrophic and neuroprotective activities, but the mechanisms underlying these effects have not been defined. Neuropilin-1 (NP-1) is a receptor for VEGF165 and placental growth factor-2 (PlGF-2), but the role of NP-1 in VEGF-dependent neurotrophic actions is unclear. Dorsal root ganglion (DRG) neurons expressed high levels of NP-1 mRNA and protein, much lower levels of KDR, and no detectable Flt-1. VEGF165 and PlGF-2 promoted DRG growth cone formation with an effect similar to that of nerve growth factor, whereas the Flt-1-specific ligand, PlGF-1, and the KDR/Flt-4 ligand, VEGF-D, had no effect. The chemorepellent NP-1 ligand, semaphorin 3A, antagonized the response to VEGF and PlGF-2. The specific KDR inhibitor, SU5614, did not affect the anti-chemorepellent effects of VEGF and PlGF-2, whereas a novel, specific antagonist of VEGF binding to NP-1, called EG3287, prevented inhibition of growth cone collapse. VEGF stimulated prostacyclin and prostaglandin E2 production in DRG cultures that was blocked by inhibitors of cyclooxygenases; the anti-chemorepellent activities of VEGF and PlGF-2 were abrogated by cyclooxygenase inhibitors, and a variety of prostacyclin analogues and prostaglandins strikingly inhibited growth cone collapse. These findings support a specific role for NP-1 in mediating neurotrophic actions of VEGF family members and also identify a novel role for prostanoids in the inhibition of neuronal chemorepulsion. Vascular endothelial growth factor (VEGF) displays neurotrophic and neuroprotective activities, but the mechanisms underlying these effects have not been defined. Neuropilin-1 (NP-1) is a receptor for VEGF165 and placental growth factor-2 (PlGF-2), but the role of NP-1 in VEGF-dependent neurotrophic actions is unclear. Dorsal root ganglion (DRG) neurons expressed high levels of NP-1 mRNA and protein, much lower levels of KDR, and no detectable Flt-1. VEGF165 and PlGF-2 promoted DRG growth cone formation with an effect similar to that of nerve growth factor, whereas the Flt-1-specific ligand, PlGF-1, and the KDR/Flt-4 ligand, VEGF-D, had no effect. The chemorepellent NP-1 ligand, semaphorin 3A, antagonized the response to VEGF and PlGF-2. The specific KDR inhibitor, SU5614, did not affect the anti-chemorepellent effects of VEGF and PlGF-2, whereas a novel, specific antagonist of VEGF binding to NP-1, called EG3287, prevented inhibition of growth cone collapse. VEGF stimulated prostacyclin and prostaglandin E2 production in DRG cultures that was blocked by inhibitors of cyclooxygenases; the anti-chemorepellent activities of VEGF and PlGF-2 were abrogated by cyclooxygenase inhibitors, and a variety of prostacyclin analogues and prostaglandins strikingly inhibited growth cone collapse. These findings support a specific role for NP-1 in mediating neurotrophic actions of VEGF family members and also identify a novel role for prostanoids in the inhibition of neuronal chemorepulsion. Vascular endothelial growth factor (VEGF) 1The abbreviations used are: VEGF, vascular endothelial growth factor; DRG, dorsal root ganglion; HUVEC, human umbilical vein endothelial cells; NP-1, neuropilin-1; PAE/KDR, porcine aortic endothelial cells expressing KDR; PAE/NP-1, porcine aortic endothelial cells expressing NP-1; PGI2, prostacyclin; sema 3A, semaphorin 3A; DMEM, Dulbecco's modified Eagle's medium; RT, reverse transcriptase; DDA, dideoxyadenosine; MES, 4-morpholineethanesulfonic acid; COX, cyclooxygenase; hVEGF, human VEGF; rat VEGF; PlGF, placental growth factor; PGE2, prostaglandin E2. is an essential mediator of vasculogenesis and angiogenesis during embryonic development and plays a central role in pathophysiological neovascularization in human disease (1Ferrara N. Gerber H.-P. LeCouter J. Nat. Med. 2003; 9: 669-676Crossref PubMed Scopus (8076) Google Scholar, 2Carmeliet P. Jain R. Nat. Med. 2003; 9: 653-660Crossref PubMed Scopus (3563) Google Scholar). A growing body of evidence indicates that VEGF also has neurotrophic and neuroprotective activities. VEGF stimulates axonal outgrowth and enhances the survival of superior cervical and dorsal root ganglion (DRG) neurons (3Sondell M. Lundborg G. Kanje M. J. Neurosci. 1999; 19: 5731-5740Crossref PubMed Google Scholar, 4Sondell M. Sundler F. Kanje M. Eur. J. Neurosci. 2000; 12: 4243-4254Crossref PubMed Scopus (340) Google Scholar), inhibits hypoxic death of cortical neurons in culture (5Jin K.L. Mao X.O. Greenberg D.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10242-10247Crossref PubMed Scopus (640) Google Scholar) and in cerebral ischemia (6Sun Y. Jin K. Xie L. Childs J. Mao X.O. Logvinova A. Greenberg D.A. J. Clin. Investig. 2003; 111: 1843-1851Crossref PubMed Scopus (1051) Google Scholar), and promotes neurogenesis in vitro and in vivo (7Jin K. Zhu Y. Sun Y. Mao X.O. Xie L. Greenberg D.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11946-11950Crossref PubMed Scopus (1309) Google Scholar). The mechanisms underlying the neuronal effects of VEGF remain largely unclear, however. VEGF exerts its biological effects through high affinity binding to two tyrosine kinase receptors, Flt-1 (VEGF-R1) and KDR (VEGF-R2), which are expressed in most vascular endothelial cells (8Neufeld G. Cohen T. Gengrinovitch S. Poltorak Z. FASEB J. 1999; 13: 9-22Crossref PubMed Scopus (3182) Google Scholar, 9Zachary I. Gliki G. Cardiovasc. Res. 2001; 49: 568-581Crossref PubMed Scopus (586) Google Scholar). KDR binds VEGF with lower affinity than Flt-1 and is also recognized by VEGF-C, VEGF-D, and VEGF-E, whereas Flt-1 is also a receptor for PlGF and VEGF-B (1Ferrara N. Gerber H.-P. LeCouter J. Nat. Med. 2003; 9: 669-676Crossref PubMed Scopus (8076) Google Scholar). After binding and activation of KDR, VEGF stimulates extracellular signal-regulated kinase activation and an array of other early signaling events followed by short and long term cellular biological effects including production of prostacyclin (PGI2) and nitric oxide, increased cell survival, cell migration, proliferation, and angiogenesis (10Waltenberger J. Claesson Welsh L. Siegbahn A. Shibuya M. Heldin C.H. J. Biol. Chem. 1994; 269: 26988-26995Abstract Full Text PDF PubMed Google Scholar, 11Laitinen M. Zachary I. Breier G. Pakkanen T. Hakkinen T. Luoma J. Abedi H. Risau W. Soma M. Laakso M. Martin J.F. Yla Herttuala S. Hum. Gene Ther. 1997; 8: 1737-1744Crossref PubMed Scopus (181) Google Scholar, 12Wheeler-Jones C. Abu-Ghazaleh R. Cospedal R. Houliston R.A. Martin J. Zachary I. FEBS Lett. 1997; 420: 28-32Crossref PubMed Scopus (238) Google Scholar, 13Xia P. Aiello L.P. Ishii H. Jiang Z.Y. Park D.J. Robinson G.S. Takagi H. Newsome W.P. Jirousek M.R. King G.L. J. Clin. Investig. 1996; 98: 2018-2026Crossref PubMed Scopus (529) Google Scholar, 14Gerber H.P. McMurtrey A. Kowalski J. Yan M. Keyt B.A. Dixit V. Ferrara N. J. Biol. Chem. 1998; 273: 30336-30343Abstract Full Text Full Text PDF PubMed Scopus (1761) Google Scholar, 15Takahashi T. Yamaguchi S. Chida K. Shibuya M. EMBO J. 2001; 20: 2768-2778Crossref PubMed Scopus (626) Google Scholar, 16Gliki G. Abu-Ghazaleh R. Jezequel S. Wheeler-Jones C. Zachary I. Biochem. J. 2001; 353: 503-512Crossref PubMed Scopus (86) Google Scholar, 17Abedi H. Zachary I. J. Biol. Chem. 1997; 272: 15442-15451Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar). The function of Flt-1 in the endothelium is unclear, but it is thought to regulate the activity of VEGF partly by acting as a decoy receptor, and in part though direct regulatory effects on KDR (1Ferrara N. Gerber H.