Batimastat (BB-94) inhibits matrix metalloproteinases of equine laminitis

A method for culturing explants of lamellar hoof was developed to investigate the process of lamellar separation that occurs in laminitis. Explants, consisting of hoof wall, dermal and epidermal lamellae and the adjacent sub-lamellar connective tissue remained intact when cultured in tissue culture medium for 2 days. However, when cultured in the presence of the matrix metalloproteinase (MMP) activator aminophenylmercuric acetate (APMA), the lamellae separated when tension was applied by pulling the hoof wall in an opposite direction to the connective tissue. The separation occurred between the epidermal basal cells and the basement membrane therefore mimicking the lesion of laminitis. Electrophoresis of culture medium from control hoof explants into gradient polyacrylamide gels co-polymerised with gelatin revealed that the explants had produced 2 gelatinases of molecular weight 92 and 72 kDa corresponding to EqMMP-9 and EqMMP-2 respectively. Minor bands of lower molecular weight were the active forms of these enzymes. The zymograms of culture medium from APMA treated explants revealed an increase in the amount of active MMPs. Equine polymorphs cultured for 2 days produced only EqMMP-9. Lamellar explant medium from horses with acute laminitis contained increased amounts of zymogen and active EqMMP-2 and EqMMP-9 particularly in explants from the fore hooves. Zymography of homogenates of normal lamellar hoof tissue revealed only EqMMP-2 and a minor active band. However, homogenates of lamellar tissue from horses with laminitis showed that EqMMP-9 was present as well as increased EqMMP-2 in both zymogen and active forms. Addition of the MMP inhibitor batimastat (BB-94) to the culture medium of APMA treated explants prevented lamellar separation. BB-94 incubated with polyacrylamide strips containing the MMPs from laminitis affected lamellar explants inhibited enzymatic activity at a concentration of 1 mmol/l. It is concluded that activation of MMPs may be responsible for the lamellar separation seen in laminitis and that MMP inhibitors may be useful clinically for preventing this process.     

Limited cleavage of extracellular matrix protein BM-40 by matrix metalloproteinases increases its affinity for collagens

The 33-kDa matrix protein BM-40 (SPARC, osteonectin) consists of an acidic N-terminal domain I, a central cysteine-rich follistatin-like module, and a C-terminal extracellular calcium-binding (EC) module. Previous studies attributed collagen IV and high affinity calcium binding of BM-40 to its EC module, which was shown by x-ray crystallography to consist of an EF-hand pair surrounded by several alpha-helical and loop segments. This module was now shown by surface plasmon resonance assay to bind with similar affinities to collagens I, III, and V. Cleavage of recombinant BM-40 and its EC module by collagenase-3, gelatinases A and B, matrilysin, and stromelysin-1 showed similar fragment patterns, whereas collagenase-1 was inactive. Some differences were, however, observed in cleavage rates and the preference of certain cleavage sites. Edman degradation of fragments demonstrated only three to four major cleavage sites in the central region of domain I and a single uniform cleavage in helix C of the EC module. Cleavage is accompanied by a 7-20-fold increase in binding activity for collagens I, IV, and V but revealed only small effects on calcium-dependent alpha-helical changes in the EC module. The data were interpreted to indicate that helix C cleavage is mainly responsible for enhancing collagen affinity by exposing the underlying helix A of the EC module. A similar activation may also occur in situ as indicated previously for tissue-derived BM-40.     

Proteolysis of extracellular matrix by invadopodia facilitates human breast cancer cell invasion and is mediated by matrix metalloproteinases

Breast cancer cell lines vary in invasive behavior and one highly invasive cell line (MDA-MB-231) proteolytically degrades extracellular matrix with invadopodia (Thompson et al. 1992, J Cell Physiol, 150, 534-44; Chen et al 1994, Breast Cancer Res Treat, 31, 217-26). Invadopodial proteolysis of extracellular matrix is thought to be necessary for invasion; however, this has not been demonstrated directly. To obtain such evidence, normal (HBL-100) and malignant (MCF-7, MDA-MB-231) breast cells were evaluated for invadopodial proteolysis of extracellular matrix and invasive behavior. We report that invadopodial proteolysis of immobilized fibronectin is positively correlated with invasion of cells into type I collagen gels. Moreover, reducing the proteolytic activity of invadopodia with the metalloproteinase inhibitor, batimastat (BB-94), also decreases invasion indicating that breast cancer cell invasion is dependent upon proteolytically active invadopodia.     

Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in HTLV-I-associated myelopathy

Matrix metalloproteinases (MMPs) have been reported to be involved in inflammatory disorders of the central nervous system (CNS). However, little is known about the role of MMPs in the pathogenesis of HTLV-I-associated myelopathy (HAM)/Tropical spastic paraparesis (TSP). To address this issue, we examined the tissue expression and localization of MMPs and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs) in the spinal cord lesions of HAM/TSP using immunohistochemistry. In addition, the blood and cerebrospinal fluid (CSF) levels of MMPs and TIMPs of the patients with HAM/TSP were determined using sandwich enzyme immunoassays (SIA) and gelatin zymography. Immunohistochemical studies revealed that collagen IV and decorin immunoreactivity on the basement membrane of CNS parenchymal vessels was partially disrupted where inflammatory mononuclear cells infiltrated in active-chronic lesions of HAM/TSP. In these lesions, MMP-2 (gelatinase A) was immunostained mainly on the surface of foamy macrophages and lymphocytes, whereas MMP-9 (gelatinase B) expression was positive in the intravascular and perivascular mononuclear cells but not on foamy macrophages. In contrast, inactive chronic lesions of the spinal cords of the HAM/TSP contained fewer MMP-2-positive or MMP-9-positive mononuclear cells than active-chronic lesions. Many parenchymal vessels had thickened vascular walls which showed increased immunoreactivity to decorin. SIA revealed that production levels of MMP-2 and MMP-9 in both blood and CSF were higher in the patients with HAM/TSP than those in non-inflammatory other neurological disease controls (ONDs). Using zymography, proMMP-9 was detected more frequently in the CSF of patients with HAM/TSP than those in ONDs. Taken together, our data indicate that MMP-2 and MMP-9 may play an important role in the blood-brain barrier breakdown and tissue remodeling in the CNS of HAM/TSP.     

Activation and novel processing of matrix metalloproteinases by a thiol-proteinase from the oral anaerobe Porphyromonas gingivalis

A critical outcome of periodontal disease is degradation of the collagenous periodontal ligament that connects teeth to bone in the dental arch. Periodontal diseases occur in response to bacterial colonization of the teeth, but their molecular pathogenesis is still speculative. One family of enzymes, known as the matrix metalloproteinases (MMPs), has been implicated in the degradation of the periodontal ligament. MMPs, which are also suspected to play a role in many other physiologic and pathologic remodeling processes, can be secreted by epithelial cells surrounding the teeth and are found in relative abundance in tissues and fluids near periodontally diseased sites. Since most MMPs are secreted as inactive zymogens which may be activated by limited proteolysis, it has been suggested that proteinases expressed by the infecting periodontal pathogens might activate latent host MMPs to initiate or accelerate degradation of the collegenous periodontal ligament. The aim of this work was to examine interactions between purified host MMPs and bacterial proteinase. In this article, we demonstrate that a proteinase isolated from the periodontopathogen Porphyromonas gingivalis can activate MMP-1, MMP-3, and MMP-9 and can catalyze the superactivation of MMP-1 by MMP-3. Activation of these MMPs is demonstrated to result from initial hydrolysis within their propeptide. Also, for MMP-1 and MMP-9, the P. gingivalis proteinase cleaves the MMP propeptide following a lysine residue at a previously unreported site which, for both MMPs, is one residue NH2-terminal to the known autocatalytic cleavage site. These data describe a mode of virulence for the periodontopathogen Porphyromonas gingivalis that involves activation of host-degradative enzymes.     

Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in reactive and neoplastic lymphoid cells

We have studied the expression of gelatinase A, gelatinase B, interstitial collagenase, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 in reactive lymphoid cells, as well as in a series of cell lines derived from neoplasms of B- and T-cell lineage. Using both Northern blot analysis and zymography, gelatinase B activity was detected by zymography in two Burkitt cell lines and in a tonsillar cell suspension, while gelatinase A and interstitial collagenase activities were not detected by either method. TIMP-1 expression was demonstrated by Northern blot analysis in the multipotential neoplastic K-562 cell line, the high grade Burkitt's B-cell lymphoma lines, isolated tonsillar B cells and at low levels in peripheral blood T cells, but was not expressed in any of the neoplastic T-cell lines or isolated peripheral blood B cells. In contrast, TIMP-2 expression was restricted to tissues containing cells of T-cell lineage with high levels being observed in the neoplastic T-cell lines and lower levels in normal peripheral blood T cells and hyperplastic tonsil. Expression of TIMP-1 and TIMP-2 was confirmed at the protein level by reverse zymography and immunofluorescence assays using antihuman TIMP polyclonal antibodies. Expression of gelatinase B by the high grade B-cell Burkitt's lymphoma cell lines is consistent with previous findings in large cell immunoblastic lymphomas and indicates that this enzyme may play an important role in high grade non-Hodgkin's lymphomas. TIMP expression correlated with cell lineage in that TIMP-1 was primarily observed in B cells and TIMP-2 was restricted to T cells.     

A prospective study of matrix metalloproteinases in proliferative vitreoretinopathy

PURPOSE: The migration, proliferation, differentiation, and adhesion of cells and other cellular functions are influenced by the surrounding extracellular matrix, in normal and wound-healing conditions. The matrix metalloproteinases (MMPs) are a family of enzymes that degrade and remodel the extracellular matrix and, thus, play a central role in the wound-healing process. Proliferative vitreoretinopathy (PVR), a wound-healing process in the retina, is a major cause of the failure of retinal detachment surgery. The role of MMPs in the pathobiology of PVR is unknown. We have investigated the presence of MMPs in the vitreous of patients with retinal detachment and the predictive value of MMPs for the future development of PVR. METHODS: A prospective study was conducted on 140 consecutive patients with a rhegmatogenous retinal detachment in whom vitrectomy was considered necessary because of a giant retinal tear and the presence of preoperative PVR, among other reasons. Vitreous samples were obtained and analyzed by zymography for the presence of MMPs. The patients were then followed up for the development of postoperative PVR (mild and severe). RESULTS: Two species of MMPs were detected in the vitreous: MMP-2 and MMP-9. MMP-2 was detected in all of the vitreous samples obtained, whereas MMP-9 was found in only 64 (47%) of 136 samples. The levels of MMPs detected were not significantly associated with the presence of preoperative PVR (P > 0.05), but they were significantly associated (P < 0.05) with the development of postoperative PVR (mild and severe). CONCLUSIONS: The results from this prospective study suggest that MMPs may be an important predictor and may also play a role in the development of postoperative PVR.     

Polarized distribution of metalloproteinases in the bovine interphotoreceptor matrix

Previous studies from this laboratory had indicated that metalloproteolytic activity was present in the interphotoreceptor matrix. This report extends those observations by providing evidence for the presence of multiple forms of metalloproteinase and for their polarized distribution within the interphotoreceptor matrix as determined by zymogram analysis on substrate-loaded gels. Retinal pigment epithelium-associated interphotoreceptor matrix, both unfractionated as well as fractions separated by gel filtration, exhibited bands of proteolytic activity on gelatin-loaded gels at about 70-75 kDa and 90 kDa, possibly due to gelatinases A and B (MMP-2 and MMP-9, respectively). In contrast, no neutral proteolytic activity was seen in retina-associated interphotoreceptor matrix unless it was first fractionated by gel filtration, whereupon a diversity of forms was exhibited, including additional major bands of proteolytic activity at about 150 kDa and 100 kDa, on gelatin-loaded gels, and at about 180 kDa, on casein-loaded gels, along with many minor species. In all cases, all proteinase activity was inhibited by chelating agents. Since these enzymes may be involved in the turnover and remodeling of components present in the interphotoreceptor matrix, many of which are distributed in a compartmentalized fashion, this unequal distribution of metalloproteinases may be correlated with substrate specificity.     

