J.F. Woessner

Evidence for metalloproteinase and metalloproteinase inhibitor imbalance in human osteoarthritic cartilage.

Cartilage specimens from tibial plateaus, obtained from 13 osteoarthritic (OA) patients and seven controls, were selected from three regions: zone A, center of fibrillated area; zone B, area adjacent to fibrillation, and zone C, remote region of plateau. Acid and neutral metalloproteinases and tissue inhibitor of metalloproteinase (TIMP) were extracted with 2 M guanidine. Methods were developed to selectively destroy either proteinases or TIMP to prevent cross-reaction during assay. Acid and neutral proteinases were elevated approximately 150% in OA; TIMP was elevated approximately 50%. A positive correlation (r = 0.50) was found between acid and neutral proteinase activities in OA, but not in controls. Both proteinases were elevated two-to threefold in zones A, B, and C. However, the self-active form of the acid metalloproteinase was elevated only in zones A and B (200%); it correlated well with the Mankin scores, whereas the total activities did not. TIMP was elevated (50%) only in zones A and B. Both the proteinase levels and the Mankin score were elevated to a greater extent in the medial, than in the lateral, compartment. Titration of TIMP against the two metalloproteinases indicates that there is a small excess of inhibitor over enzymes in normal cartilage. In OA, TIMP does not increase to the same extent as the proteinases; the resultant excess of proteinases over TIMP may contribute to cartilage breakdown.

Metalloproteases of human articular cartilage that digest cartilage proteoglycan at neutral and acid pH.

Extracts of human articular cartilage contain proteases capable of degrading the proteoglycan component of cartilage matrix at neutral and acid pH. These enzymes have been partially purified by ion exchange chromotography and characterized by disc electrophoresis, inhibition patterns, and action of proteoglycan. Three distinct metalloproteases are described. A neutral protease that digests proteoglycan subunit optimally at pH 7.25 has been purified up to 900-fold. It is strongly inhibited by o-phenanthroline, alpha-2-macroglobulin, and egg white, and to a lesser extent by D-penicillamine and EDTA. Inhibition by chelating agents is reversed by cobalt, zinc, and ferrous ions. Two acid metalloproteases, distinct from cathespins B1, D, and F, digest proteoglycan subunit at pH 4.5 and 5.5. Both are inhibited by o-phenanthroline and activity is restored by cobalt, zinc, or ferrous ions. With electron microscopy, it was found that cartilage slices were depleted of ruthenium red-staining matrix proteoglycan after incubation in vitro with a partially purified cartilage extract at neutral pH. Sedimentation, gel chromatography, sodium dodecyl sulfate-gel electrophoresis, and immuno-diffusion studies of digests of isolated proteoglycan fraction produced by the partially purified cartilage extract at neutral and acid pH confirmed that the cartilage enzymes act only on the protein component of proteoglycan subunit, producing fragments with 5 to 12 chondroitin sulfate chains. The link proteins were not digested.

Localization of collagenase in the growth plate of rachitic rats.

In the transition from proliferation to hypertrophic cell zones in the growth plate, there is an increase in chondrocyte volume and a corresponding decrease in collagen content to accommodate the enlarging cells. It is postulated that collagenase accounts for this collagen loss. To test this hypothesis, tibial growth plates were obtained from normal rats, rachitic rats deficient in vitamin D and phosphate, and rats after 48 and 72 h of healing from rickets. Collagenase was quantitated by a pellet assay based on the release of solubilized collagen from the endogenous insoluble collagen in the tissue homogenates. A fourfold greater collagen release and a concomitant sixfold greater hypertrophic cell volume were measured in rachitic growth plates compared with normal age-matched controls. During healing of rickets, collagenase activity and hypertrophic cell volume returned almost to control levels. Rachitic growth plates were dissected into the juxtaepiphyseal 1/3 and the juxtametaphyseal 2/3. The latter portion contained greater than 95% of the hypertrophic cells and 86% of the collagenase. The collagen-degrading activity was extracted from this region and was shown to be a true collagenase by its production of typical A fragments of tropocollagen produced by collagenase action. The enzyme was activated by aminophenylmercuric acetate and trypsin and was inhibited by EDTA, 1,10-phenanthroline, and a tissue inhibitor of metalloproteinases from human articular cartilage. Inhibitors of aspartic, cysteine, and serine proteases had no effect. Micropuncture fluids aspirated from rachitic cartilage contained latent collagenase activity, indicating an extracellular localization. Negative tests for hemoglobin in the rachitic cartilage samples indicated that there was no contamination by capillaries and that this was not a source of collagenase. It is concluded that extracellular collagenase accounts for the loss of cartilage matrix in the hypertrophic zone, and that this process may be distinct from that of capillary invasion.

