phenylmercuric-acetate and Osteoarthritis

Specific modifications of the proteoglycan (PG) structure of osteoarthritic (OA) dog cartilage in relation to endogenous metalloprotease activity were examined using murine anti-proteoglycan monoclonal antibodies (MoAbs). OA lesions were induced over a period of 8 weeks in crossbred dogs (Pond-Nuki model). The articular cartilage was removed and homogenized in a Tris buffer, pH 7.5, and then divided into four groups: direct PG extraction, no addition, presence of 1 mM p-aminophenyl mercuric acetate (APMA), and presence of 1 mM APMA and 10 mM o-phenanthroline, incubated for 42 h at 37 degrees C followed by PG extraction. MoAbs reactive with PG protein and carbohydrate epitopes included 1C6, 3B3, 5D4, D1B2, and m4D6. The results showed marked alterations induced by APMA activation of the endogenous metalloproteases. PG changes were apparent at at least three sites: one was either in the hyaluronic acid-binding region or between the hyaluronic-binding region and the G2 globular domain, another was between the keratan-sulfate-rich domain and the chondroitin sulfate-attachment domain, and a third was in the chondroitin sulfate-attachment domain. Constitutive metalloprotease activity resulted in less marked PG alterations with preservation of functional PG aggregation to hyaluronan.

Topics: Animals; Antibodies, Monoclonal; Cartilage, Articular; Chromatography, High Pressure Liquid; Dogs; Enzyme Activation; Enzyme-Linked Immunosorbent Assay; Epitopes; Glycosaminoglycans; Hydroxylamines; Immunohistochemistry; Metalloendopeptidases; Microscopy, Electron; Osteoarthritis; Phenylmercuric Acetate; Proteoglycans; Radioimmunoassay; Time Factors

One of the best studied animal models of osteoarthritis is a dog model in which the anterior cruciate ligament of the hind limb stifle joint is transected (Pond-Nuki model). To determine whether stromelysin might play a role in this model, it was necessary to purify the enzyme for production of suitable probes. In the present study, dog synovial fibroblasts were stimulated to express a metalloproteinase that was demonstrated to be canine prostromelysin by Northern blot, protein purification and amino-terminal sequence analyses. Unlike rabbit synoviocytes, passaged dog synoviocytes did not express stromelysin mRNA in response to recombinant human IL-1, but expressed stromelysin mRNA only upon treatment with dog monocyte-conditioned medium (dMCM). The aminophenylmercuric acetate (APMA)-activatable metalloproteinase present in the culture supernatants of stimulated dog synoviocytes was purified using a combination of ion-exchange and dye matrix affinity chromatography. The purified canine metalloproteinase co-migrated on reducing SDS-PAGE with recombinant human prostromelysin-1 as a doublet with apparent molecular masses of 54 and 56 kDa. Similar to APMA-activated human prostromelysin-1, the APMA-activated canine metalloproteinase was inhibited by 1,10-phenanthroline or recombinant human tissue inhibitor of metalloproteinase (TIMP). The amino-terminal sequences of the canine pro- and APMA-activated enzymes were compared with those of human, rabbit and rat stromelysin. The striking homologies among the sequences demonstrated that the purified canine metalloproteinase was indeed canine prostromelysin. A rabbit anti-canine prostromelysin polyclonal antiserum was generated and used to localize the enzyme within cultured dog synoviocytes and articular cartilage stimulated with dMCM. The reagents developed in this study should be useful for examining the expression and distribution of prostromelysin in canine models of osteoarthritis.

Topics: Amino Acid Sequence; Animals; Cartilage, Articular; Cells, Cultured; Collagenases; Culture Media; Cytokines; Disease Models, Animal; Dogs; Enzyme Induction; Enzyme Precursors; Fibroblasts; Glycoproteins; Humans; Leukocytes, Mononuclear; Metalloendopeptidases; Molecular Sequence Data; Osteoarthritis; Phenanthrolines; Phenylmercuric Acetate; Rabbits; Rats; Recombinant Proteins; Sequence Homology; Species Specificity; Stimulation, Chemical; Synovial Membrane; Tissue Inhibitor of Metalloproteinases

There is increasing evidence that the proteoglycan-degrading neutral metalloproteinase, stromelysin, is a key enzyme in the pathogenesis of osteoarthrosis. Equine synovial lining cells were stimulated in vitro to produce stromelysin, and phenylbutazone, flunixin, betamethasone, sodium hyaluronate and polysulphated glycosaminoglycan (PSGAG) were tested for their ability to inhibit the action of this enzyme on 14C-labelled casein substrate. Only PSGAG possessed inhibitory activity at concentrations likely to be achieved therapeutically in the equine fetlock joint.

Topics: Animals; Anti-Inflammatory Agents; Anti-Inflammatory Agents, Non-Steroidal; Betamethasone; Clonixin; Dose-Response Relationship, Drug; Enzyme Activation; Glycosaminoglycans; Horse Diseases; Horses; Hyaluronic Acid; Matrix Metalloproteinase 3; Matrix Metalloproteinase Inhibitors; Osteoarthritis; Phenylbutazone; Phenylmercuric Acetate; Trypsin

The neutral, metal dependent, proteoglycan degrading enzymes (NMPEs) in human osteoarthritic knee cartilage homogenates were activated by p-aminophenylmercuric acetate (APMA). The resultant effect on the structure of newly synthesised and already existing sulphated proteoglycan was measured. Newly synthesised and already existing proteoglycan aggregated to hyaluronic acid was reduced (p less than 0.01, p less than 0.05 respectively) when measured by chromatography on Sepharose CL-2B eluted with associative buffer. The APMA activated enzyme affected both the newly synthesised and already existing proteoglycan aggregate similarly (r = 0.79, p less than 0.001). Treatment of cartilage homogenates with APMA and 1,10-phenanthroline (10 mM) showed that the amount of aggregated proteoglycan was at the control level. The hydrodynamic size of the proteoglycan monomer (A1D1) was also reduced by treatment of cartilage homogenates with APMA. Reaggregation experiments with fraction A1D1 and exogenous hyaluronic acid and link protein showed a similar defect in forming proteoglycan aggregates. These data showed that activation of the NMPEs altered the structure of proteoglycan in two ways. The most consistent change was a reduction in the ability of proteoglycan to form aggregates with hyaluronic acid. This was likely to have occurred via a cleavage of the core protein in or around the hyaluronic acid binding globular domain. A second alteration probably includes a limited proteolytic cleavage in the remainder of the core protein.

Topics: Aged; Aged, 80 and over; Cartilage, Articular; Centrifugation, Density Gradient; Chromatography, Gel; Enzyme Activation; Female; Humans; Hyaluronic Acid; Knee Joint; Macromolecular Substances; Male; Metalloendopeptidases; Middle Aged; Organ Culture Techniques; Osteoarthritis; Phenylmercuric Acetate; Proteoglycans