Methods for analysis of matrix metalloproteinase regulation of neutrophil-endothelial cell adhesion

Recent evidence indicates novel role for matrix metalloproteinases (MMPs), in particular gelatinase A (MMP-2), in the regulation of vascular biology that are unrelated to their well-known proteolytic breakdown of matrix proteins. We have previously reported that MMP-2 can modulate vascular reactivity by cleavage of the Gly32-Leu33 bound in big endothelin-1 (ET-1) yielding a novel vasoactive peptide ET-11–32]. These studies were conducted to investigate whether gelatinolytic MMPs could affect neutrophil-endothelial cell attachment. ET-11–32] produced by MMP-2 up-regulated CD11b/CD18 expression on human neutrophils, thereby promoted their adhesion to cultured endothelial cells. ET-11–32] evoked release of gelatinase B (MMP-9), which in turn cleaved big ET-1 to yield ET-11–32], thus revealing a self-amplifying loop for ET-11–32] generation. ET-11–32] was rather resistant to cleavage by neutrophil proteases and further metabolism of ET-11–32] was not a prerequisite for its biological actions on neutrophils. The neutrophil responses to ET-11–32] were mediated via activation of ETA receptors through activation of the Ras/Raf-1/MEK/ERK signaling pathway. These results suggest a novel role for gelatinase A and B in the regulation of neutrophil functions and their interactions with endothelial cells. Here we describe the methods in detail as they relate to our previously published work. Published: October 28, 2002