Collagen development in heart and skin of the chick embryo

Abstract 1. 1.|The development of the collagenous framework was studied in the chick-embryo heart and skin. Collagen was detected in the heart on the 3rd day of development. The heart growth and collagen growth from the 5th to the 21st day of development can be described by continuous exponental functions. However, collagen increases more rapidly than heart weight as described by the relationship: Collagen = K (heart wet wt.)1.61 2. 2.|A mechanical interpretation of the data suggests that the increasing proportion of collagen in the enlarging heart may be related to the increasing mechanical strength requirements of the enlarging heart. 3. 3.|The amount of soluble collagen in the heart was found to be almost directly proportional to the net rate of collagen synthesis in the heart. 4. 4.|Collagen growth in the skin does not follow a continuous exponential time curve. Rather, there is an abrupt increase in skin collagen concentration between the 12th and 17th days of development. 5. 5.|This discontinuity in the synthesis of skin collagen points up the difficulty in attempting to relate soluble collagen content to the rate of growth of the organism or tissue.

Characterization of the metalloproteinase inhibitor produced by bovine articular chondrocyte cultures.

Primary cultures of bovine articular chondrocytes release a latent metalloproteinase which is activated by incubation with organomercurials to degrade proteoglycans. All the enzyme present in the culture medium is latent and binds to columns of heparin-Sepharose. The yield of activity from the heparin-Sepharose columns (measured after organomercurial treatment) is approximately 300-1000% depending on the chondrocyte culture batch. Recombination of column fractions shows that the increase in activity is due to the separation of an inhibitor of the metalloproteinase by the chromatographic step. The metalloproteinase inhibitor has a molecular weight of approximately 35000 (determined by Bio-Gel P-60 chromatography) and binds reversibly to columns of concavalin A-Sepharose. It is relatively heat stable (30 min at 60 degrees C) and resistant to inactivation by trypsin (2 h, 37 degrees C, 10 microgram/ml trypsin). The inhibitor is active against rat uterine collagenase and gelatinase but does not affect bacterial metalloproteinases such as thermolysin and Clostridium histolyticum collagenase.

Production of collagenase and tissue inhibitor of metalloproteinases (TIMP) by rat growth plates in culture

Growth plate cartilage from normal and vitamin D-phosphate deficient (-VDP) rats was cultured to study the production of collagenase and tissue inhibitor of metalloproteinases (TIMP) in vitro. All tissues secreted latent collagenase into the medium at a constant rate during the 5 days in culture. Microdissected-VDP growth plates, containing predominatly hypertrophic cells, released up to 8-fold more collagenase into the medium than either intact-VDP or normal growth plates. TIMP was also secreted during the culture, but its rate of production was not as dependent on tissue type as collagenase. The tissue level of collagenase and TIMP before culture was compared with that found in conditioned medium and remnant tissue after culture. During the 5 day culture period microdissected-VDP growth plates, containing predominatly hypertrophic cells, produced 3-times more collagenase/microgram DNA over the starting level than either intact-VDP or normal growth plates. TIMP was never found in tissues after they had been cultured, but was present in all tissues before culture except those containing predominatly hypertrophic cells. The amount of TIMP required to block collagenase was calculated. Growth plates in culture produced enough TIMP to block all collagenase found in the medium and remnant tissue, while extracts of uncultured intact -VDP growth plates, and those divided to contain hypertrophic cells, had an excess of collagenase over TIMP. The results suggest that hypertrophic cells produce far more collagenase than other cells in the growth plate, but all cell types have about the same capacity to synthesize TIMP. As a result, increased collagenase synthesis by hypertrophic cells may surpass increases in TIMP synthesis and lead to collagen removal. This would allow for thinning of the longitudinal septa and expansion of the hypertrophic cells.

Ascorbic acid stimulates the resorption of canine articular cartilage induced by a factor derived from activated rabbit macrophages

SummaryArticular cartilage explants from the knees of mongrel dogs release 5–10% of their proteoglycan content spontaneously when cultured for 4 days in serum-free modified Biggers medium. A factor synthesized and secreted by lipopolysaccharide-stimulated rabbit macrophages can stimulate this release of proteoglycan by 2 to 3-fold. The release of proteoglycan in response to macrophage factor is maximal in the presence of 1.5–50 μg/ml l-ascorbic acid. In the absence of ascorbate, or with high levels of ascorbate (150 μg/ml), the effect of the factor is diminished by 50%. d-isoascorbate, reduced glutathione, or dithiothreitol cannot substitute for l-ascorbate in producing this effect, while dehydroascorbate can.