Abstract
Hepatocyte growth factor (HGF) is a pleiotropic factor that plays an important role in complex biological processes such as embryogenesis, tissue regeneration, cancerogenesis, and angiogenesis. HGF promotes cell proliferation, survival, motility, and morphogenesis through binding to its receptor, a transmembrane tyrosine kinase encoded by the MET proto-oncogene (c-met). Structurally speaking, HGF is a polypeptide related to the enzymes of the blood coagulation cascade. Thus, it comprises kringle domains that in some other proteins have been shown to be responsible for the anti-angiogenic activity. To check whether the isolated kringles of HGF were able to inhibit angiogenesis, we produced them as recombinant proteins and compared their biological activity with that of the recombinant HGF N-terminal domain (N). We showed that (i) none of the isolated HGF kringle exhibits an anti-angiogenic activity; (ii) N is a new anti-angiogenic polypeptide; (iii) the inhibitory action of N is not specific toward HGF, because it antagonized the angiogenic activity of other growth factors, such as fibroblast growth factor-2 and vascular endothelial growth factor; and (iv) in contrast with full-length HGF, N does not bind to the c-met receptor in vitro, but fully retains its heparin-binding capacity. Our results suggest that N inhibits angiogenesis not by disrupting the HGF/c-met interaction but rather by interfering with the endothelial glycosaminoglycans, which are the secondary binding sites of HGF.
Highlights
The hepatocyte growth factor (HGF)1 [1], known as Scatter factor [2], was originally described as a potent mitogen
We showed that (i) none of the isolated Hepatocyte growth factor (HGF) kringle exhibits an anti-angiogenic activity; (ii) N is a new anti-angiogenic polypeptide; (iii) the inhibitory action of N is not specific toward HGF, because it antagonized the angiogenic activity of other growth factors, such as fibroblast growth factor-2 and vascular endothelial growth factor; and (iv) in contrast with full-length HGF, N does not bind to the c-met receptor in vitro, but fully retains its heparin-binding capacity
More recently the prothrombin kringle-2 domain has been posed of the HGF receptor (c-met) extracellular domain fused to human IgG1 Fc; HUVEC, human umbilical vein endothelial cell; Human aortic smooth muscle cells (hASMCs), human aortic smooth muscle cell; NHEK, human normal epidermal keratinocyte; SPR, surface plasmon resonance; GAG, glycosaminoglycan; PBS, phosphate-buffered saline
Summary
Materials—The bacterial expression vectors pET15b and pET21b(ϩ), the Escherichia coli expression strain BL21(DE3), and tetracycline hydrochloride were purchased from Novagen (VWR International, Fontenay-sous-Bois, France). Following an overnight incubation with stirring, the protein solutions were concentrated using Ultrafree-15 Centrifugal Filter Devices and dialyzed against PBS containing 10% glycerol for 2 days with at least three changes of buffer. The rising concentrations (10 –5000 nM) of purified recombinant HGF-derived proteins were added to some wells, and HUVECs were incubated for a further 24 h. HUVECs (passage 2) isolated by trypsin-EDTA treatment and suspended in the M199 medium supplemented with 2.5% FBS, were added at a final concentration of 5 ϫ 105 cells/ml in a 1 mg/ml collagen solution. Iodinated proteins were purified by chromatography on PD-10 columns equilibrated with 25 mM Tris-HCl, pH 7.4, containing 0.4 M NaCl, 5 mM EDTA, and 0.25% AlbuMAX and concentrated using the Ultrafree-4 Centrifugal Filter Device. Statistical significance was evaluated by analysis of variance followed by Bonferroni/ Dunn analysis
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