-P. LeCouter J. Nat. Med. 2003; 9: 669-676Crossref PubMed Scopus (8076) Google Scholar, 18Zeng H. Dvorak H.F. Mukhopadhyay D. J. Biol. Chem. 2001; 276: 26969-26979Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). Neuropilin-1 (NP-1) is a non-tyrosine kinase receptor for VEGF165, the heparin-binding PlGF-2 isoform, VEGF-B, and VEGF-E (19Soker S. Takashima S. Miao H.Q. Neufeld G. Klagsbrun M. Cell. 1998; 92: 735-745Abstract Full Text Full Text PDF PubMed Scopus (2115) Google Scholar, 20Migdal M. Huppertz B. Tessler S. Comforti A. Shibuya M. Reich R. Baumann H. Neufeld G. J. Biol. Chem. 1998; 273: 22272-22278Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 21Makinen T. Olofsson B. Karpanen T. Hellman U. Soker S. Klagsbrun M. Eriksson U. Alitalo K. J. Biol. Chem. 1999; 274: 21217-21222Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). NP-1 was first identified as a receptor for semaphorin 3A (sema 3A), a member of a family of polypeptides involved in axonal guidance and patterning (22He Z. Tessier-Lavigne M. Cell. 1997; 90: 739-751Abstract Full Text Full Text PDF PubMed Scopus (987) Google Scholar, 23Kolodkin A.L. Levengood D.V. Rowe E.G. Tai Y.T. Giger R.J. Ginty D.D. Cell. 1997; 90: 753-762Abstract Full Text Full Text PDF PubMed Scopus (1019) Google Scholar), and is expressed in endothelial cells, several tumor cell types, and in certain types of neuron including DRG, olfactory, and optic nerves (19Soker S. Takashima S. Miao H.Q. Neufeld G. Klagsbrun M. Cell. 1998; 92: 735-745Abstract Full Text Full Text PDF PubMed Scopus (2115) Google Scholar, 24Fujisawa H. Kitsukawa T. Kawakami A. Takagi S. Shimizu M. Hirata T. Cell Tissue Res. 1997; 290: 465-470Crossref PubMed Scopus (81) Google Scholar). NP-2 has a similar domain structure to NP-1 with 44% amino acid identity and exhibits a distinct expression pattern in the developing nervous system (23Kolodkin A.L. Levengood D.V. Rowe E.G. Tai Y.T. Giger R.J. Ginty D.D. Cell. 1997; 90: 753-762Abstract Full Text Full Text PDF PubMed Scopus (1019) Google Scholar, 25Chen H. Chedotal A. He Z. Goodman C.S. Tessier-Lavigne M. Neuron. 1997; 19: 547-559Abstract Full Text Full Text PDF PubMed Scopus (576) Google Scholar). Sema 3A binds and induces neuronal growth cone collapse specifically through NP-1, whereas sema 3B, 3C, 3E, and 3F recognize both neuropilins, acting as NP-1 antagonists and NP-2 agonists (25Chen H. Chedotal A. He Z. Goodman C.S. Tessier-Lavigne M. Neuron. 1997; 19: 547-559Abstract Full Text Full Text PDF PubMed Scopus (576) Google Scholar, 26Chen H. He Z. Bagri A. Tessier-Lavigne M. Neuron. 1998; 21: 1283-1290Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 27Giger R.J. Urquhart E.R. Gillespie S.K. Levengood D.V. Ginty D.D. Kolodkin A.L. Neuron. 1998; 21: 1079-1092Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 28Takahashi T. Nakamura F. Jin Z. Kalb R.G. Strittmatter S.M. Nat. Neurosci. 1998; 1: 487-493Crossref PubMed Scopus (186) Google Scholar). Neuropilins also play essential roles in angiogenesis. Overexpression of NP-1 in mice results in increased capillary formation, vasodilatation, and malformation of the heart (29Kitsukawa T. Shimono A. Kawakami A. Kondoh H. Fujisawa H. Development. 1995; 121: 4309-4318Crossref PubMed Google Scholar), whereas mice deficient in NP-1 exhibit both defects in embryonic axonal patterning and an array of vascular abnormalities including defective development of large vessels and impaired neural and yolk sac vascularization (30Kawasaki T. Kitsukawa T. Bekku Y. Matsuda Y. Sanbo M. Yagi T. Fujisawa H. Development. 