A magnetic resonance imaging study of joint degeneration in the guinea pig knee.
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Animals↗
Biomedical subjects
J A Tyler
Publications and source records attributed to J A Tyler.
Upregulation of insulin-like growth factor I gene expression in the lesions of osteoarthritic human articular cartilage.
Expression of insulin-like growth factor I (IGF-I)mRNA and IGF-I protein was studied in human osteoarthritic and young articular cartilage by in situ hybridisation and immunohistochemistry. In situ hybridisation showed that relatively low amounts of IGF-I mRNA signal were present in anatomically normal regions of osteoarthritic and young cartilage. In fibrillated osteoarthritic cartilage, however, the signal intensity was significantly higher than in non-fibrillated cartilage. Particularly high levels of IGF-I mRNA were present in the surface cell clones of more advanced lesions, the amount of signal being about four to five times greater than in anatomically normal cartilage. The amount of message varied with cartilage depth. In young cartilage there was less IGF-I mRNA in the superficial zone than in the middle and deep zones. In fibrillated regions of osteoarthritic joints the amount of message in surface cells was greater than in deeper regions. A specific human IGF-I antibody was used to show the presence intracellularly of IGF-I protein in osteoarthritic and young cartilage. Raised levels of IGF-I message in osteoarthritic chondrocytes may represent an attempt at increased matrix repair, operating by an autocrine/paracrine mechanism.
Adolescent↗
Effect of interleukin-1 and insulin like growth factor-1 on the release of proteoglycan components and hyaluronan from pig articular cartilage in explant culture.
The turnover of proteoglycans was investigated in articular cartilage in explant culture by analysing the components released into the culture medium. The effect of IL-1 alpha on the release of fragments derived from different proteoglycan domains and hyaluronan (HA) was determined over 4 days in culture. The effect of IGF-1 (100 ng/ml) on matrix degradation of proteoglycan and its ability to inhibit the effects of IL-1 (10 ng/ml) was also assessed. The rate of release of G1 and G2 globular domains of proteoglycans into the culture medium was determined by radioimmunoassay. In unstimulated control cartilage there was a greater release of proteoglycan G2 domain than of G1 domain suggesting that cleavage occurred between them and that some G1 was preferentially retained bound in the matrix. Compared with control cartilage IL-1 stimulated the release of all proteoglycan components and hyaluronan. IL-1 had a greater effect on the release of G1 than on G2 domain, but also resulted in some net loss of these proteins (approximately 45% as detected in the immunoassays). In explants treated with both IL-1 and IGF-1 there was much less release of proteoglycan fragments and evidence for less extensive degradation. IGF-1 was particularly affective in preventing any increase in HA release and also preventing the apparent loss of G1 and G2 domains. It also partially inhibited the release of G1 and G2 domains and the sulphated glycosaminoglycan fragments. IGF-1 was therefore an effective antagonist of IL-1 action on cartilage. It is not known at what level it blocks the chondrocyte response to IL-1, but it clearly results in the suppression of matrix degradative activity.
Animals↗
Insulin-like growth factor 1 can decrease degradation and promote synthesis of proteoglycan in cartilage exposed to cytokines.
A model system of explanted cartilage has been used in vitro to determine whether insulin-like growth factor 1 (IGF 1), which promotes matrix formation is effective in the presence of cytokines such as interleukin 1 (IL1) and tumour necrosis factor (TNF), which induce net matrix depletion. IGF 1 induced a dose-dependent 2.5-fold stimulation of proteoglycan synthesis, with a half-maximal dose of 25 ng/ml. A similar relative increase occurred in response to IGF 1 (10-100 ng/ml) in cartilage cultured also with IL1 or TNF (5-500 pM). There was no detectable qualitative change in the average molecular size or charge of the aggregating proteoglycan synthesized by explants exposed to IGF 1 alone or with IL1 or TNF. The increased production of prostaglandin E2, which is initiated when IL1 or TNF bind to the chondrocytes, was the same in the presence or absence of IGF 1. The time taken for 50% of pre-labelled proteoglycan to be released from the explants (t1/2) increased in the presence of IGF 1 (100 ng/ml) from 21 to 32 days in control cultures and from 8 to 26 days in cartilage cultured with IL1 (50 pM). It is concluded that IGF 1 enhances the synthesis of aggregating proteoglycan in cartilage exposed to cytokines and can directly decrease both the basal and the cytokine-stimulated degradation of proteoglycan in cartilage.
