Abstract
Nerve growth factor (NGF) is a well known neurotropic and neurotrophic agonist in the nervous system, which recently was shown to also induce angiogenic effects in endothelial cells (ECs). To measure NGF effects on the migration of cultured ECs, an important step in neoangiogenesis, we optimized an omnidirectional migration assay using human aortic endothelial cells (HAECs) and validated the assay with human recombinant basic fibroblast growth factor (rhbFGF) and human recombinant vascular endothelial growth factor (rhVEGF). The potencies of nerve growth factor purified from various species (viper, mouse, and recombinant human) to stimulate HAEC migration was similar to that of VEGF and basic fibroblast growth factor (bFGF) (EC50 of ∼0.5 ng/ml). Recombinant human bFGF was significantly more efficacious than either viper NGF or rhVEGF, both of which stimulated HAEC migration by ∼30% over basal spontaneous migration. NGF-mediated stimulation of HAEC migration was completely blocked by the NGF/TrkA receptor antagonist K252a [(8R*,9S*,11S*)-(|)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,-8H,11H-2,7b,11a-triazadibenzo(a,g)cycloocta(c,d,e)trindene-1-one] (30 nM) but not by the VEGF/Flk receptor antagonist SU-5416 [3-[(2,4-dimethylpyrrol-5-yl) methylidenyl]-indolin-2-one] (250 nM), indicating a direct effect of NGF via TrkA receptor activation on HAEC migration. Viper NGF stimulation of HAEC migration was additively increased by either rhVEGF or rhbFGF, suggesting a potentiating interaction between their tyrosine kinase receptor signaling pathways. Viper NGF represents a novel pharmacological tool to investigate possible TrkA receptor subtypes in endothelial cells. The ability of NGF to stimulate migration of HAEC cells in vitro implies that this factor may play an important role in the cardiovascular system besides its well known effects in the nervous system.
Footnotes
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This study was supported in part by grants from the National Aeronautics and Space Administration (to P.I.L.), the Nanotechnology Institute of Southeastern Pennsylvania (to P.I.L.), The Stein family Foundation (to P.I.L. and P.L.), and the Calhoun Endowment (to P.I.L., F.D.A., A.R., and J.-P.D.). This study was carried out in partial fulfillment of the requirements for a Master of Sciences in Biomedical Engineering degree (J.-P.D.).
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doi:10.1124/jpet.105.093252.
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ABBREVIATIONS: EC, endothelial cell; VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor; NGF, nerve growth factor; HAEC, human aortic endothelial cell; RAOEC, rat aortic endothelial cell; RAMEC, rat adrenal medullary endothelial cell; OM, omnidirectional migration; FGF, fibroblast growth factor; rhVEGF, human recombinant vascular endothelial growth factor; rhbFGF, recombinant human basic fibroblast growth factor; Flk-1/KDR, subtype of VEGF receptors (VEGFR2); SU-5416, 3-[(2,4-dimethylpyrrol-5-yl) methylidenyl]-indolin-2-one; K252a, (8R*,9S*,11S*)-(|)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo(a,g)cycloocta(c,d,e)trindene-1-one; PBS, phosphate-buffered saline; DMSO, dimethyl sulfoxide; rhNGF, recombinant human nerve growth factor; mNGF, mouse nerve growth factor; vNGF, viper nerve growth factor; GM, growth medium; FBS, fetal bovine serum; EM, experimental medium; ANOVA, analysis of variance.
- Received July 25, 2005.
- Accepted August 23, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
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