batimastat and Inflammation

Osteopontin (OPN) is a chemotactic factor which can be cleaved to the pro-inflammatory form by matrix metalloproteinases (MMPs). To test the hypothesis that OPN can modulate inflammatory liver injury during cholestasis, wild-type (WT) C57BL/6 and OPN knockout (OPN-KO) mice underwent bile duct ligation (BDL). OPN-KO mice showed significant reduction in liver injury (plasma ALT and necrosis) and neutrophil recruitment compared with WT animals at 24h but not 72h after BDL. In WT mice, a 4-fold increase in hepatic MMP-3 mRNA and elevated MMP activities and cleaved OPN levels were observed in bile. WT mice subjected to BDL in the presence of the MMP inhibitor BB-94 showed reduced liver injury, less neutrophil extravasation and diminished levels of cleaved OPN in bile. Thus, during obstructive cholestasis, OPN released from biliary epithelial cells could be cleaved by MMPs in bile. When the biliary system leaks, cleaved OPN enters the parenchyma and attracts neutrophils. In the absence of OPN, other chemoattractants, e.g. chemokines, mediate a delayed inflammatory response and injury. Taken together, our data suggest that OPN is the pro-inflammatory mediator that initiates the early neutrophil-mediated injury phase during obstructive cholestasis in mice.

Topics: Alanine Transaminase; Animals; Bile Acids and Salts; Bile Ducts; Cholestasis; Inflammation; Ligation; Male; Matrix Metalloproteinase 3; Mice; Mice, Inbred C57BL; Neutrophil Infiltration; Osteopontin; Phenylalanine; Thiophenes

The chemokine fractalkine is considered as unique since it exists both as membrane-bound adhesion molecule and as shed soluble chemoattractant. Here the hypothesis was tested whether placental fractalkine can be shed and released into the maternal circulation. Immunohistochemical staining of human first trimester and term placenta sections localized fractalkine at the apical microvillous plasma membrane of the syncytiotrophoblast. Gene expression analysis revealed abundant upregulation in placental fractalkine at term, compared to first trimester. Fractalkine expression and release were detected in the trophoblast cell line BeWo, in primary term trophoblasts and placental explants. Incubation of BeWo cells and placental explants with metalloprotease inhibitor Batimastat inhibited the release of soluble fractalkine and at the same time increased the membrane-bound form. These results demonstrate that human placenta is a source for fractalkine, which is expressed in the syncytiotrophoblast and can be released into the maternal circulation by constitutive metalloprotease dependent shedding. Increased expression and release of placental fractalkine may contribute to low grade systemic inflammatory responses in third trimester of normal pregnancy. Aberrant placental metalloprotease activity may not only affect the release of placenta derived fractalkine but may at the same time affect the abundance of the membrane-bound form of the chemokine.

Topics: Adult; Cell Line; Cell Membrane; Chemokine CX3CL1; Female; Gene Expression Regulation, Enzymologic; Humans; Immunohistochemistry; Inflammation; Metalloproteases; Microvilli; Phenylalanine; Placenta; Pregnancy; Pregnancy Trimester, First; Thiophenes; Trophoblasts

Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, involves severe muscle degeneration, inflammation, fibrosis, and early death in afflicted boys. Matrix metalloproteinases (MMPs) are extracellular proteases that cause tissue degradation in several disease states. In this study, we tested the hypothesis that the expression levels of various MMPs are abnormally increased and that their inhibition will ameliorate muscle pathogenesis in animal models of DMD. Our results show that the transcript levels of several MMPs are significantly up-regulated, whereas tissue inhibitors of MMPs are down-regulated, in dystrophic muscle of mdx mice. Chronic administration of batimastat (BB-94), a broad spectrum peptide inhibitor of MMPs, reduced necrosis, infiltration of macrophages, centronucleated fibers, and the expression of embryonic myosin heavy chain in skeletal muscle of mdx mice. Batimastat also reduced the expression of several inflammatory molecules and augmented the levels of sarcolemmal protein beta-dystroglycan and neuronal nitric oxide in mdx mice. In addition, muscle force production in isometric contraction was increased in batimastat-treated mdx mice compared with those treated with vehicle alone. Furthermore, inhibition of MMPs using batimastat reduced the activation of mitogen-activated protein kinases and activator protein-1 in myofibers of mdx mice. Our study provides the novel evidence that the expression of MMPs is atypically increased in DMD, that their inhibition ameliorates pathogenesis, and that batimastat could prove to be a significant candidate for DMD therapy.

Topics: Animals; Disease Models, Animal; Dystrophin; Fibrosis; Gene Expression Regulation; Humans; Inflammation; Male; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Mice; Mice, Inbred C57BL; Mice, Inbred mdx; Mitogen-Activated Protein Kinases; Molecular Sequence Data; Muscle, Skeletal; Muscular Dystrophy, Duchenne; Phenylalanine; Protease Inhibitors; Thiophenes; Transcription Factor AP-1

We have isolated a novel soluble factor(s), neutrophil activator of matrix metalloproteinases (NAM), secreted by unstimulated normal human peripheral blood neutrophils that causes the activation of cell secreted promatrix metalloproteinase-2 (proMMP-2). Partially purified preparations of NAM have been isolated from the conditioned media of neutrophils employing gelatin-Sepharose chromatography and differential membrane filter centrifugation. NAM activity, as assessed by exposing primary human umbilical vein endothelial cells (HUVEC) or HT1080 cells to NAM followed by gelatin zymography, was seen within one hour. Tissue inhibitor of metalloproteinase-2 (TIMP-2) and hydroxamic acid derived inhibitors of MMPs (CT1746 and BB94) abrogated the activation of proMMP-2 by NAM, while inhibitors of serine and cysteine proteases showed no effect. NAM also produced an increase in TIMP-2 binding to HUVEC and HT1080 cell surfaces that was inhibited by TIMP-2, CT1746, and BB94. Time-dependent increases in MT1-MMP protein and mRNA were seen following the addition of NAM to cells. These data support a role for NAM in cancer dissemination.

Topics: Adult; Amides; Calcimycin; Cathepsin G; Cathepsins; Cells, Cultured; Culture Media, Conditioned; Endothelium, Vascular; Enzyme Activation; Enzyme Precursors; Humans; Inflammation; Ionomycin; Matrix Metalloproteinase 14; Matrix Metalloproteinase 2; Neoplasm Invasiveness; Neoplasm Metastasis; Neutrophils; Oligopeptides; Pancreatic Elastase; Phenylalanine; Protease Inhibitors; Proteins; RNA, Messenger; Serine Endopeptidases; Substrate Specificity; Tetradecanoylphorbol Acetate; Thiophenes; Tissue Inhibitor of Metalloproteinase-2; Umbilical Veins

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.

Topics: Animals; Culture Media; Culture Techniques; Electrophoresis, Polyacrylamide Gel; Extracellular Matrix; Foot Diseases; Gelatinases; Hoof and Claw; Horse Diseases; Horses; Inflammation; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Metalloendopeptidases; Phenylalanine; Protease Inhibitors; Thiophenes

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Arthritis, Experimental; Indomethacin; Inflammation; Isomerism; Metalloendopeptidases; Phenylalanine; Radiography; Rats; Thiophenes; Time Factors