Regeneration of soft tissues is promoted by MMP1 treatment after digit amputation in mice.

The ratio of matrix metalloproteinases (MMPs) to the tissue inhibitors of metalloproteinases (TIMPs) in wounded tissues strictly control the protease activity of MMPs, and therefore regulate the progress of wound closure, tissue regeneration and scar formation. Some amphibians (i.e. axolotl/newt) demonstrate complete regeneration of missing or wounded digits and even limbs; MMPs play a critical role during amphibian regeneration. Conversely, mammalian wound healing re-establishes tissue integrity, but at the expense of scar tissue formation. The differences between amphibian regeneration and mammalian wound healing can be attributed to the greater ratio of MMPs to TIMPs in amphibian tissue. Previous studies have demonstrated the ability of MMP1 to effectively promote skeletal muscle regeneration by favoring extracellular matrix (ECM) remodeling to enhance cell proliferation and migration. In this study, MMP1 was administered to the digits amputated at the mid-second phalanx of adult mice to observe its effect on digit regeneration. Results indicated that the regeneration of soft tissue and the rate of wound closure were significantly improved by MMP1 administration, but the elongation of the skeletal tissue was insignificantly affected. During digit regeneration, more mutipotent progenitor cells, capillary vasculature and neuromuscular-related tissues were observed in MMP1 treated tissues; moreover, there was less fibrotic tissue formed in treated digits. In summary, MMP1 was found to be effective in promoting wound healing in amputated digits of adult mice.

Site-specific targeting of antibody activity in vivo mediated by disease-associated proteases.

As a general strategy to selectively target antibody activity in vivo, a molecular architecture was designed to render binding activity dependent upon proteases in disease tissues. A protease-activated antibody (pro-antibody) targeting vascular cell adhesion molecule 1 (VCAM-1), a marker of atherosclerotic plaques, was constructed by tethering a binding site-masking peptide to the antibody via a matrix metalloprotease (MMP) susceptible linker. Pro-antibody activation in vitro by MMP-1 yielded a 200-fold increase in binding affinity and restored anti-VCAM-1 binding in tissue sections from ApoE⁻/⁻ mice ex vivo. The pro-antibody was efficiently activated by native proteases in aorta tissue extracts from ApoE⁻/⁻, but not from normal mice, and accumulated in aortic plaques in vivo with enhanced selectivity when compared to the unmodified antibody. Pro-antibody accumulation in aortic plaques was MMP-dependent, and significantly inhibited by a broad-spectrum MMP inhibitor. These results demonstrate that the activity of disease-associated proteases can be exploited to site-specifically target antibody activity in vivo.

N-alpha-PGP and PGP, potential biomarkers and therapeutic targets for COPD.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a common respiratory disorder for which new diagnostic and therapeutic approaches are required. Hallmarks of COPD are matrix destruction and neutrophilic airway inflammation in the lung. We have previously described two tri-peptides, N-alpha-PGP and PGP, which are collagen fragments and neutrophil chemoattractants. In this study, we investigate if N-alpha-PGP and PGP are biomarkers and potential therapeutic targets for COPD. METHODS: Induced sputum samples from COPD patients, healthy controls and asthmatics were examined for levels of N-alpha-PGP and PGP using mass spectrometry and for the ability to generate PGP de novo from collagen. Proteases important in PGP generation in the lung were identified by the use of specific inhibitors in the PGP generation assay and by instillation of proteases into mouse lungs. Serum levels of PGP were compared between COPD patients and controls. RESULTS: N-alpha-PGP was detected in most COPD sputum samples but in no asthmatics or controls. PGP was detected in a few controls and in all COPD sputum samples, where it correlated with levels of myeloperoxidase. COPD sputum samples had the ability to generate N-alpha-PGP and PGP de novo from collagen. PGP generation by COPD sputum was blocked by inhibitors of matrix metalloproteases (MMP's) 1 and 9 and prolyl endopeptidase. MMP's 1 and 9 and prolyl endopeptidase acted synergistically to generate PGP in vivo when instilled into mouse lungs. Serum levels of PGP were also significantly higher in COPD patients than in controls CONCLUSION: N-alpha-PGP and PGP may represent novel diagnostic tests and biomarkers for COPD. Inhibition of this pathway may provide novel therapies for COPD directed at the chronic, neutrophilic, airway inflammation which underlies disease progression.