Activation of neutrophil respiratory burst by cytokines and chemoattractants. Regulatory role of extracellular matrix glycoproteins

OBJECTIVE AND DESIGN: We investigated the in vitro responsiveness of neutrophils adherent to fibronectin (FN) and laminin (LM), toward natural pro-inflammatory and/or phagocyte-activating agents. MATERIALS AND METHODS: Neutrophils from normal volunteers were layered on polystyrene wells precoated or not with FN and/or LM and tested for their ability of responding to eleven pro-inflammatory mediators by evaluation of superoxide anion (O2-) production and adherence. Results, expressed as mean +/-1SEM, were evaluated by non-parametric analyses (Mann-Whitney U-test or Kruskal-Wallis non-parametric ANOVA analysis) RESULTS: Precoating polystyrene wells with LM or FN prevented the plastic-induced neutrophil (O2-) production. Among eleven agents, tumor necrosis factor-alpha (TNF, 3.0+/-0.3 nmoles (O2-)/5 x 10(4) neutrophils/180 min, p < 0.001), granulocyte-macrophage colony stimulating factor (GM-CSF, 2.1+/-0.3 nmoles (O2-)/5 x 10(4) neutrophils/180 min, p < 0.05) and formyl-peptides (fMLP, 2.5+/-0.5 nmoles (O2-)/5 x 10(4) neutrophils/180min, p < 0.01) caused massive (O2-) production by neutrophils adherent to FN. None of the mediators was capable of triggering (O2-) production by neutrophils adherent to LM. LM, mixed with FN to coat wells, caused a dose-dependent inhibition of the oxidative burst triggered by TNF (IC50 LM: 0.84+/-0.03 microg, mean+/-1 SEM), GM-CSF (IC50 LM: 0.36+/-0.16micro/g, mean+/-1SEM) and fMLP (IC50 LM: 0.54+/-0.008 microg, mean+/-1 SEM). To the contrary, fMLP (85.5+/-27.7%), TNF (163.1+/-67.5%), and GM-CSF (121.8+/-66.4%) caused a significant augmentation of neutrophil adherence to LM, suggesting that LM-mediated inhibition of neutrophil oxidative metabolism does not depend on the concomitant LM-induced inhibition of neutrophil adherence. Finally, neither solid-phase FN nor LM affected (O2-) production by neutrophils in response to immune complexes. CONCLUSIONS: Extracellular matrix glycoproteins dictate the response of neutrophils to soluble mediators but not to immune complexes. This appears to be a biologically meaningful mechanism to localise the risk of cellular reactions to mediators that are able to diffuse easily from tissue sites of generation and become widely distributed in body fluids during inflammatory diseases.     

Inhibition of matrix metalloproteinases attenuates anti-Thy1.1 nephritis

An understanding of the olfactory system of any animal must account for how odor mixtures are perceived and processed. The present experiments apply associationist models to the study of how elements are processed in binary odorant mixtures. Using experimental designs for Proboscis Extension Conditioning of honey bees, I show that learning about a pure odorant element is frequently affected by its occurrence in a mixture with a second odorant. Presence of a background odor when an odorant is associated with sucrose reinforcement decreases the rate and/or asymptotic level of associative strength that accumulates to that odorant. This interaction is in part due to synthetic qualities that arise in sensory transduction and initial processing. In addition, it involves an attention-like processing system like that involved in overshadowing. Therefore, a model that includes representations of the component and configural qualities of odorants in mixtures is needed to provide a more complete account of learning about odor mixtures.     

