IN-VIVO ACTIVATION OF MET TYROSINE KINASE BY HETERODIMERIC HEPATOCYTEGROWTH-FACTOR MOLECULE PROMOTES ANGIOGENESIS

Hepatocyte growth factor (HGF) is a powerful motogen and mitogen for e pithelial cells. The factor is a 90-kD heterodimer composed of an a ch ain containing four kringle motifs and a beta 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 art icle we discuss (1) the molecular domains of HGF required to activate in vitro and in vivo endothelial cells, studied by use of molecular mu tants, and (2) the characteristics of the angiogenic response to HGF i n an experimental model system of implanted reconstituted basement mem brane (Matrigel). Two groups of mutants were made and used in vitro an d in vivo: one with deletions of kringle domains and one with substitu tion 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 ct chain did not induce proliferation of endothelial c ells 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 n mol/L) activated a little endothelial cell motogenic response that was 60% lower than that elicited by HGF. Substitution of Arg 489 with Gin 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 t he 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 u sed at equimolar, biologically active HGF concentrations. A little ang iogenic response was observed at a concentration 10-fold higher than t hat of HGF. The uncleavable single-chain molecule was devoid of activi ty. The transcript of the HGF receptor was present in the Matrigel plu g containing HGF, and the angiogenic response involved its activation, as shown by the agonist effect elicited by a monoclonal antibody agai nst the extracellular domain of the receptor. Furthermore, [3-(1,4,-di hydroxytetralyl)-methylene-2-oxindole], a novel tyrosine kinase inhibi tor effective on the HGF receptor, inhibited HGF-induced angiogenesis. During the formation of the new vessels, HGF induces expression of ot her angiogenic factors and chemokines: these include placental growth factor, vascular endothelial growth factor, KC, JE, macrophage inflamm atory protein-2, and HGF itself. A neutralizing antibody to vascular e ndothelial growth factor partially prevented the angiogenesis induced by HGF. The results of this study demonstrate that the angiogenic resp onse induced by HGF in vivo is elicited by stimulation of the HGF rece ptor: requires the presence of both alpha and beta chains, and is ampl ified by other molecules, including vascular endothelial growth factor .