© 1995 American Heart Association, Inc.
Articles |
In Vivo Activation of met Tyrosine Kinase by Heterodimeric Hepatocyte Growth Factor Molecule Promotes Angiogenesis
From the Dipartimento di Genetica, Biologia e Chimica Medica (F.S., M.A., E.G., F.B.), the Institute for Cancer Research and Treatment (A.F., M.P., G.G., P.M.C.), and the Laboratorio di Immunopatologia (G.C.), Università di Torino, and the Cattedra di Nefrologia, II Facoltà di Medicina, Università di Pavia, Varese (G.C.), Italy.
Correspondence to Dr F. Bussolino, Dipartimento di Genetica, Biologia e Chimica Medica, Università di Torino, Via Santena 5bis, 10126 Torino, Italy.
Abstract Hepatocyte growth factor (HGF) is a
powerful motogen and mitogen for epithelial cells. The factor is a
90-kD heterodimer composed of an 
chain containing four kringle
motifs and a ß 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 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 and
ß chains, and is amplified by other molecules, including vascular
endothelial growth factor.
Key Words: growth factors • angiogenesis
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