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-1[1-32]. These studies were conducted to investigate whether gelatinolytic MMPs could affect neutrophil-endothelial cell attachment. ET-1[1-32] produced by MMP-2 up-regulated CD11b/CD18 expression on human neutrophils, thereby promoted their adhesion to cultured endothelial cells. ET-1[1-32] evoked release of gelatinase B (MMP-9), which in turn cleaved big ET-1 to yield ET-1[1-32], thus revealing a self-amplifying loop for ET-1[1-32] generation. ET-1[1-32] was rather resistant to cleavage by neutrophil proteases and further metabolism of ET-1[1-32] was not a prerequisite for its biological actions on neutrophils. The neutrophil responses to ET-1[1-32] were mediated via activation of ET(A)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.
Department of Biochemistry, University of Alberta. Medical Sciences Building 3-08, Edmonton Alberta, Canada T6G 2S2. Canada.Research Center, Maisonneuve-Rosemont Hospital and Department of Medicine, University of Montréal. Montréal Québec, Canada H1T 2M4. Canada.Mass Spectrometry Facility, Department of Chemistry, University of Alberta. Edmonton Alberta, Canada T6G 2S2. Canada.Perinatal Research Center, University of Alberta. Edmonton Alberta, Canada T6G 2S2. Canada. janos.g.filep@umontreal.ca
Carlos
C
Fernandez-Patron
Christine
C
Zouki
Randy M.
RM
Whittal
John S.D.
JS
Chan
Sandra T.
ST
Davidge
J áNosG
Filep J