Endothelial tubulogenesis within fibrin gels specifically requires the activity of membrane-type-matrix metalloproteinases (MT-MMPs).
Macro- and microvascular endothelial cells (EC) formed tubular structures when cultured within a 3D fibrin matrix, a process that was enhanced by vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), hepatocyte growth factor/scatter factor (HGF/SF) and an angiogenic cocktail composed of nine angiogenic factors. Endothelial tubulogenesis was also increased in co-culture with tumour cells such as U87 glioma cells, but not with non-tumorigenic cell types such as Madin-Darby canine kidney (MDCK) epithelial cells. VEGF/FGF-2-stimulated tube formation was dependent on metalloproteinase function [it is inhibited by the addition of tissue inhibitor of metalloproteinases-2 (TIMP-2)], whereas aprotinin, E64 [trans-epoxysuccinyl-L-leucylamido (4-guanidino)-butane] and pepstatin had no effect. In addition, TIMP-4 also inhibited tubulogenesis, but TIMP-1 or the C-terminal haemopexin domain of matrix metalloproteinase-2 (MMP-2) (PEX) and an anti-MMP-2 function-blocking antibody were unable to block tube formation. This suggests that MMP-2 and other soluble MMPs are not essential for tubulogenesis in fibrin gels, instead TIMP-1-insensitive MMPs, such as members of the membrane type-MMPs (MT-MMP) sub-group (MT1-, MT2-, MT3- or MT5-MMP), are required for this process. Further support for a role for MT1-MMP in endothelial tubulogenesis is that recombinant Y36G N-terminal TIMP-2 mutant protein, which retains an essentially unaltered apparent inhibition constant (K(i)(app)) for several MMPs compared to wild-type N-TIMP-2 but is a 40-fold poorer inhibitor of MT1-MMP, was unable to block tubulogenesis. Furthermore, when EC were cultured within fibrin gels, the mRNA levels of several MMPs (including MT1-MMP, MT2-MMP, MT3-MMP and MMP-2) increased during tubulogenesis. Therefore MT-MMPs and specifically MT1-MMP are likely candidates for involvement during endothelial tubulogenesis within a fibrin matrix, and thus their blockade may be a viable strategy for inhibition of angiogenesis.
Lafleur
Marc A
MA
School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ, UK.
Handsley
Madeleine M
MM
Knäuper
Vera
V
Murphy
Gillian
G
Edwards
Dylan R
DR
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D018920:Coculture Techniques; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000254:growth & development*; D005337:Fibrin / Q000378:metabolism*; D005782:Gels; D006133:Growth Substances / Q000494:pharmacology; D006801:Humans; D053517:Matrix Metalloproteinase 15; D053513:Matrix Metalloproteinase 16; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018919:Neovascularization, Physiologic / Q000502:physiology*; D011480:Protease Inhibitors / Q000378:metabolism; D013997:Time Factors; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics / Q000378:metabolism; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000235:genetics / Q000378:metabolism
D005782:Gels; D006133:Growth Substances; C507098:MMP15 protein, human; C507102:MMP16 protein, human; D011480:Protease Inhibitors; D019715:Tissue Inhibitor of Metalloproteinase-1; D019716:Tissue Inhibitor of Metalloproteinase-2; D005337:Fibrin; D053517:Matrix Metalloproteinase 15; D053513:Matrix Metalloproteinase 16; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases