title: Inhibition of FGF-2-mediated chemotaxis of murine brain capillary endothelial cells by cyclic RGDfV peptide through blocking the redistribution of c-Src into focal adhesions.
abstract: The alpha(v)beta(3) integrin is essential for fibroblast growth factor (FGF)-induced angiogenesis in vivo. However, the role of this integrin in FGF-2-mediated cellular responses by cultured endothelial cells is largely unknown. Cyclic RGDfV (cRGDfV) peptide is widely used to inhibit the binding of alpha(v)beta(3) integrin to vitronectin. To investigate the role of this integrin in FGF-2-mediated cellular responses, we used immortalized murine brain capillary endothelial cells, denoted IBE cells. Because IBE cells proliferate and migrate in response to FGF-2-treatment, when cultured on fibronectin-coated surface, we first examined the inhibitory activity of this peptide on the binding of alpha(v)beta(3) integrin to fibronectin as well as vitronectin. Solid phase binding assay revealed that cRGDfV peptide strongly inhibited the binding of purified alpha(v)beta(3) integrin to vitonectin- and fibronectin-coated plastic surfaces at a concentration of 50 microM. cRGDfV peptide at 50 microM inhibited spreading as well as adhesion of IBE cells on vitronectin-coated plastic surface but not on fibronectin. On fibronectin-coated substrata, cRGDfV at 50 microM attenuated FGF-2-mediated chemotaxis, but not FGF-2-induced proliferation, of IBE cells. We have previously demonstrated that mitogen-activated protein kinase (MAPK) activation within focal adhesions through c-Src activity was involved in FGF-2-induced chemotaxis of IBE cells. Treatment of cells with cRGDfV peptide was associated with reduced c-Src activity without tyrosine dephosphorylation. Immunofluorescent staining showed that cRGDfV inhibited redistribution of c-Src into focal adhesions. MAPK activation by FGF-2 within focal adhesions was also attenuated in the presence of cRGDfV peptide. Our results indicated that cRGDfV peptide inhibited redistribution of c-Src into focal adhesions, leading to impaired MAPK activation within focal adhesions and motility in FGF-2-treated endothelial cells.
authors.affiliation: Department of Urology, Nagasaki University School of Medicine, 1-7-1, Sakamoto, Nagasaki, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Shono
authors.forename: Y
authors.initials: Y
authors.lastname: Mochizuki
authors.forename: H
authors.initials: H
authors.lastname: Kanetake
authors.forename: S
authors.initials: S
authors.lastname: Kanda
mesh_terms: D020533:Angiogenesis Inhibitors / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000098:blood supply; D002196:Capillaries / Q000166:cytology; D002633:Chemotaxis / Q000187:drug effects*; D003315:Cornea / Q000098:blood supply; D004730:Endothelium, Vascular / Q000187:drug effects*; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D005353:Fibronectins / Q000378:metabolism; D022001:Focal Adhesions / Q000187:drug effects*; D051379:Mice; D018919:Neovascularization, Physiologic / Q000187:drug effects; D010456:Peptides, Cyclic / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D019030:Receptors, Vitronectin / Q000378:metabolism
chemical_list: D020533:Angiogenesis Inhibitors; D005353:Fibronectins; D010456:Peptides, Cyclic; D019030:Receptors, Vitronectin; C092113:cyclo(arginyl-glycyl-aspartyl-phenylalanyl-valyl); D016222:Fibroblast Growth Factor 2; D016392:Proto-Oncogene Proteins pp60(c-src)