VEGF release by MMP-9 mediated heparan sulphate cleavage induces colorectal cancer angiogenesis
Introduction
Colorectal carcinomas (CRC) are characterised by enhanced VEGF expression and the corresponding high microvascular densities, indicating increased angiogenic activity and leading to worse patient survival.1, 2 Therapy using anti-VEGF antibodies improves CRC patient survival, emphasising VEGF as a major angiogenic factor.3, 4, 5, 6 VEGF expression is up-regulated by hypoxia and various tumour-related cytokines including Transforming Growth Factor-β, Interleukin-1β, Platelet-derived Growth Factor and Epidermal Growth Factor.7, 8 At least six human VEGF isoforms are known, ranging in length from 121 to 206 amino acids, from which VEGF165 is the most predominant. Except for VEGF121 all isoforms contain a heparin-binding domain mediating adhesion to the extracellular matrix (ECM) by interaction with heparan sulphate proteoglycans (HSPGs).9 Binding of larger isoforms to the ECM provides a reservoir of biologically active VEGF. Consequently, the local release of soluble VEGF is a key factor in angiogenesis. Various proteinases have been studied as mediators of VEGF cleavage and/or release, including members of the matrix metalloproteinase (MMP) family like MMP-2,10, 11 MMP-7,12 MMP-913 and MMP-14.14, 15 Most of the experiments showing the involvement of proteinases in VEGF-release were done using animal models and simple in vitro systems. Because interspecies differences have been described for several substrate/MMP combinations,16 we investigated the role of the gelatinases MMP-2 and MMP-9 in VEGF-bioavailability in human CRC. First, the concentration and localisation of VEGF, MMP-2 and MMP-9 were determined in tissues and plasma of CRC patients. Next, we used a 3-dimensional ECM-producing human colon cancer model system to evaluate the role of several MMPs in VEGF release. Recombinant MMPs and conditioned media from neutrophils and myofibroblasts, cell-types associated with the angiogenic switch, were evaluated for their MMP content and VEGF-releasing capacity. VEGF functionality was determined in a 3-dimensional human endothelial cell sprouting assay, resembling in vivo angiogenesis. This study shows the importance of especially MMP-9 in the release of biologically active VEGF165 from the ECM, mainly by the cleavage of HSPGs, leading to the angiogenic switch in CRC.
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Patient material
Pre-operative plasma and tissue specimens from 46 patients (29 ♂, 17 ♀) undergoing resection for primary CRC at the department of Oncologic Surgery, Leiden University Medical Centre, were collected as described before.17 Tissues were homogenised in Tris/Tween-80 and the protein concentrations were determined as described previously.18, 19 For immunohistochemistry, tissue specimens were fixed in 4% paraformaldehyde, dehydrated in graded alcohol and xylene and embedded in paraffin. All human
VEGF and MMP-2 and -9 levels in CRC and in the circulation
Fig. 1 shows the concentrations of VEGF and MMP-9 in CRC and the corresponding mucosa. VEGF and MMP-9 levels were significantly enhanced in tumours (both P < 0.0001, n = 46 pairs) and were mutually correlated (R = 0.405, P < 0.0005, n = 92). Tissue MMP-2 levels were not enhanced in CRC compared to normal mucosa (18.4 versus 18.3 ng/mg protein, n = 46 pairs) and did not correlate with the tumour VEGF concentration. Tumour levels of MMP-2 and MMP-9 correlated weakly with each other (R = 0.250, P = 0.019), but
Discussion
The ability of VEGF to induce angiogenesis depends on the presence of active, mobile isoforms within the microenvironment. After production and release from the cells, VEGF bioavailability is regulated via cleavage and/or release by proteolytic activity in combination with the acidic pH present in the tumour microenvironment.26 heparanases,27, 28 plasmin,9, 29 urokinase,30, 31 phoshatidyl-inositol phosholipase C9 and MMPs11, 12, 13, 14, 15 have been shown to cleave larger VEGF isoforms into
Conflict of interest statement
None declared.
Acknowledgements
We thank Mrs. C. Coomans from the Centre for Human Genetics (Leuven University, Belgium) for providing the HSPG antibody, Dr. I.M. Teepe-Twiss (Department of Clinical Pharmacy and Toxicology, LUMC) for providing Bevacuzimab, E. Dreef, Dr. A. Gorter and Prof Dr. P.C.W. Hogendoorn (Department of Pathology, LUMC) for immunohistochemical support and J.M. van der Zon (Department of Gastroenterology-Hepatology, LUMC) for excellent technical assistance.
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These authors contributed equally to the work.