1999; 126: 4895-4902Crossref PubMed Google Scholar). Inactivation of both NP-1 and NP-2 causes a more severe failure of embryonic vascularization resulting in death at E8.5 (31Takashima S. Kitakaze M. Asakura M. Asanuma H. Sanada S. Tashiro F. Niwa H. Miyazaki J.J. Hirota S. Kitamura Y. Kitsukawa T. Fujisawa H. Klagsbrun M. Hori M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3657-3662Crossref PubMed Scopus (327) Google Scholar). However, despite the strong evidence that NP-1 is a major receptor for VEGF165, its roles in the biological functions of this factor remain poorly understood. In this study, we sought to define the receptors and intracellular mechanisms underlying VEGF-dependent inhibition of DRG growth cone collapse. DRG neurons expressed high levels of NP-1 mRNA and protein and a low level of KDR. VEGF164/165 and PlGF-2, ligands for NP-1, both inhibited growth cone collapse with similar efficacy, whereas ligands specific for either Flt-1 or KDR and Flt-4 had no effect. The chemoattractant effects of VEGF164/165 and PlGF-2 were unaffected by KDR inhibition but were blocked by a specific inhibitor of VEGF binding to NP-1. The results also show that the inhibition of growth cone collapse by VEGF family NP-1 ligands is dependent upon cyclooxygenase-derived prostanoid production and that prostanoids exert striking anti-chemorepellent effects on DRG neurons. These findings demonstrate key roles for NP-1 and prostanoid generation in mediating neuroprotective effects of VEGF family members in DRG neurons. Materials—Recombinant human VEGF165 (hVEGF165), rat VEGF164 (rVEGF164), PlGF-1, PlGF-2, sema 3A, and VEGF-D were obtained from R & D Systems. Iloprost and cicaprost were the gift of Dr. Fiona McDonald (Schering AG, Berlin, Germany). PGE2, NS-398, SC-560, indomethacin, SQ22536, and 2′,5′-dideoxyadenosine (DDA) were from Calbiochem. Carbaprostacyclin, PGE1, sulprostone, and butaprost were from Cayman Chemical Inc. M199 and Ham's F-12 medium were from Invitrogen. Endothelial cell basal medium and DMEM, 25 mm HEPES, pH 7.3, were from Clonetics and Sigma, respectively. EG3287 was synthesized as described previously (32, Selwood, D., Zachary, I., and Loehr, M. (October 9, 2003) U. S. Patent WO03082918Google Scholar) and by Bachem Inc. and was >90% pure. Full details of the design, synthesis, and biological characterization of EG3287 will be described elsewhere. 2H. Jia, A. Bagherzadeh, M. Löhr, L. Cheng, D. Selwood, and I. Zachary, manuscript in preparation. All other reagents used were of the purest grade available. Cell Culture—Human umbilical vein endothelial cells (HUVECs) were purchased from TCS CellWorks Ltd. (Buckingham, UK) and cultured in endothelial cell basal medium supplemented with 10% fetal bovine serum, 10 ng/ml human epidermal growth factor, 12 μg/ml bovine brain extract, 50 μg/ml gentamicin sulfate, and 50 ng/ml amphotericin-8. Porcine aortic endothelial cells expressing NP-1 (PAE/NP-1) were provided by Dr. Shay Soker. The cells were grown in Ham's F-12 medium containing 10% fetal bovine serum and 25 μg/ml hygromycin B. PAE cells expressing either KDR (PAE/KDR) or Flt-1 (PAE/Flt-1) were provided by Professor Lena Claesson-Welsh and grown in Ham's F-12 medium containing 10% fetal bovine serum and 250 μg/ml gentamicin G418. Dorsal root ganglion (DRG) explants from newborn rats were cultured as described (33Williams R.S. Cheng L. Mudge A.W. Harwood A.J. Nature. 2002; 417: 292-295Crossref PubMed Scopus (558) Google Scholar, 34Cheng L. Mudge A.W. Neuron. 