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Originally published In Press as doi:10.1074/jbc.M402488200 on May 4, 2004

J. Biol. Chem., Vol. 279, Issue 29, 30654-30661, July 16, 2004
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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*

Lili Cheng{ddagger}, Haiyan Jia{ddagger}§, Marianne Löhr¶, Azadeh Bagherzadeh{ddagger}§, David I. R. Holmes{ddagger}§, David Selwood¶, and Ian Zachary{ddagger}||

From the {ddagger}Department of Medicine and §Ark Therapeutics Ltd., The Rayne Institute, University College London, 5 University Street, London WC1E 6JJ and Wolfson Institute for Biomedical Research, University College London, London WC1E 6BT, United Kingdom

Received for publication, March 4, 2004 , and in revised form, April 30, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Vascular endothelial growth factor (VEGF)1 is an essential mediator of vasculogenesis and angiogenesis during embryonic development and plays a central role in pathophysiological neovascularization in human disease (1, 2). 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 (3, 4), inhibits hypoxic death of cortical neurons in culture (5) and in cerebral ischemia (6), and promotes neurogenesis in vitro and in vivo (7). 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 (8, 9). 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 (1). 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 (10–17). 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 (1, 18). Neuropilin-1 (NP-1) is a non-tyrosine kinase receptor for VEGF165, the heparin-binding PlGF-2 isoform, VEGF-B, and VEGF-E (19–21). 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 (22, 23), and is expressed in endothelial cells, several tumor cell types, and in certain types of neuron including DRG, olfactory, and optic nerves (19, 24). 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 (23, 25). 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 (25–28). Neuropilins also play essential roles in angiogenesis. Overexpression of NP-1 in mice results in increased capillary formation, vasodilatation, and malformation of the heart (29), 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 (30). Inactivation of both NP-1 and NP-2 causes a more severe failure of embryonic vascularization resulting in death at E8.5 (31). 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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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) and by Bachem Inc. and was >90% pure. Full details of the design, synthesis, and biological characterization of EG3287 will be described elsewhere.2 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 (33, 34) 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 (34). 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 F1{alpha}, 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 {chi}2 test. A value of p < 0.05 was taken as statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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.



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  		FIG. 1.
Expression of VEGF receptors in DRG neurons. A, RNA was extracted from DRG neurons (DRG), DRG-associated fibroblasts (FIB), DRG-associated Schwann cells (SC), brain hippocampal cells (HIP), or from HUVECs (HUV), and expression of KDR, Flt-1, NP-1, and NP-2 was determined by specific RT-PCR, using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a reference gene. B, protein was extracted from the cell types indicated in A, and VEGF receptors were detected by immunoblotting. DRG neurons express predominantly NP-1 with little detectable expression of KDR or Flt-1 receptors. The results are representative of two independent experiments.

 
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 (20). 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 (35), 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).



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  		FIG. 2.
VEGF164/165 and PlGF-2 inhibit DRG growth cone collapse. A, DRG neurons were incubated for 24 h either in the absence of serum or other factors (control, C), or with 50 ng/ml nerve growth factor, 10 ng/ml rat VEGF164 (rVEGF), 25 ng/ml human VEGF165 (hVEGF), 25 ng/ml PlGF-2, 25 ng/ml PlGF-1, 1 µg/ml human (hVEGF-D) or mouse VEGF-D (mVEGF-D), and growth cone collapse was determined. The values for growth cone collapse shown here and in subsequent figures represent mean percentages ± S.E. of growth cone collapse calculated as described under "Experimental Procedures." *, p < 0.000004 for treatments versus control. Here and in subsequent figures, the dashed line represents the control level of growth cone collapse in the absence of exogenously added factors. B, representative photographs showing the effects of rVEGF164, sema 3A, and rVEGF164 plus sema 3A on DRG growth cones. VEGF increases growth cone area and partially reverses the chemorepulsive effect of sema 3A. Arrowheads indicate growth cones in the presence of rVEGF164 plus sema 3A. C, DRG neurons were incubated for 24 h in the absence of serum or other factors (C), or with 10 ng/ml rVEGF164 (rV) 25 ng/ml hVEGF165 (hV), or 25 ng/ml PlGF-2 (P), and then incubated with either vehicle or the indicated concentrations of recombinant sema 3A for 30 min. The induction of growth cone collapse by sema 3A is strongly inhibited by VEGF and PlGF-2. The results shown are representative of four independent experiments.

