MAB3321 Sigma-AldrichAnti-MMP-13 Antibody, clone 181-15A12

Anterior cruciate ligament (ACL) degeneration is observed in most osteoarthritis (OA)-affected knee joints. However, the specific spatial and temporal relations of these changes and their association with extracellular matrix (ECM) degeneration are not well understood. The objective of this study was to characterize the patterns and relations of aging-related and OA-associated changes in ACL cells and the ECM.Human knee joints from 80 donors (age 23 through 94) were obtained at autopsy. ACL degeneration was assessed histologically by using a quantitative scoring system. Tissue sections were analyzed for cell density, cell organization, ECM components, ECM-degrading enzymes and markers of differentiation, proliferation, and stem cells.Total cell number in normal ACL decreased with aging but increased in degenerated ACL, because of the formation of perivascular cell aggregates and islands of chondrocyte-like cells. Matrix metalloproteinase (MMP)-1, -3, and -13 expression was reduced in aging ACL but increased in degenerated ACL, mainly in the chondrocyte-like cells. Collagen I was expressed throughout normal and degenerated ACL. Collagen II and X were detected only in the areas with chondroid metaplasia, which also expressed collagen III. Sox9, Runt-related transcription factor 2 (Runx2), and scleraxis expression was increased in the chondrocyte-like cells in degenerated ACL. Alpha-smooth muscle actin (α-SMA), a marker of myofibroblasts and the progenitor cell marker STRO-1, decreased with aging in normal ACL. In degenerated ACL, the new cell aggregates were positive for α-SMA and STRO-1.ACL aging is characterized by reduced cell density and activation. In contrast, ACL degeneration is associated with cell recruitment or proliferation, including progenitor cells or myofibroblasts. Abnormally differentiated chondrocyte-like cell aggregates in degenerated ACL produce abnormal ECM and may predispose to mechanical failure.

The discoidin domain receptors (DDRs) are receptor tyrosine kinases that upon binding to collagens undergo receptor phosphorylation, which in turn activates signal transduction pathways that regulate cell-collagen interactions. We report here that collagen-dependent DDR1 activation is partly regulated by the proteolytic activity of the membrane-anchored collagenases, MT1-, MT2-, and MT3-matrix metalloproteinase (MMP). These collagenases cleave DDR1 and attenuate collagen I- and IV-induced receptor phosphorylation. This effect is not due to ligand degradation, as it proceeds even when the receptor is stimulated with collagenase-resistant collagen I (r/r) or with a triple-helical peptide harboring the DDR recognition motif in collagens. Moreover, the secreted collagenases MMP-1 and MMP-13 and the glycosylphosphatidylinositol-anchored membrane-type MMPs (MT4- and MT6-MMP) have no effect on DDR1 cleavage or activation. N-terminal sequencing of the MT1-MMP-mediated cleaved products and mutational analyses show that cleavage of DDR1 takes place within the extracellular juxtamembrane region, generating a membrane-anchored C-terminal fragment. Metalloproteinase inhibitor studies show that constitutive shedding of endogenous DDR1 in breast cancer HCC1806 cells is partly mediated by MT1-MMP, which also regulates collagen-induced receptor activation. Taken together, these data suggest a role for the collagenase of membrane-type MMPs in regulation of DDR1 cleavage and activation at the cell-matrix interface.

To investigate whether the reduction of discoidin domain receptor 2 (DDR-2), a cell membrane tyrosine kinase receptor for native type II collagen, attenuates the progression of articular cartilage degeneration in mouse models of osteoarthritis (OA).

AIMS: The matrix metalloproteinase (MMP)/tissue inhibitor of metalloproteinase (TIMP) system has a major role in tumour invasion and metastasis. Roles in pathways involved in early tumour development are also being identified for this system, and the aim of this study was to define the expression profile of the major MMPs and TIMPs in colorectal polyp cancers.

Siberian hamsters adapt to seasonal changes by reducing their reproductive function during short days (SD). SD exposure reduces uterine mass and reproductive capacity, but underlying cellular mechanisms remain unknown. Because matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are important in uterine development, parturition, and postpartum remodeling, their expression in uterine tissue from Siberian hamsters undergoing photoperiod-mediated reproductive regression and recrudescence was investigated. Female hamsters were exposed to long day (LD, 16L:8D, controls) or SD (8L:16D) for 3-12 weeks (regression); a second group was exposed to SD or LD for 14 weeks and then transferred to LD for 0-8 weeks (recrudescence). Hamsters were euthanized, uteri collected, and homogenates analyzed by gelatin zymography or Western blotting for MMP and TIMP protein levels. Uterine weight decreased (67-75%) at SD weeks 12-14 and increased post-LD transfer (PT) reaching LD values by PT week 2. MMP-2, but not MMP-9 activity was reduced by SD week 12 or 14 but increased to LD levels at PT week 2. MMP-3 expression increased at SD week 9 compared to other SD and LD groups. MMP-14 and -13 protein levels decreased at SD week 3 but returned to LD levels by SD week 6. During recrudescence, MMP-3 (PT weeks 0-2), MMP-13 (PT week 4), and MMP-14 (PT weeks 2, 4) protein levels were higher than LD. TIMP-1 and 2 were present at low levels. Significant and differential variations in uterine MMP activity/expression during photoperiod-induced regression and recrudescence were observed. These changes likely reflect increases in tissue remodeling during both the adaptation to SD and the restoration of reproductive function.

