In Vivo Activation ofmetTyrosine Kinase by Heterodimeric Hepatocyte Growth Factor Molecule Promotes Angiogenesis

Abstract

Hepatocyte growth factor (HGF) is a powerful motogen and mitogen for epithelial cells. The factor is a 90-kD heterodimer composed of an α chain containing four kringle motifs and a β chain showing structural homologies with serine proteases. It is, however, devoid of enzymatic activity. Recently, it has been reported that HGF activates migration and proliferation of endothelial cells and is angiogenic. In this article we discuss (1) the molecular domains of HGF required to activate in vitro and in vivo endothelial cells, studied by use of molecular mutants, and (2) the characteristics of the angiogenic response to HGF in an experimental model system of implanted reconstituted basement membrane (Matrigel). Two groups of mutants were made and used in vitro and in vivo: one with deletions of kringle domains and one with substitution at the cleavage site of the HGF precursor. In vitro, HGF variants containing only the first two (HGF-NK2) or the first three kringles (HGF-NK3) of the α chain did not induce proliferation of endothelial cells even if used at a concentration 160-fold higher than that optimal for HGF (0.05 nmol/L). High concentrations of these mutants (4 to 8 nmol/L) activated a little endothelial cell motogenic response that was 60% lower than that elicited by HGF. Substitution of Arg 489 with Gln 489 in the HGF precursor generated an uncleavable single-chain factor, unable to induce either endothelial cell migration or proliferation. In vivo, HGF induced a dose-dependent angiogenic response, which was enhanced by heparin. Optimal HGF concentration (0.42 nmol/L) induced the appearance of clusters of migrating endothelial cells after 2 days. Canalized vessels appeared after 4 days, and the angiogenic response was completed within 6 days with full vascularization of the implanted Matrigel plug. HGF-NK2 and HGF-NK3 did not induce angiogenesis when used at equimolar, biologically active HGF concentrations. A little angiogenic response was observed at a concentration 10-fold higher than that of HGF. The uncleavable single-chain molecule was devoid of activity. The transcript of the HGF receptor was present in the Matrigel plug containing HGF, and the angiogenic response involved its activation, as shown by the agonist effect elicited by a monoclonal antibody against the extracellular domain of the receptor. Furthermore, [3-(1,4,-dihydroxytetralyl)-methylene-2-oxindole], a novel tyrosine kinase inhibitor effective on the HGF receptor, inhibited HGF-induced angiogenesis. During the formation of the new vessels, HGF induces expression of other angiogenic factors and chemokines: these include placental growth factor, vascular endothelial growth factor, KC, JE, macrophage inflammatory protein–2, and HGF itself. A neutralizing antibody to vascular endothelial growth factor partially prevented the angiogenesis induced by HGF. The results of this study demonstrate that the angiogenic response induced by HGF in vivo is elicited by stimulation of the HGF receptor, requires the presence of both α and β chains, and is amplified by other molecules, including vascular endothelial growth factor.