1996; 16: 309-319Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar) on 24-well tissue culture plates precoated with poly-d-lysine (10 μg/ml) and laminin (10 μg/ml) in serum-free DMEM/F-12 medium supplemented with 100 μg/ml transferrin, 16 μg/ml putrescine, 5 μg/ml insulin, 50 ng/ml thyroxine, 50 ng/ml triiodothyronine, 39 ng/ml sodium selenite, 100 μg/ml crystallized bovine serum albumin, and with or without nerve growth factor (50 ng ml-1, mouse 7 S form, Alomone Labs). Cytosine arabinofuranoside (10 μm) was added after 1 day in culture to kill non-neuronal cells. To obtain DRG neuronal cell cultures, DRG were dissected into L15 medium and then incubated at 37 °C in EBSS containing 0.025% trypsin, 0.1% collagenase, and 0.004% DNase; enzymes were inactivated by addition of 10% fetal calf serum, and the suspension was centrifuged. The cell pellet was resuspended in growth medium and filtered through nylon mesh (20-μm pore size) to obtain a single cell suspension. Sciatic nerves were dissected from P7 rats, enzymatically dissociated to single cells, and filtered through 20-μm gauze, and the Schwann cells were purified by immunopanning to remove OX42-positive blood cells and Thy+ fibroblasts as described previously (34Cheng L. Mudge A.W. Neuron. 1996; 16: 309-319Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). These cells were plated on poly-d-lysine and laminin-coated dishes and grown in serum-free, defined medium. Fibroblasts were from Thy+ panning dishes and plated on PDL-coated dishes; growth medium was DMEM/F-12 plus 10% fetal calf serum. Primary cultures of hippocampal neurons were prepared from P4 rats and dissociated in calcium/magnesium-free Hanks' solution containing trypsin, grown in neurobasal medium with 200 mm glutamine, newborn serum, and B27 supplement. Neuronal Growth Cone Collapse—The growth factors and chemicals to be tested were added after 1 day, and explants were cultured for a further 24 h or at the times indicated before fixation. Recombinant sema 3A protein was routinely added only for 30 min prior to fixation. Explants or cells were loaded with the fluorescent dye calcein (Molecular Probes) for 30 min and then fixed with 4% paraformaldehyde and 0.2% glutaraldehyde in cytoskeletal buffer containing 10 mm MES buffer, pH 6.1, with 138 mm KCl, 3 mm MgCl2, and 2 mm EGTA buffer; aldehydes were quenched with sodium borohydride. In some experiments, cells were labeled with monoclonal antibody to acetylated tubulin (Sigma, clone 6-11 B-1) followed by anti-mouse immunoglobulin and then biotin/streptavidin conjugated to Texas Red. Random fields were scored at the perimeter of the axonal halo that grows out from the explanted cell bodies. Collapsed growth cones had no calcein-labeled lamellae extending beyond the microtubules. Experiments were performed by using triplicate coverslips for each treatment, and data were pooled from three to five different cultures. Western Blotting—Cells were lysed in lysis buffer containing 64 mm Tris-HCl, pH 6.8, 0.2 mm Na3VO4, 2% SDS, 10% glycerol, and 0.1 mm 4-(2-aminoethyl)benzenesulfonyl fluoride. Samples were separated by 7.5% SDS-PAGE and transferred to nitrocellulose. Western blots were probed with antibodies to NP-1, NP-2, KDR, and Flt-1 (all from Santa Cruz Biotechnology). RNA Isolation and Semi-quantitative RT-PCR Analysis—Total RNA was isolated using the RNeasy mini kit (Qiagen Ltd.). During RNA purification an on-column DNase digestion was performed using the RNase-free DNase set (Qiagen Ltd.) to remove residual chromosomal DNA. First strand cDNA was synthesized from 2 μg of each total RNA sample using the Superscript first strand synthesis system for RT-PCR (Invitrogen). PCRs were performed on a PTC-100 programmable thermal controller (MJ Research, Inc.) using the TaqPCR master mix kit (Qiagen Ltd.) and custom-made rat or human gene-specific oligonucleotide primers (MWG Biotec). The PCR cycle number required for amplification to be within