 
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 (36), 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) 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.


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  		TABLE I
VEGF exon 7/8 peptides Boldface letters indicate aminobutyric acid.

 



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  		FIG. 3.
Specific inhibition of 125I-VEGF165 binding to NP-1 by the dicyclic peptide EG3287. A, effects of peptides corresponding to VEGF exons 7 and 8 on 125I-VEGF165 binding to PAE/NP-1 cells. The peptide sequence and positions of intramolecular disulfide bonds of EG3287 are shown. B, EG3287 specifically inhibits 125I-VEGF165 binding to PAE/NP-1 cells. Confluent PAE/NP-1, PAE/KDR, PAE/Flt-1, and HUVECs were incubated with 125I-VEGF165 at 0.1 nM in the presence of the indicated concentrations of EG3287. Values represent mean percentages ± S.E. of specific 125I-VEGF165 binding calculated from triplicate determinations. Similar results were obtained from three independent experiments. The IC50 values for inhibition of EG3287 125I-VEGF165 binding to PAE/NP-1 cells and HUVECs were 3 and 26 µM, respectively.

 
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).



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  		FIG. 4.
Antagonism of the anti-chemorepulsive effects of rVEGF164 and PlGF-2 by EG3287. A, DRG neurons were incubated for 24 h in the absence of serum or other factors (C), or with 10 ng/ml rVEGF164 or 25 ng/ml PlGF-2, and in the absence or presence of 30 µM EG3287 as indicated. The NP-1 antagonist EG3287 blocked the inhibition of growth cone collapse by rVEGF164 or PlGF-2. The results shown are representative of three independent experiments. B, representative photographs showing the effects of 30 µM EG3287 and EG3287 plus rVEGF164 on DRG growth cones. The arrowheads indicate growth cones in the presence of EG3287 plus rVEGF164. C, DRG neurons were incubated for 24 h in the presence of the indicated concentrations of EG3287 either alone (3287) or with 10 ng/ml rVEGF164 or 25 ng/ml PlGF-2. EG3287 increases growth cone collapse and inhibits the anti-chemorepulsive effects of rVEGF164 and PlGF-2 within the concentration range 10–100 µM. The results shown are representative of three independent experiments.

 
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 (37–40) and also mediate biological functions of VEGF (12, 16). 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).



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  		FIG. 5.
Inhibition of growth cone collapse by VEGF164 and PlGF-2 are dependent on COX-derived prostanoid production. A, production of PGI2 and PGE2 was measured in DRG cultures treated for 20 h with either no addition (C), 10 ng/ml rVEGF164 (V), 10 ng/ml PlGF-2 (P), in the absence or presence of 2 µM indomethacin (Indo), 5 µM NS-398 (NS), 100 nM SC-560 (SC), or 5 µM NS-398 plus 100 nM SC-560 as indicated. The results shown are representative of three independent experiments. B, DRG growth cone collapse was measured after incubation for 24 h in the absence of serum or other factors (C), or with 10 ng/ml rVEGF164 (V) or 10 ng/ml PlGF-2 (P), and in the absence or presence of either 2 µM indomethacin (Indo), 5 µM NS-398, 100 nM SC-560, or 5 µM NS-398 plus 100 nM SC-560 as indicated.

 
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).