The activation and function of host matrix metalloproteinases in dentin matrix breakdown in caries lesions

Matrix metalloproteinases (MMPs) are a family of enzymes which, in concert, are capable of degrading collagen. We investigated whether human MMPs could participate in the degradation of dentin organic matrix after demineralization. We performed Western blot analyses using MMP-specific antibodies to identify MMPs in human dental caries lesions. Enzymography and functional activity assays, with 125I-labeled gelatin as substrate or quantitating the degradation of type I collagen, were used to determine the activity of purified and salivary gelatinolytic (MMP-2 and MMP-9) and collagenolytic (MMP-8) enzymes with and without acid-activation in pHs relevant to caries. Respective analyses were done with caries-related bacteria. We performed electron microscope analyses to assess the degradative activity of sterilized salivary host MMPs on demineralized human dentin. Human MMP-2, MMP-8, and MMP-9 were identified in demineralized dentinal lesions. The latent purified forms of these enzymes were activated at low pH (4.5), followed by neutralization, mimicking the conditions during caries progression. Incubation of human saliva at low pH followed by neutralization resulted in a four-fold increase in the gelatinolytic activity. No gelatinolytic or collagenolytic activity was observed in bacterial samples. The activated enzymes in saliva degraded demineralized dentin organic matrix in vitro. These results demonstrate the pH-dependent activation mechanism of MMPs, which may have a distinct role in different physiological and pathological conditions. They further demonstrate that host MMPs, activated by bacterial acids, have a crucial role in the destruction of dentin by caries.     

Function of proteoglycans in the extracellular matrix

Estimating phylogenies from DNA sequence data has become the major methodology of molecular phylogenetics. To date, molecular phylogenetics of the vertebrates has been very dependent on mtDNA, but studies involving mtDNA are limited because the several genes comprising the mt-genome are inherited as a single linkage group. The only apparent solution to this problem is to sequence additional genes, each representing a distinct linkage group, so that the resultant gene trees provide independent estimates of the species tree. There exists the need to find novel gene sequences which contain enough phylogenetic information to resolve relationships between closely related species. A possible source is the nuclear-encoded introns, because they evolve more rapidly than exons. We designed primers to amplify and sequence the 7 intron from the beta-fibrinogen gene for a recently evolved group, the woodpeckers. We sequenced the entire intron for 10 specimens representing five species. Nucleotide substitutions are randomly distributed along the length of the intron, suggesting selective neutrality. A preliminary analysis indicates that the phylogenetic signal in the intron is as strong as that in the mitochondrial encoded cytochrome b (cyt b) gene. The topology of the beta-fibrinogen tree is identical to that of the cyt b tree. This analysis demonstrates the ability of the 7 intron of beta-fibrinogen to provide well resolved, independent gene trees for recently evolved groups and establishes it as a source of sequences to be used in other phylogenetic studies.     

Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in the mouse uterus during the peri-implantation period

In this study the SEB-activated LAK cytotoxicity was identified and characterized in human peripheral blood lymphocytes (PBMC). After 3 days of SEB stimulation, the PBMC acquired a cytotoxicity against traditional LAK targets, K-562 and Daudi, beside that human glomerular endothelial cells (HGEC) were effectively lysed. The magnetic separation of SEB-stimulated CD5+ T cells revealed that the dominant LAK cytotoxicity remained in the CD5- lymphocyte fraction. The major part of the SEB-generated cytotoxicity of CD5- cells could be blocked with specific antibodies to IL-2 and IFN-gamma. The IFN-gamma pretreatment of HGEC reduced the target sensitivity, but because of the upregulation of MHC class II on HGEC surface, these cells were able to present SEB to CD5+ cells. These results suggested that in bacterial superantigen-mediated infection, the non-T (NK cells-derived) LAK cells might have a primary pathogenic role, and the adverse effect of IFN-gamma, that was massively secreted from superantigen-stimulated cells, requires greater consideration.