Hepatocyte growth factor-induced endothelial cell motility is mediated by the upregulation of inducible nitric oxide synthase expression

Objectives: Hepatocyte growth factor (HGF) is an angiogenic mitogen which stimulates migration in various cell types and has been shown to induce the production of nitric oxide (NO) in epithelial cells. Conflicting data exist on the effect of NO on endothelial cell migration. The aim of this study was to investigate a possible role for NO in HGF-stimulated endothelial cell motility. Methods: The study was performed primarily using an endothelial cell line derived from adult human saphenous vein. Transient transfection experiments were additionally performed using an adult human coronary artery endothelial cell line, Nitric oxide synthase expression was examined by western blot analysis. Time-lapse digital image microscopy was used to measure cell motility. A DNA construct was used in transient transfections to over-express inducible nitric oxide synthase (iNOS) as an N-terminal fusion to enhanced green fluorescent protein (EGFP). Results: HGF upregulated the expression of iNOS but not constitutive endothelial nitric oxide synthase (eNOS). Treatment of cells with the specific iNOS inhibitor 1400 W revealed that functional iNOS was required for HGF-stimulated endothelial cell motility, HGF-induced iNOS expression was partially abrogated in the presence of the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor LY294002, but not the Sre kinase inhibitor, PP1. Endothelial cell motility increased significantly (P<0.0001) in the presence of the exogenous NO donor spermine-NO and cells expressing the iNOS-EGFP fusion protein exhibited significantly greater (P=0.0038) motility than those expressing EGFP alone. Conclusions: These combined data show that elevated NO production is sufficient to stimulate endothelial cell motility and link HGF and NO. both previously implicated in modulating motility, in a common signalling pathway. (C) 2002 Elsevier Science B.V. All rights reserved.