Controlled release of matrix metalloproteinase 1 with or without skeletal myoblasts transplantation improves cardiac function of rat hearts with chronic myocardial infarction.

Skeletal myoblast transplantation has been applied clinically for severe ischemic cardiomyopathy. Matrix metalloproteinase 1 (MMP-1) reduces fibrosis and prevents the progress of heart failure. We hypothesized that MMP-1 administration to the infarct area enhances the efficacy of skeletal myoblast transplantation. The controlled release of MMP-1 improved cardiac functions of rats with chronic myocardiac infarction with or without transplantation of skeletal myoblasts. Improvement in cardiac function and small fibrotic area inside the infarcted area were observed compared with those of myoblast transplantation. In conclusion, controlled release of MMP-1 was effective in cardioprotection in postmyocardial infarction although the combination with cell transplantation showed the similar effect.

Matrix metalloproteinase-1 treatment of muscle fibrosis.

The onset of scarring after injury may impede the regeneration and functional recovery of skeletal muscle. Matrix metalloproteinase-1 (MMP-1) hydrolyzes type I collagen and thus may improve muscle regeneration by resolving fibrotic tissue. We examined the effect of recombinant human MMP-1 on fibrosis in the lacerated gastrocnemius muscle of NOD/scid mice, allowing treatment potential to be ascertained in isolation from immune response. The efficacy of proMMP-1 and active MMP-1 were compared with or without poly(ethylene glycol) (PEG) modification, which was intended to increase the enzyme's stability. Active MMP-1 was most effective in reducing fibrosis, although treatment with proMMP-1 was also beneficial relative to controls. PEG-modified MMP-1 had minimal activity in vivo, despite retaining activity towards a thioester substrate. Moreover, the modified enzyme was inactivated by trypsin and subtilisin at rates comparable to that of native MMP-1. These results and those of computational structural studies suggest that modification occurs at the C-terminal hemopexin domain of MMP-1, which plays a critical role in collagen turnover. Site-specific modifications that spares catalytic and substrate binding sites while protecting susceptible proteolytic digestion sites may be beneficial. We conclude that active MMP-1 can effectively reduce muscle scarring and that its activity is related to the ability of the enzyme to digest collagen, thereby facilitating remodeling of the injured muscle.

Matrix metalloproteinase-1 therapy improves muscle healing.

Muscle undergoes time-dependent phases of healing after injury, which ultimately results in residual fibrosis in the injured area. The use of exogenous matrix metalloproteinases (MMPs) may improve recovery after muscle injury by promoting the digestion of existing fibrous tissue and releasing local growth factors. In the current experiment, bilateral gastrocnemius (GM) lacerations were created in severe combined immunodeficient mice. Twenty-five days after injury (peak posttraumatic fibrosis), C2C12 cells (myoblasts) transduced with the LacZ reporter gene were injected with exogenous MMP-1 into the right GMs at the site of injury; the cells were also injected along with PBS (control) at the site of injury in the left GMs. The muscle tissues were examined histologically via X-gal, hemotoxylin and eosin, and Masson's trichrome staining. The MMP-treated limbs contained more regenerating myofibers than did the control limbs (MMP 170+/-96 fibers, control 62+/-51 fibers; P<0.001). Less fibrous tissue was observed within MMP-treated muscles (MMP: 24+/-11%, control: 35+/-15%; P<0.01). These results suggest that the direct injection of MMP-1 into the zone of injury during fibrosis can enhance muscle regeneration by increasing the number of myofibers and decreasing the amount of fibrous tissue.