Matrix metalloproteinase-9 (MMP-9) in vivo substrates in left ventricle remodeling process

Matrix metalloproteinase-9 MMP-9 is involved in the early stages of wound healing, including the
inflammatory reaction that follows myocardial infarction and neovascularization. However, its
overexpression in the infarct zone leads to deleterious effects. Understanding MMP-9 function and
modulation of its activity provides an opportunity to prevent excessive remodeling of the left ventricle.
To assess the role of MMP-9 in remodeling process we employed a broad search of in vivo substrates.
Based on comparative analysis of MMP-9 null and wild type mice, several peptides mimicking putative
substrates were synthesized. The cleavage sites in the substrates were identified using high
performance liquid chromatography and mass spectrometry. Peptide mapping studies revealed MMP-9
cleavage sites in several proteins, potential biomarkers of excessive remodeling. Specifically,
osteopontin, thrombospondin and C-terminal telopeptide regions of type I collagen were susceptible to
proteolysis by MMP-9. The best target for MMP-9 was fibronectin, which has multiple cleavage sites in
its sequence. In addition to in vivo substrate screening, a selective triple-helical peptide inhibitor MMP-
9i has been designed, synthesized, and utilized as an MMP-9 probe. The sequence of inhibitor was
derived from the known MMP-9 substrate type V collagen. In the MMP-9i construct, the G~V scissile
bond has been replaced with phosphinate moiety that mimics the transition state of hydrolysis but
cannot be cleaved. MMP-9i's effect on MMP-9 activity in serum was tested in a mouse model. The
administration of MMP-9i resulted in 30 loss of MMP-9 activity suggesting that MMP-9i can be utilized
to regulate activity of MMP-9 in vivo.