Tokmina-Roszyk, Dorota

Matrix Metalloproteinase-28 (MMP-28) is the newest and least characterized member of MMP family. To date several potential substrate candidates for MMP-28 have been proposed but no in vivo substrates for this enzyme were confirmed. In the central nervous system (CNS) MMP-28 is believed to be important factor during myelination of the developing nervous system as well as during remyelination that follows neuronal injury. On the other hand, MMP-28 has been found in actively demyelinating lesions in both experimental autoimmune encephalopathy (EAE) and multiple sclerosis patients suggesting its possible role in pathological events associated with autoimmune neurodegenerative processes. In addition, MMP-28 has been linked to modulation of immune response and activation of macrophages which presents another role of this enzyme in autoimmune pathologies. In the study described herein, MMP-28 has been shown to affect myelin composition and appearance, mitochondrial protein content, and vesicular transport proteins. Moreover, the decrease in myelin basic protein quantity observed in healthy MMP-28KO animals affected the myelin staining intensity in various brain regions including corpus callous. Cellular energetic studies did not reveal differences in mitochondrial function in MMP-28KO animals and no difference in reactive oxygen species was observed. In the EAE model, MMP-28 deletion increased the occurrence of atypical form of EAE characterized by increased inflammation of arbor vitae of the brain. In addition, MMP-28 deletion decreased the inflammatory infiltrates present in brains obtained from EAE animals. Lastly, MMP-28 has been shown to affect cellular energetics and activation of bone marrow derived macrophages during the initial stages and after 24 h activation. In addition, MMP-28 deletion increased proinflammatory cytokines and receptors CD86 and iNOS found in M1 polarized macrophages.

Matrix metalloproteinases MMPs are a family of proteolytic enzymes that mediate the degradation of
various components of the extracellular matrix. Their functions are essential for normal physiological
processes such as wound healing, but their dysregulation is associated with various pathologies
including autoimmune diseases such as Multiple Sclerosis MS. Experimental Autoimmune
Encephalomyelitis EAE is a well-established murine model of MS that is mediated by CD4 T-cells.
These cells penetrate the blood-brain-barrier BBB, recruit other immune cells, initiate destruction of the
myelin sheath, and cause axonal loss. MMP-9 is a hallmark enzyme in progression of MS that is
required for penetration of the BBB and generation of autoantigens in EAE. In addition, recent studies
have demonstrated that MMP-9 contributes to normal intracellular function of various cell types
including antigen activated T-cells; however, the intracellular role of MMP-9 in immune cell activation
during EAE pathogenesis is not known. In this study, we used a highly selective MMP-9 triple-helical
peptide inhibitor THPI that is a phosphinate transition state analog to examine antigen specific T-cell
responses. We found that selective inhibition of MMP-9 can mitigate pathogenic T-cell activity and
cellular trafficking as well as the clinical severity of EAE, suggesting that selective MMP-9 inhibition in
MS can be a potent therapeutic option.

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.