Destructive remodeling of extracellular matrix has been shown to be present in aneurysmal abdominal aorta. We used real-time quantitative reverse transcriptase polymerase chain reaction to determine the relative expression of matrix metalloproteinase-13 (MMP13) in aortic tissue samples from patients who underwent abdominal aortic aneurysm repair operations (n = 36) and from nonaneurysmal autopsy samples (n = 20). The assays were carried out simultaneously in the same reaction tubes for ribosomal 18S RNA to correct for different amounts of input RNA. MMP13 was expressed in all parts of aorta and its expression was elevated in aneurysmal sac. In further studies using MMP13-specific antibody we demonstrated that MMP13 protein was present in the aneurysmal wall.

Proteases play fundamentally important roles in normal physiology and disease pathology. Methods for detection of active proteolysis may greatly aid in the diagnosis of disease progression, and suggest modes of therapeutic intervention. Most assays for proteolytic potential are limited by a lack of specificity and/or quantification. We have developed a solid-phase activity assay for members of the matrix metalloproteinase (MMP) family that is specific and can be used to quantify active enzyme concentration. The assay has two principal components: a capture antibody that immobilizes the MMP without perturbing the enzyme active site, and a fluorescence resonance energy transfer substrate for monitoring proteolysis at low enzyme concentrations. The assay was standardized for MMP-1, MMP-3, MMP-13, and MMP-14. The efficiency of the assay was found to be critically dependent upon the quality of the antibodies, the use of substrates exhibiting high specific activities for the enzymes, and enzyme samples that are fresh. The assay was applied to studies of constitutive and induced MMP activity in human melanoma cells. Analysis of several melanoma cell lines, and comparison with prior studies, correlated higher constitutive MMP-13 activity with higher levels of the cell surface receptor CD44. Ligands to two different melanoma cell surface receptors (the alpha2beta1 integrin or CD44) were found to induce different proteolytic profiles, suggesting that the extracellular matrix can modulate melanoma invasion. Overall, the solid-phase MMP activity assay was found to be valuable for analysis of protease activity in cellular environments. The solid-phase assay is suitably flexible to allow studies of virtually any proteolytic enzyme for which appropriate substrates and antibodies are available.

We compared the incorporation of bone allografts with or without vancomycin in tibial defects of 18 pigs. High-quality radiographs, histological examination, immunological expression of metalloproteinase-13 (MMP-13) and transforming growth factor-beta 2 (TGFbeta2) indicated that there was no significant difference in bone allograft incorporation between up to 220 times the MIC (minimum inhibitory concentration) in bone allografts with 1 g of vancomycin in each 300 g of allograft or without this supplement.

BACKGROUND: Matrix metalloproteinase (MMP) activation contributes to tissue remodeling in several disease states, and increased MMP activity has been observed in left ventricular (LV) failure. The present study tested the hypothesis that MMP inhibition would influence LV remodeling and function in developing LV failure. METHODS AND RESULTS: LV size and function were measured in 5 groups of rats: (1) obese male spontaneously hypertensive heart failure rats (SHHF) at 9 months (n=10), (2) SHHF at 13 months (n=12), (3) SHHF rats treated with an MMP inhibitor during months 9 to 13 (PD166793 5 mg. kg(-1). d(-1) PO; n=14), (4) normotensive Wistar-Furth rats (WF) at 9 months (n=12), and (5) WF at 13 months (n=12). Plasma concentrations of the MMP inhibitor (116+/-11 micromol/L) reduced in vitro LV myocardial MMP-2 activity by approximately 100%. LV function and geometry were similar in WF rats at 9 and 13 months. LV peak +dP/dt was unchanged at 9 months in SHHF but by 13 months was reduced in the SHHF group compared with WF (3578+/-477 versus 5983+/-109 mm Hg/s, P/=0.05). LV volume measured at an equivalent ex vivo pressure (10 mm Hg) was increased in SHHF at 9 months compared with WF (443+/-12 versus 563+/-33 mL, P/=0.05) and increased further by 13 months (899+/-64 mL, P/=0.05). LV myocardial MMP-2 activity was increased by approximately 2-fold in SHHF at 9 and 13 months. With MMP inhibition, LV peak +dP/dt was similar to WF values and LV volume was reduced compared with untreated SHHF values (678+/-28 mL, P/=0.05). CONCLUSIONS: MMP activity contributes to LV dilation and progression to LV dysfunction in a rodent HF model, and direct MMP inhibition can attenuate this process.