the exponential phase was experimentally determined for each primer pair. First strand cDNAs were normalized with respect to expression of glyceraldehyde-3-phosphate dehydrogenase (sense 5′-ACCACAGTCCATGCCATCAC-3′ and antisense 5′-TCCACCACCCTGTTGCTGTA-3′). Subsequently, first strand cDNAs were used to assess expression of neuropilin 1 (sense 5′-TCAGGACCACACAGGAGATG-3′ and antisense 5′-CTGGCTTCCTGGAGATGTTC-3′), neuropilin 2 (human/mouse/rat neuropilin 2 primer pair, R & D Systems), KDR (sense 5′-TAAGGGCATGGAGTTCTTGG-3′ and antisense 5′-AGGAAACAGGTGAGGTAGGC-3′), and Flt-1 (sense 5′-TGCAAGGAACCTCAGACAAG-3′ and antisense 5′-GCAGTATTCCACGATCACCA-3′). PCR products were resolved on 1% agarose gels and visualized by ethidium bromide staining. 125I-VEGF165 Binding—Confluent endothelial cells in 24-well plates were washed twice with phosphate-buffered saline. At 4 °C, various concentrations of peptides, as indicated, diluted in binding medium (DMEM, 25 mm HEPES, pH 7.3, containing 0.1% bovine serum albumin) were added, followed by addition of the indicated concentration of 125I-VEGF165 (1200–1800 Ci/mmol, Amersham Biosciences). After 2 h of incubation at 4 °C, the medium was aspirated and washed four times with cold phosphate-buffered saline. The cells were lysed with 0.25 m NaOH, 0.5% SDS solution, and the bound radioactivity of the lysates was measured. Nonspecific binding was determined in the presence of 100-fold excess unlabeled VEGF165. PGI2 and PGE2 assay—Prostaglandin E2 (PGE2) and the stable PGI2 metabolite, 6-keto-prostaglandin F1α, were measured in DRG culture supernatants using specific enzyme immunoassays kits (Amersham Biosciences). Statistical Analysis—Statistical analysis of data was performed using the Prism (version 3.0) statistical package. Differences in the frequency of growth cone collapse between two groups were assessed by χ2 test. A value of p < 0.05 was taken as statistically significant. The expression of the VEGF receptors, KDR, Flt1, NP-1, and NP-2, was examined in DRG neurons by RT-PCR and Western blot and compared with that in HUVECs, an endothelial cell type that naturally expresses all four receptors abundantly. Cultured DRG neurons, DRG-associated fibroblasts, Schwann cells, and hippocampal neurons all strongly expressed mRNA for NP-1 at a level similar to that in HUVECs and co-expressed NP-2 at a lower level (Fig. 1A). KDR mRNA was detectable in DRG neurons but at a much lower level than in HUVECs, and Flt-1 expression was not detectable (Fig. 1A). KDR and Flt-1 mRNAs were not detected in the other neuronal cell types examined. Western blot analysis showed that DRG-derived neurons expressed a NP-1-immunoreactive band of Mr ∼130,000 that co-migrated with the major NP-1 band present in HUVECs and at a similar level (Fig. 1B). In contrast, NP-2 was strongly expressed in HUVECs but not detectable in DRG extracts. Immunoreactive bands corresponding to KDR and Flt-1 could not be detected in DRG neurons but were strongly expressed in HUVECs. Treatment of cultured DRG explants with rVEGF164 in the absence of other factors reduced growth cone collapse from ∼60 to ∼40%, an effect very similar to that of nerve growth factor (Fig. 2A). In addition to reducing the percentage of collapsed growth cones, it was also observed that rVEGF164 induced a noticeable increase in the area of individual growth cones (Fig. 2B). Similar effects were produced by hVEGF165 and the heparin-binding PlGF isoform, PlGF-2, that binds to both Flt-1 and NP-1 (20Migdal M. Huppertz B. Tessler S. Comforti A. Shibuya M. Reich R. Baumann H. Neufeld G. J. Biol. Chem. 1998; 273: 22272-22278Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). In contrast, PlGF-1, a specific ligand for Flt-1 that is unable to bind NP-1, caused no decrease in growth cone collapse. In addition, VEGF-D, a ligand for KDR and Flt-4 (35Achen M.G. Jeltsch M. Kukk E. Makinen T. Vitali A. Wilks A.F. Alitalo K. Stacker S.