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

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. 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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 and a of and PlGF-2, for NP-1, both inhibited growth cone collapse with similar whereas specific for Flt-1 KDR and had no effect. The effects of and PlGF-2 were by KDR inhibition but were blocked by a specific of VEGF binding to The results also that the inhibition of growth cone collapse by VEGF family NP-1 is production and that prostanoids anti-chemorepellent effects on DRG These findings roles for NP-1 and in mediating neuroprotective effects of VEGF family members in DRG human VEGF165 rat PlGF-1, PlGF-2, sema 3A, and were and were the of PGE2, and were and were and were cell and DMEM, were and was as and M. U. S. Scholar) and by and was Full of the and biological of A. M. L. D. and I. in other used were of the Cell umbilical vein endothelial cells were and in endothelial cell with human growth factor, and aortic endothelial cells expressing NP-1 were by The cells were in and B. cells expressing KDR Flt-1 were by and in and Dorsal root ganglion (DRG) were as L. 2002; PubMed Scopus Google Scholar, L. Neuron. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) on culture with and in with and with nerve growth factor was in culture to DRG neuronal cell DRG were and at in and were by of and the was The cell was in growth and through to a cell nerves were to cells, and through and the cells were by to cells and as L. Neuron. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). These cells were on and and in were and on growth was cultures of neurons were and in in with and growth and to were and were for a at the sema 3A was for to cells were with the for and with and in with and were with In cells were with to followed by and to were at the of the axonal that the cell growth had no the were by for and were to were in and were by and to were with to NP-1, KDR, and Flt-1 and was the an was the to was of the first system for were on a the and rat human The for to the was for were with to expression of and first were used to expression of and KDR and and Flt-1 and were on and by endothelial cells in were with of as in binding were followed by of the of After of at the was and with The cells were with and the of the was binding was in the of and E2 and the were in DRG culture specific of was the in the of growth cone collapse two were by A of was as The expression of the VEGF receptors, KDR, NP-1, and was in DRG neurons by and and with that in an endothelial cell that receptors DRG cells, and neurons expressed mRNA for NP-1 at a similar to that in and NP-2 at a lower KDR mRNA was detectable in DRG neurons but at a much lower than in and Flt-1 expression was not detectable KDR and Flt-1 were not in the other neuronal cell types that neurons expressed a of that with the major NP-1 in and at a similar In NP-2 was expressed in but not detectable in DRG to KDR and Flt-1 not in DRG neurons but were expressed in of DRG with in the of other growth cone collapse to an effect similar to that of nerve growth factor In to the of growth it was also that a in the of growth effects were by and the heparin-binding PlGF isoform, PlGF-2, that binds to both Flt-1 and NP-1 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 PlGF-1, a specific for Flt-1 that is to NP-1, no in growth cone collapse. In VEGF-D, a for KDR and M. T. A. Alitalo K. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar), had no effect on growth cone collapse at to it was that at KDR extracellular signal-regulated kinase cell migration, and in not sema 3A growth cone collapse in DRG in a and with PlGF-2 inhibited the effect of and sema 3A The effects of were not by a specific of the KDR tyrosine SU5614, whereas this blocked a of signaling events and biological in not specifically the role of NP-1 receptors in DRG several by VEGF identified as the major S. S. H. H. Klagsbrun M. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar), were for effects on binding to a of a and M. U. S. 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The role of prostanoids in neurotrophic growth cone activity has not been of DRG neurons with and PlGF-2 increased production of and that was inhibited by the cyclooxygenase inhibitor, and specific inhibitors of and In and PlGF-2, increased production of and and for and PlGF-2 and and and for and PlGF-2 the and production was in DRG cultures and was also by inhibitors production was for the inhibition of growth cone collapse by and PlGF-2, DRG were with these in the of an in the of growth cone collapse and prevented inhibition of collapse by PlGF-2 and the effects of and PlGF-2 and a more inhibition of the response to and PlGF-2 The inhibition of anti-chemorepellent effects of VEGF by inhibitors to of prostanoids growth cone collapse. The prostacyclin analogues and a in the of growth by an in the of growth A and In the and PGE2, inhibited growth cone whereas the and receptor and the had effect. to results with and PlGF-2, antagonized the effects of and sema 3A inhibition of growth cone collapse by was blocked by a of and the inhibitors did not the effect of that the of prostacyclin inhibition of of the of the effects of PlGF-2, and that a more inhibition of growth cone collapse had a effect and its effect was whereas inhibition of growth cone collapse by PlGF-2 was detectable and a in The biological effects of prostacyclin and its analogues are activation of domain receptors by the protein, to activation and of intracellular The role of this in mediating effects of prostacyclin on growth was two inhibitors, and in and both inhibited the in the of growth in a but had no effect on collapse. evidence that NP-1 binds VEGF and is for vascular the role of this receptor in VEGF biological functions has not been defined. The support the that the effects of VEGF in DRG neurons are by NP-1 and are to the VEGF tyrosine kinase receptors, KDR DRG neurons expressed high levels of mRNA and for NP-1 but expression of KDR and no detectable PlGF-2, an of the factor PlGF that binds NP-1 and but not KDR, inhibited growth cone whereas PlGF-1, which binds VEGF-D, a for KDR and inhibition of DRG neuronal collapse. the specific KDR did not the effect of VEGF, whereas EG3287, a specific antagonist of VEGF binding to NP-1, blocked inhibition of growth cone collapse a similar to that for inhibition of binding to VEGF inhibited sema growth cone collapse in DRG with VEGF and sema 3A for binding to NP-1 as H.Q. Soker S. L. Klagsbrun M. J. 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In and PlGF-2 production in DRG neuronal The inhibition of the effects of VEGF by inhibitors indicates that this neuronal response to VEGF is on inhibitors also increased growth cone collapse in the of sema 3A, that production also play a role in the of growth cone The that prostanoids play a role in outgrowth of DRG neurons is by the inhibition of growth cone collapse and increased growth cone by a variety of specific and The inhibition of the in growth cone collapse by inhibitors of indicates that and NP-1 has been to as a for VEGF165 in endothelial cells, and NP-1 is for vascular of the role of this receptor in biological functions of VEGF has The results a specific role for NP-1 in mediating effects of VEGF in DRG neurons of The antagonist of VEGF binding to NP-1, EG3287, a for the role of NP-1 in biological functions of VEGF more findings also a role for prostanoids in the of growth cone collapse that is essential for the of these findings for the mechanisms underlying neuronal functions of VEGF, the function of NP-1, and