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  		FIG. 6.
Prostacyclin and prostaglandins are inhibitors of DRG growth cone collapse. A, growth cone collapse in DRG neurons was determined after incubation for 24 h in the absence of serum or other factors (C), or with 10 ng/ml rVEGF164, 50 nM iloprost (Ilo), 10 µM carbaprostacyclin (cPC), 100 nM cicaprost (Cica), 1 or 5 µg/ml PGE1, 1 or 10 µM PGE2, 0.5 µM sulprostone (Sulp), or 10 µM butaprost (Buta). *, p < 0.00002 for treatments versus control. The results shown are representative of four independent experiments. B, representative photographs of control and iloprost-treated DRG growth cones. Iloprost caused a marked increase in the area of individual growth cones. C, DRG neurons were incubated for 24 h in the absence of serum or other factors (C) or in the presence of 50 nM iloprost (Ilo), and then were incubated with either vehicle or the indicated concentrations of recombinant sema 3A for 30 min. The induction of growth cone collapse by sema 3A is inhibited by iloprost. D, DRG neurons were incubated for 24 h in the absence of serum or other factors (C), or with 10 ng/ml rVEGF164, or 50 nM iloprost (Ilo) in the absence or presence of 5 µM NS-398 plus 100 nM SC-560 as indicated. COX inhibition blocks the anti-chemorepulsive effect of VEGF but does not prevent inhibition of growth cone collapse by iloprost. The results shown are representative of two independent experiments.

 
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, and its effect was maximal after 6 h, whereas inhibition of growth cone collapse by either rVEGF164 or PlGF-2 was detectable after 2 h and reached a maximum in 8–10 h. The biological effects of prostacyclin and its analogues are mediated via activation of seven transmembrane domain IP receptors coupled by the heterotrimeric G protein, Gs, to adenylyl cyclase activation and generation of intracellular cyclic AMP. The role of this pathway in mediating effects of prostacyclin on growth cones was investigated using two selective adenylyl cyclase inhibitors, SQ22536 and DDA. As shown in Fig. 7B, SQ22536 and DDA both inhibited the iloprost-mediated reduction in the percentage of collapsed growth cones in a concentration-dependent manner but had no effect on basal collapse.



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  		FIG. 7.
Inhibition of growth cone collapse by prostacyclin is rapid and mediated via adenylyl cyclase. A, growth cone collapse was determined after DRG neurons were treated for the times indicated with 50 nM iloprost ({blacksquare}), 10 ng/ml rVEGF164 (•), or 10 ng/ml PlGF-2 ({blacktriangleup}). Iloprost caused a detectable decrease in growth cone collapse after 30 min and 2 h, whereas the anti-chemorepulsive effects of rVEGF164 and PlGF-2 were evident after only 2–4 h. B, growth cone collapse was determined after DRG neurons were incubated for 24 h in the absence of serum or other factors (C) or with 50 nM iloprost (Ilo), and in the absence or presence of the indicated concentrations of the adenylyl cyclase inhibitors, SQ22536 (SQ), or DDA. SQ22536 and DDA did not affect basal growth cone collapse but inhibited the anti-chemorepulsive effect of iloprost. The results shown are representative of 2 independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Despite abundant evidence that NP-1 binds VEGF and is required for normal vascular development, the role of this receptor in VEGF biological functions has not been defined. The data presented here support the conclusion that the anti-chemorepulsive effects of VEGF in DRG neurons are mediated by NP-1 and are unlikely to involve the VEGF tyrosine kinase receptors, KDR or Flt1. First, DRG neurons expressed high levels of mRNA and protein for NP-1 but displayed very low level expression of KDR and no detectable Flt1 expression. Second, PlGF-2, an isoform of the VEGF-related factor PlGF that binds NP-1 and Flt1, but not KDR, inhibited growth cone collapse, whereas neither PlGF-1, which binds Flt1 only, nor VEGF-D, a ligand for KDR and Flt4, caused any inhibition of DRG neuronal collapse. Third, the specific KDR inhibitor SU5614 did not reduce the anti-chemorepulsive effect of VEGF, whereas EG3287, a specific peptide antagonist of VEGF binding to NP-1, blocked VEGF-mediated inhibition of growth cone collapse within a concentration range very similar to that for inhibition of 125I-VEGF165 binding to PAE/NP-1 cells. Furthermore, VEGF inhibited sema 3A-induced growth cone collapse in DRG explants, consistent with competition between VEGF and sema 3A for binding to NP-1 as reported previously (41), and the inhibitory effect of VEGF on sema 3A-induced chemorepulsion was blocked by EG3287. These findings indicate that inhibition of basal DRG neuronal growth cone collapse and VEGF antagonism of the chemorepellent activity of sema 3A are NP-1-mediated functions. The finding that EG3287 markedly increased basal growth cone collapse in the absence of exogenous VEGF or sema 3A suggests that binding of this peptide may also inhibit constitutive functions of NP-1 that are essential for maintaining growth cone integrity, a possibility that warrants further investigation.