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 548-553Crossref PubMed Scopus (1030) Google Scholar), had no effect on growth cone collapse at concentrations from 50 ng/ml up to 1 μg/ml; it was verified that VEGF-D at 1 μg/ml induced KDR phosphorylation, extracellular signal-regulated kinase activation, cell migration, and tubulogenesis in HUVECs (results not shown). Recombinant sema 3A protein induced growth cone collapse in cultured DRG explants in a concentration-dependent manner, and preincubation with either rVEGF164, hVEGF165, or PlGF-2 inhibited the chemorepulsive effect of 100 and 500 ng/ml sema 3A (Fig. 2C). The anti-chemorepulsive effects of rVEGF164 or mPlGF-2 were not reduced by a specific inhibitor of the KDR tyrosine kinase, SU5614, whereas this inhibitor blocked a range of VEGF-induced signaling events and biological responses in HUVECs (results not shown). To examine specifically the role of NP-1 receptors in DRG neurons, several peptides encoded by VEGF exons 7 and/or 8 (Table I), previously identified as the major NP-1-binding region (36Soker S. Gollamudi Payne S. Fidder H. Charmahelli H. Klagsbrun M. J. Biol. Chem. 1997; 272: 31582-31588Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar), were tested for their effects on 125I-VEGF binding to NP-1. At a concentration of 100 μm, a dicyclic exon 7-derived peptide (32, Selwood, D., Zachary, I., and Loehr, M. (October 9, 2003) U. S. Patent WO03082918Google Scholar) corresponding to residues 138–165 of VEGF165 comprising the COOH-terminal residues of exon 7 and all of exon 8 (designated EG3287) inhibited 125I-VEGF165 binding to PAE/NP-1 (Fig. 3A). EG3287 inhibited binding of radiolabeled 125I-VEGF165 to PAE/NP-1 with an IC50 of 3 μm and 95% inhibition of binding at 100 μm but had no effect on 125I-VEGF165 binding to PAE/KDR or PAE/Flt1 cells (Fig. 3B). EG3287 also inhibited 125I-VEGF165 to HUVECs with reduced potency (IC50 26 μm) and caused ∼70% inhibition of specific binding at 100 μm, consistent with expression of KDR and Flt-1 receptors in these cells (Fig. 3B). Preparations of EG3287 retained full activity, as determined by inhibition of 125I-VEGF165 binding to NP-1, for up to 12 months with storage at -20 °C.Table IVEGF exon 7/8 peptides Boldface letters indicate aminobutyric acid. Open table in a new tab EG3287 reduced the anti-chemorepulsive effects of rVEGF164 and PlGF-2 within the concentration range 10–100 μm (Fig. 4, A–C) and also blocked the ability of VEGF to inhibit sema 3A-induced growth cone collapse (results not shown). EG3287 co-incubation of sema 3A with a submaximum concentration of the antagonist (30 μm) caused some increase in growth cone collapse above that obtained with either EG3287 or sema 3A alone, but the effect of the two combined was not additive (results not shown). Incubation of DRG explants with EG3287 alone caused a marked and concentration-dependent increase in growth cone collapse with a detectable effect at 10 μm and a maximum effect at 100 μm similar to the effect of sema 3A (Fig. 4C). The results presented in Figs. 1, 2, 3, 4 indicated that the chemoattractant and anti-chemorepulsive effects of VEGF in DRG growth cones were mediated primarily through the NP-1 receptor with little involvement of Flt-1 or the major VEGF signaling receptor, KDR. Given that NP-1 does not have a known signaling function, we next examined other signaling pathways that could be responsible for mediating VEGF-dependent inhibition of DRG growth cone collapse. Prostaglandins exert direct effects on sensory neurons via specific EP and IP receptors (37Smith J.A. Amagasu S.M. Eglen R.M. Hunter J.C. Bley K.R. Br. J. Pharmacol. 