Objective. To determine the expression of matrix metalloproteinase 9/gelatinase B (MMP-9) in synovial fluid (SF), plasma, and synovial tissue from individuals with rheumatoid arthritis (RA), inflammatory arthritis (IA), and osteoarthritis (OA), using specific monoclonal antibody reagents.Methods. Gelatinolytic activity in the SF and plasfna of patients with RA, IA, and OA was assessed by gelatin zymography. A mouse monoclonal antiserum, 277.13, which selectively recognizes soluble latent forms of human MMP-9, was used to quantitate MMP-9 levels Ih patient synovial efisions, plasma, and synovial tissue with a capture sandwich enzyme-linked immunosorbent assay (ELISA). Fifty-one SF samples (31 RA, 9 OA, 11 IA) were analyzed. Immunolocalization of MMP-9 in RA, OA, and normal synovium was investigated using MMP-9-specific antisera.Results. MMP-9 antigen levels in synovial e hsions were elevated 67-fold in RA samples compared with OA samples. In addition, although MMP-9 antigen levels in IA synovial efisions were 2.7-fold less than the values in RA samples, they were elevated 34-fold over the values in OA samples. These data indicate an association between increased MMP-9 levels and inflammatory arthritis. A predominant 92-kd gelatinolytic activity (specifically inhibited by EDTA) was evident in RA and IA samples, but no activity was observed in OA
Glycosidases have been demonstrated to be elevated in the interstitial fluid of tumors, sera of animals and patients with tumors, and in some tumor tissue as compared to normal adjacent tissue. Elevations of serum beta-N-acetylglucosaminidase and beta-glucuronidase most commonly have been found to occur and these enzymes have been shown to be secreted into the extracellular medium by many different tumor cell types in vitro. The mechanism of cellular release of these hydrolytic enzymes probably involves tumor lysosomal exocytosis. Increased tumor glycosidase levels may promote increased tumor cell shedding from primary tumors, local invasion and perhaps be responsible directly, or indirectly for structural changes in tumor cell surface glycoconjugates. These cell surface changes could facilitate tumor cell thrombus formation, secondary site implantation and attachment in the microcirculation to endothelial cells and/or subendothelial basement membrane components. Other studies have demonstrated a correlation between metastatic cell potential and increased endoglycosidase and polysaccharide lyase activity. Generally, metastatic tumor cell variants have been found to be more invasive and capable of degrading proteoglycan basement membrane components, in part due to these increased levels of degradative enzymes. Hence, it is of considerable interest to develop inhibitors against these enzymes. Initial studies with glucuronidase inhibitors in the therapy of bladder tumors have been promising and with the advent of better agents and the use of appropriate in vitro metastatic models it may be possible to design and develop agents which interfere in various metastatic events and limit tumor progression.
The cell-surface localization and site of activation of type IV collagenases/gelatinases (matrix metalloproteinases, MMP) in bovine pulmonary microvascular endothelial (BPMVE) cells was examined. Sucrose density centrifugation of plasma membranes and immunofluorescent staining of whole cells indicated association of 72 kDa (MMP-2) and 96 kDa (MMP-9) type IV collagenase/gelatinases with the plasma membrane. Incubation of the BPMVE cells with rhodaminated MMP-9 demonstrated colocalization with beta 1-integrin, indicating incorporation into the focal contacts. The focal contacts were extracted with saponin, and associated proteolytic activity was examined by zymography. The focal contacts contained latent MMP-2, and stimulation of the cells with cytochalasin D or with 8-bromoadenosine 3',5'-cyclic monophosphate with 3-isobutyl-1-methylxanthine increased both latent and activated MMP-9 in the focal contacts. Addition of these stimuli in unconditioned culture medium did not produce this effect, indicating that the MMP-9 in focal contact extracts was derived from previously secreted enzyme. The activated metalloproteinase degraded extracellular matrix collagens and was inhibited by 1,10-phenanthroline. These findings indicate that endothelial cells release MMP into the extracellular milieu and then concentrate and activate MMP-9 from medium at the focal contacts.
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