The biological functions of PlGF isoforms are poorly understood, and effects of PlGFs in primary neuronal cells have not previously received much attention. The finding that PlGF-2 had a marked anti-chemorepulsive effect in DRG neurons, similar to the effect of VEGF, indicates that NP-1-binding PlGF isoforms can elicit biological effects independently of Flt-1, and the finding raises the possibility that PlGF acting through NP-1 may have neurotrophic properties in vivo.

The most well established biological role for NP-1 is in mediating chemorepulsive effects of sema 3A in neuronal axons, but because NP-1 recognizes a variety of semaphorins that display biological effects antagonistic to those of sema 3A and is also able to bind VEGF, a potent chemoattractant, it is likely that the function of NP-1 is not restricted to chemorepulsion in sensory neurons. An implication of the present study is that NP-1 is a bifunctional receptor capable of mediating either chemorepellent activity or neurite outgrowth depending on the stimulating ligand. Neuropilins are not thought to be sufficient to relay a biological signal alone, partly because of the lack of well defined signaling motifs in their cytoplasmic domains. Interaction between NP-1 and plexin A is required for sema 3A-triggered growth cone collapse (42), although identification of the post-receptor signals involved in mediating the chemorepellent activity of sema 3A has so far proved elusive. Because VEGF and PlGF-2 prevented growth cone collapse via NP-1 in the absence of KDR and Flt-1 receptors, it is unclear whether these ligands are acting to inhibit NP-1-mediated collapse and/or are stimulating an alternative signaling pathway via NP-1 that promotes neurite outgrowth. The fact that VEGF stimulated growth cone activity in the absence of other exogenous factors suggests that NP-1 may mediate a VEGF signaling response leading to inhibition of growth cone collapse, but both the nature of this response and how VEGF binding to NP-1 is able to transduce a biological signal in DRG neurons remain to be determined. Candidates for mediation of NP-1-dependent VEGF DRG neuronal signaling are plexins, and a recently identified PSD-95/Dlg/ZO-1 domain protein, NP-1-interacting protein-1 (synectin or RGS-GAIP-interacting protein), binds to a carboxyl-terminal PSD-95/Dlg/ZO-1 domain binding motif in NP-1 (43).

The results of this study also reveal a previously unknown role for COX-derived prostanoids in mediating anti-chemorepulsion in DRG neurons. IP receptors for prostacyclins and EP receptors for PGE1 and PGE2 are strongly expressed in DRG neurons (39, 44), and prostacyclin analogues and PGE1 and PGE2 stimulate a variety of responses in sensory neurons, including adenylyl cyclase activity, cyclic AMP production (45), sensitization to bradykinin (46, 47), and neuropeptide release (39, 45, 47). COX-derived prostanoids are thought to have important physiological roles in the mediation of pain and inflammation (48), and IP receptor-deficient mice display impaired nociception, anti-thrombosis, and inflammatory responses (38). VEGF induces PGI2 production in endothelial cells via extracellular signal-regulated kinase-mediated activation of cytosolic phospholipase A2 (12) and induction of COX-2 (49), and PGI2 and other prostaglandins have been implicated in mediating biological responses to VEGF, including increased vasopermeability and angiogenesis (50, 51). DRG neurons exhibited significant basal production of PGI2 that was blocked by COX inhibitors, indicating that growth cones are sites of active prostanoid production and consistent with the observation that DRG growth cones possess high levels of arachidonic acid, the precursor for prostanoid biosynthesis (52). In addition, VEGF164 and PlGF-2 enhanced COX-dependent prostanoid production in DRG neuronal cultures. The inhibition of the anti-chemorepulsive effects of VEGF by COX inhibitors indicates that this neuronal response to VEGF is dependent on VEGF-stimulated prostanoid production. COX inhibitors also increased basal growth cone collapse in the absence of exogenous sema 3A, suggesting that constitutive COX-mediated prostanoid production may also play a role in the maintenance of basal growth cone function. The contention that prostanoids play a key role in outgrowth of DRG sensory neurons is further supported by the marked inhibition of growth cone collapse and increased growth cone size induced by a variety of specific PGI2 analogues, PGE1 and PGE2. The inhibition of the PGI2-mediated reduction in growth cone collapse by inhibitors of adenylyl cyclase indicates that PGI2 acts via IP receptor-mediated cyclic AMP generation and downstream signaling.