1998; 124: 513-523Crossref PubMed Scopus (84) Google Scholar, 38Murata T. Ushikubi F. Matsuoka T. Hirata M. Yamasaki A. Sugimoto Y. Ichikawa A. Aze Y. Tanaka T. Yoshida N. Ueno A. Oh-ishi S. Narumiya S. Nature. 1997; 388: 678-682Crossref PubMed Scopus (690) Google Scholar, 39Southall M.D. Vasko M.R. J. Biol. Chem. 2001; 276: 16083-16091Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 40Rowlands D.K. Kao C. Wise H. Br. J. Pharmacol. 2001; 133: 13-22Crossref PubMed Scopus (24) Google Scholar) and also mediate biological functions of VEGF (12Wheeler-Jones C. Abu-Ghazaleh R. Cospedal R. Houliston R.A. Martin J. Zachary I. FEBS Lett. 1997; 420: 28-32Crossref PubMed Scopus (238) Google Scholar, 16Gliki G. Abu-Ghazaleh R. Jezequel S. Wheeler-Jones C. Zachary I. Biochem. J. 2001; 353: 503-512Crossref PubMed Scopus (86) Google Scholar). The role of prostanoids in regulating neurotrophic or growth cone activity has not been examined previously, however (Fig. 5A). Treatment of DRG neurons with rVEGF164 and PlGF-2 increased production of PGI2 and PGE2 that was inhibited by the nonspecific cyclooxygenase (COX) inhibitor, indomethacin, and specific inhibitors of COX-1, SC560, and COX-2, NS-398 (Fig. 5A). In three independent experiments, rVEGF164 and PlGF-2, respectively, increased production of PGI2 1.7- and 1.6-fold (p < 0.014 and 0.019 for rVEGF164 and PlGF-2 versus control), and PGE2 2.4- and 1.6-fold (p < 0.015 and 0.022 for rVEGF164 and PlGF-2 versus control) above the control, unstimulated level. Significant basal PGI2 and PGE2 production was detected in DRG cultures and was also markedly reduced by COX inhibitors (Fig. 5A). To examine whether endogenous prostanoid production was necessary for the inhibition of growth cone collapse by VEGF164 and PlGF-2, DRG explants were treated with these factors in the presence of COX inhibitors. Indomethacin caused an increase in the basal level of growth cone collapse and prevented inhibition of collapse induced by rVEGF164 or PlGF-2 (Fig. 5B). SC560 and NS-398 similarly reduced the anti-chemorepulsive effects of rVEGF164 and PlGF-2 and caused a more marked inhibition of the response to rVEGF164 and PlGF-2 when added together (Fig. 5B). The inhibition of anti-chemorepellent effects of VEGF by COX inhibitors prompted us to examine whether addition of exogenous prostanoids could prevent growth cone collapse. The prostacyclin analogues iloprost, cicaprost, and carbaprostacyclin all caused a striking decrease in the percentage of collapsed growth cones, accompanied by an increase in the size of growth cones (Fig. 6, A and B). In addition, the prostaglandins, PGE1 and PGE2, markedly inhibited growth cone collapse, whereas the EP3 and EP1 receptor agonist, sulprostone, and the EP2 agonist butaprost had little effect. Similar to results obtained with VEGF164/165 and PlGF-2, iloprost antagonized the chemorepulsive effects of 100 and 500 ng/ml sema 3A (Fig. 6C). Although inhibition of growth cone collapse by rVEGF164 was blocked by a combination of NS-398 and SC-560, the COX inhibitors did not reduce the anti-chemorepulsive effect of iloprost, indicating that the addition of exogenous prostacyclin circumvented inhibition of COX-derived prostanoid biosynthesis (Fig. 6D). Comparison of the time dependence of the anti-chemorepulsive effects of rVEGF164, PlGF-2, and iloprost showed that iloprost caused a more rapid inhibition of growth cone collapse (Fig. 7A). Iloprost had a marked anti-repellent effect after 30 min,

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