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Dissecting the Role of Matrix Metalloproteinases (MMP) and Integrin _vß₃ in Angiogenesis In vitro: Absence of Hemopexin C Domain Bioactivity, but Membrane-Type 1-MMP and _vß₃ Are Critical

¹ Department of Morphology, University Medical Center, Geneva, Switzerland; ² Oncology Research and ³ Medicinal Chemistry, Merck KGaA, Darmstadt, Germany; ⁴ Centre for Blood Research, Department of Oral Biological and Medical Sciences, and Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada; ⁵ Celltech, Slough, Berkshire, United Kingdom; ⁶ School of Biological Sciences, University of East Anglia, Norwich, United Kingdom; and ⁷ NetCare Molecular Medicine Institute, Unitas Hospital, and Department of Immunology, Faculty of Health Sciences, University of Pretoria, Pretoria, South Africa

Requests for reprints: Michael S. Pepper, NetCare Molecular Medicine Institute, Unitas Hospital, Clifton Avenue, 0140 Lyttleton, Pretoria, South Africa. Phone: 27-12-677-8504; Fax: 27-12-677-8505; E-mail: mpepper{at}doctors.netcare.co.za.

Matrix metalloproteinase (MMP)-2 and its hemopexin C domain autolytic fragment (also called PEX) have been proposed to be crucial for angiogenesis. Here, we have investigated the dependency of in vitro angiogenesis on MMP-mediated extracellular proteolysis and integrin _vß₃–mediated cell adhesion in a three-dimensional collagen I model. The hydroxamate-based synthetic inhibitors BB94, CT1399, and CT1847 inhibited endothelial cell invasion, as did neutralizing anti–membrane-type 1-MMP (MT1-MMP) antibodies and tissue inhibitor of MMP (TIMP)-2 and TIMP-3 but not TIMP-1. This confirmed the pivotal importance of MT1-MMP over other MMPs in this model. Invasion was also inhibited by a nonpeptidic antagonist of integrin _vß₃, EMD 361276. Although PEX strongly inhibited pro-MMP-2 activation, when contaminating lipopolysaccharide was neutralized, PEX neither affected angiogenesis nor bound integrin _vß₃. Moreover, no specific binding of pro-MMP-2 to integrin _vß₃ was found, whereas only one out of four independently prepared enzymatically active MMP-2 preparations could bind integrin _vß₃, and this in a PEX-independent manner. Likewise, integrin _vß₃–expressing cells did not bind MMP-2-coated surfaces. Hence, these findings show that endothelial cell invasion of collagen I gels is MT1-MMP and _vß₃- dependent but MMP-2 independent and does not support a role for PEX in _vß₃ integrin binding or in modulating angiogenesis in this system.

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