NP-1 has been proposed to act principally as a "docking" molecule for VEGF165 in endothelial cells, and although NP-1 is required for normal vascular development, elucidation of the precise role of this receptor in biological functions of VEGF has proved elusive. The results presented here demonstrate a key specific role for NP-1 in mediating anti-chemorepulsive effects of VEGF in DRG neurons independent of KDR. The peptide antagonist of VEGF binding to NP-1, EG3287, should be a valuable tool for investigating the role of NP-1 in biological functions of VEGF more generally. Our findings also reveal a previously unsuspected role for COX-derived prostanoids in the regulation of growth cone collapse that is essential for the anti-repellent action of VEGF. Together, these findings will be helpful for elucidating the mechanisms underlying neuronal functions of VEGF, the function of NP-1, and neurite outgrowth.


   FOOTNOTES
 
* This work was supported by British Heart Foundation Grant BS/94001 (I. Z.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| To whom correspondence should be addressed: Dept. of Medicine, The Rayne Institute, University College London, 5 University St., London WC1E 6JJ, UK. Tel.: 20-7679-6620; Fax: 20-7679-6212; E-mail: i.zachary{at}ucl.ac.uk.

1 The 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. Back

2 H. Jia, A. Bagherzadeh, M. Löhr, L. Cheng, D. Selwood, and I. Zachary, manuscript in preparation. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Ferrara, N., Gerber, H.-P., and LeCouter, J. (2003) Nat. Med. 9, 669-676[CrossRef][Medline] [Order article via Infotrieve]
  2. Carmeliet, P., and Jain, R. (2003) Nat. Med. 9, 653-660[CrossRef][Medline] [Order article via Infotrieve]
  3. Sondell, M., Lundborg, G., and Kanje, M. (1999) J. Neurosci. 19, 5731-5740[Abstract/Free Full Text]
  4. Sondell, M., Sundler, F., and Kanje, M. (2000) Eur. J. Neurosci. 12, 4243-4254[CrossRef][Medline] [Order article via Infotrieve]
  5. Jin, K. L., Mao, X. O., and Greenberg, D. A. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 10242-10247[Abstract/Free Full Text]
  6. Sun, Y., Jin, K., Xie, L., Childs, J., Mao, X. O., Logvinova, A., and Greenberg, D. A. (2003) J. Clin. Investig. 111, 1843-1851[CrossRef][Medline] [Order article via Infotrieve]
  7. Jin, K., Zhu, Y., Sun, Y., Mao, X. O., Xie, L., and Greenberg, D. A. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 11946-11950[Abstract/Free Full Text]
  8. Neufeld, G., Cohen, T., Gengrinovitch, S., and Poltorak, Z. (1999) FASEB J. 13, 9-22[Abstract/Free Full Text]
  9. Zachary, I., and Gliki, G. (2001) Cardiovasc. Res. 49, 568-581[Abstract/Free Full Text]
  10. Waltenberger, J., Claesson Welsh, L., Siegbahn, A., Shibuya, M., and Heldin, C. H. (1994) J. Biol. Chem. 269, 26988-26995[Abstract/Free Full Text]
  11. Laitinen, M., Zachary, I., Breier, G., Pakkanen, T., Hakkinen, T., Luoma, J., Abedi, H., Risau, W., Soma, M., Laakso, M., Martin, J. F., and Yla Herttuala, S. (1997) Hum. Gene Ther. 8, 1737-1744[Medline] [Order article via Infotrieve]
