Dissecting the role of matrix metalloproteinases (MMP) and integrin αvβ3 in anglogenesis In vitro:: Absence of hemopexin c domain bioactivity, but membrane-type 1-MMP and αvβ3 are critical

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 alpha(v)beta(3)-mediated cell adhesion in a three-dimensional collagen I model. The hydroxamate-based synthetic inhibitors 111394, 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 alpha(v)beta(3), EMD 361276. Although PEX strongly inhibited pro-MMP-2 activation, when contaminating lipopolysaccharide was neutralized, PEX neither affected angiogenesis nor bound integrin alpha(v)beta(3). Moreover, no specific binding of pro-MMP-2 to integrin alpha(v)beta(3) Was found, whereas only one out of four independently prepared enzymatically active MMP-2 preparations could bind integrin alpha(v)beta(3), and this in a PEX-independent manner. Likewise, integrin alpha(v)beta(3)-expressing cells did not bind MMP-2-coated surfaces. Hence, these findings show that endothelial cell invasion of collagen I gels is MTI-MMP and alpha(v)beta(3)-dependent but MMP-2 independent and does not support a role for PEX in alpha(v)beta(3) integrin binding or in modulating angiogenesis in this system.