Dissecting the Role of Matrix Metalloproteinases (MMP) and Integrin αvβ3 in AngiogenesisIn vitro: Absence of Hemopexin C Domain Bioactivity, but Membrane-Type 1-MMP and αvβ3 Are Critical

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Abstract
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 αvβ3–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 αvβ3, EMD 361276. Although PEX strongly inhibited pro-MMP-2 activation, when contaminating lipopolysaccharide was neutralized, PEX neither affected angiogenesis nor bound integrin αvβ3. Moreover, no specific binding of pro-MMP-2 to integrin αvβ3 was found, whereas only one out of four independently prepared enzymatically active MMP-2 preparations could bind integrin αvβ3, and this in a PEX-independent manner. Likewise, integrin αvβ3–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 αvβ3- dependent but MMP-2 independent and does not support a role for PEX in αvβ3 integrin binding or in modulating angiogenesis in this system.

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