  12. Wheeler-Jones, C., Abu-Ghazaleh, R., Cospedal, R., Houliston, R. A., Martin, J., and Zachary, I. (1997) FEBS Lett. 420, 28-32[CrossRef][Medline] [Order article via Infotrieve]
  13. Xia, P., Aiello, L. P., Ishii, H., Jiang, Z. Y., Park, D. J., Robinson, G. S., Takagi, H., Newsome, W. P., Jirousek, M. R., and King, G. L. (1996) J. Clin. Investig. 98, 2018-2026[Medline] [Order article via Infotrieve]
  14. Gerber, H. P., McMurtrey, A., Kowalski, J., Yan, M., Keyt, B. A., Dixit, V., and Ferrara, N. (1998) J. Biol. Chem. 273, 30336-30343[Abstract/Free Full Text]
  15. Takahashi, T., Yamaguchi, S., Chida, K., and Shibuya, M. (2001) EMBO J. 20, 2768-2778[CrossRef][Medline] [Order article via Infotrieve]
  16. Gliki, G., Abu-Ghazaleh, R., Jezequel, S., Wheeler-Jones, C., and Zachary, I. (2001) Biochem. J. 353, 503-512[CrossRef][Medline] [Order article via Infotrieve]
  17. Abedi, H., and Zachary, I. (1997) J. Biol. Chem. 272, 15442-15451[Abstract/Free Full Text]
  18. Zeng, H., Dvorak, H. F., and Mukhopadhyay, D. (2001) J. Biol. Chem. 276, 26969-26979[Abstract/Free Full Text]
  19. Soker, S., Takashima, S., Miao, H. Q., Neufeld, G., and Klagsbrun, M. (1998) Cell 92, 735-745[CrossRef][Medline] [Order article via Infotrieve]
  20. Migdal, M., Huppertz, B., Tessler, S., Comforti, A., Shibuya, M., Reich, R., Baumann, H., and Neufeld, G. (1998) J. Biol. Chem. 273, 22272-22278[Abstract/Free Full Text]
  21. Makinen, T., Olofsson, B., Karpanen, T., Hellman, U., Soker, S., Klagsbrun, M., Eriksson, U., and Alitalo, K. (1999) J. Biol. Chem. 274, 21217-21222[Abstract/Free Full Text]
  22. He, Z., and Tessier-Lavigne, M. (1997) Cell 90, 739-751[CrossRef][Medline] [Order article via Infotrieve]
  23. Kolodkin, A. L., Levengood, D. V., Rowe, E. G., Tai, Y. T., Giger, R. J., and Ginty, D. D. (1997) Cell 90, 753-762[CrossRef][Medline] [Order article via Infotrieve]
  24. Fujisawa, H., Kitsukawa, T., Kawakami, A., Takagi, S., Shimizu, M., and Hirata, T. (1997) Cell Tissue Res. 290, 465-470[CrossRef][Medline] [Order article via Infotrieve]
  25. Chen, H., Chedotal, A., He, Z., Goodman, C. S., and Tessier-Lavigne, M. (1997) Neuron 19, 547-559[CrossRef][Medline] [Order article via Infotrieve]
  26. Chen, H., He, Z., Bagri, A., Tessier-Lavigne, M. (1998) Neuron 21, 1283-1290[CrossRef][Medline] [Order article via Infotrieve]
  27. Giger, R. J., Urquhart, E. R., Gillespie, S. K., Levengood, D. V., Ginty, D. D., and Kolodkin, A. L. (1998) Neuron 21, 1079-1092[CrossRef][Medline] [Order article via Infotrieve]
  28. Takahashi, T., Nakamura, F., Jin, Z., Kalb, R. G., and Strittmatter, S. M. (1998) Nat. Neurosci. 1, 487-493[CrossRef][Medline] [Order article via Infotrieve]
  29. Kitsukawa, T., Shimono, A., Kawakami, A., Kondoh, H., and Fujisawa, H. (1995) Development 121, 4309-4318[Abstract]
  30. Kawasaki, T., Kitsukawa, T., Bekku, Y., Matsuda, Y., Sanbo, M., Yagi, T., and Fujisawa, H. (1999) Development 126, 4895-4902[Abstract]
  31. Takashima, 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., and Hori, M. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 3657-3662[Abstract/Free Full Text]
  32. Selwood, D., Zachary, I., and Loehr, M. (October 9, 2003) U. S. Patent WO03082918
  33. Williams, R. S., Cheng, L., Mudge, A. W., and Harwood, A. J. (2002) Nature 417, 292-295[CrossRef][Medline] [Order article via Infotrieve]
  34. Cheng, L., and Mudge, A. W. (1996) Neuron 16, 309-319[CrossRef][Medline] [Order article via Infotrieve]
  35. Achen, M. G., Jeltsch, M., Kukk, E., Makinen, T., Vitali, A., Wilks, A. F., Alitalo, K., and Stacker, S. A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 548-553[Abstract/Free Full Text]
  36. Soker, S., Gollamudi Payne, S., Fidder, H., Charmahelli, H., and Klagsbrun, M. (1997) J. Biol. Chem. 272, 31582-31588[Abstract/Free Full Text]
  37. Smith, J. A., Amagasu, S. M., Eglen, R. M., Hunter, J. C., and Bley, K. R. (1998) Br. J. Pharmacol. 124, 513-523[CrossRef][Medline] [Order article via Infotrieve]
  38. Murata, T., Ushikubi, F., Matsuoka, T., Hirata, M., Yamasaki, A., Sugimoto, Y., Ichikawa, A., Aze, Y., Tanaka, T., Yoshida, N., Ueno, A., Oh-ishi, S., and Narumiya, S. (1997) Nature 388, 678-682[CrossRef][Medline] [Order article via Infotrieve]
  39. Southall, M. D., and Vasko, M. R. (2001) J. Biol. Chem. 276, 16083-16091[Abstract/Free Full Text]
  40. Rowlands, D. K., Kao, C., and Wise, H. (2001) Br. J. Pharmacol. 133, 13-22[CrossRef][Medline] [Order article via Infotrieve]
  41. Miao, H. Q., Soker, S., Feiner, L., Alonso, J. L., Raper, J. A., and Klagsbrun, M. (1999) J. Cell Biol. 146, 233-242[Abstract/Free Full Text]
  42. Tamagnone, L., Artigiani, S., Chen, H., He, Z., Ming, G. I., Song, H., Chedotal, A., Winberg, M. L., Goodman, C. S., Poo, M., Tessier-Lavigne, M., and Comoglio, P. M. (1999) Cell 99, 71-80[CrossRef][Medline] [Order article via Infotrieve]
  43. Cai, H., and Reed, R. R. (1999) J. Neurosci. 19, 6519-6527[Abstract/Free Full Text]
  44. Oida, H., Namba, T., Sugimoto, Y., Ushikubi, F., Ohishi, H., Ichikawa, A., and Narumiya, S. (1995) Br. J. Pharmacol. 116, 2828-2837[Medline] [Order article via Infotrieve]
  45. Hingtgen, C. M., Waite, K. J., and Vasko, M. R. (1995) J. Neurosci. 15, 5411-5419[Abstract]
  46. Nicol, G. D., and Cui, M. (1994) J. Physiol. (Lond.) 480, 485-492[Medline] [Order article via Infotrieve]
  47. Vasko, M. R., Campbell, W. B., and Waite, K. J. (1994) J. Neurosci. 14, 4987-4997[Abstract]
  48. Davies, P., Bailey, P. J., Goldenberg, M. M., and Ford-Hutchinson, A. W. (1984) Annu. Rev. Immunol. 2, 335-357[CrossRef][Medline] [Order article via Infotrieve]
  49. Hernandez, G. L., Volpert, O. V., Iniguez, M. A., Lorenzo, E., Martinez-Martinez, S., Grau, R., Fresno, M., and Redondo, J. M. (2001) J. Exp. Med. 193, 607-620[Abstract/Free Full Text]
  50. Murohara, T., Horowitz, J. R., Silver, M., Tsurumi, Y., Chen, D., Sullivan, A., and Isner, J. M. (1998) Circulation 97, 99-107[Abstract/Free Full Text]
  51. Matsumoto, H., Ma, W. G., Daikoku, T., Zhao, X., Paria, B. C., Das, S. K., Trzaskos, J. M., and Dey, S. K. (2002) J. Biol. Chem. 277, 29260-29267[Abstract/Free Full Text]
  52. Negre-Aminou, P., and Pfenninger, K. H. (1993) J. Neurochem. 60, 1126-1136[CrossRef][Medline] [Order article via Infotrieve]

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