Animals↗
Inhibition of cartilage proteoglycan synthesis by interleukin I.
We have investigated the mechanism of inhibition of cartilage proteoglycan by interleukin 1. Proteoglycan synthesis was inhibited using lower doses of interleukin 1 than those required to cause cartilage resorption. There was no significant effect on DNA or total protein synthesis. Gel electrophoresis showed a direct inhibitory effect on core protein synthesis while pulse-chase experiments using radiolabelled sulphate showed no alteration in the rate of intracellular transport and secretion of completed proteoglycan. Chondrocytes incubated with cycloheximide showed a first-order decrease in rate of uptake of radiolabelled sulphate (t1/2 = 25 mins) but interleukin 1 induced inhibition showed a delay of at least 1 hr, consistent with a requirement to deplete intracellular pools of protein before effects on post-translational events could be observed. Foetal and neonatal cartilage responded to the cytokine in a similar way to adult cartilage.
Aging↗
Synthesis of type II collagen is decreased in cartilage cultured with interleukin 1 while the rate of intracellular degradation remains unchanged.
The effect of interleukin 1 (IL1) on the synthesis and degradation of collagen was examined in explants of cultured cartilage. IL1 induced a reversible, dose-dependent (10-100 pM) inhibition of type II collagen synthesis. The proportion of collagen produced decreased selectively from 7% to 1.2% of total protein after 72 h exposure to IL1 (100pM). There was no change in the rate of degradation of newly synthesized collagen. Analysis of newly synthesized material showed that the type II collagen synthesized in the presence of IL1 had the same characteristics as that extracted from unstimulated cartilage. The relative amounts of type II procollagen mRNA were estimated by Northern blot hybridization. The levels were decreased in cartilage cultured with IL1 to a similar extent as that seen for the type II collagen protein. Exposure to IL1 (10-350pM) for 3 days did not induce increased resorption of extracellular collagen in the cultured explants. These data demonstrate that decreased collagen production in cartilage exposed to IL1 is due primarily to decreased amounts of type II procollagen mRNA.
Animals↗
Articular cartilage cultured with interleukin 1. Increased release of link protein, hyaluronate-binding region and other proteoglycan fragments.
Pig articular cartilage was maintained in culture for 3 days with and without porcine interleukin 1. The proteoglycans remaining in the cartilage and those released into the medium were analysed by using radioimmunoassays for the hyaluronate-binding region, link protein and keratan sulphate. In interleukin 1-treated cultures after 3 days there was 38% release of total glycosaminoglycans into the medium, 18% release of binding region, 14% release of link protein and 20% release of keratan sulphate epitope, whereas in control cultures the proportions released were much less (16, 9, 10 and 7% respectively). Characterization of the proteoglycans in the media after 1.5 days and 3 days of culture showed that interleukin 1 promoted the release of proteoglycan of large average size and also the release of link protein and of low-Mr binding region which was unattached to proteoglycan. Both the link protein and binding region released were able to bind to exogenously added hyaluronate, whereas the proteoglycan in the medium was not. The proteoglycans extracted from cultured cartilage were similar to those from fresh cartilage: they contained a high proportion of aggregating proteoglycans and some low-Mr binding region. The proportion of this binding region extracted from the interleukin 1-treated cartilage was increased. The presence of interleukin 1 in the cultures therefore appeared to increase the rate of proteolytic degradation of proteoglycan in the matrix and to lead to a more rapid loss of intact binding region, of link protein and of large proteoglycan fragments into the medium.
Animals↗
Articular cartilage cultured with catabolin (pig interleukin 1) synthesizes a decreased number of normal proteoglycan molecules.
A homogeneous preparation of catabolin from pig leucocytes caused a reversible dose-dependent (0.01-1 nM) decrease in the synthesis of proteoglycan in slices of pig articular cartilage cultured in serum-free medium. The monomers that were synthesized and secreted in the presence of catabolin had the same average hydrodynamic size and ability to aggregate as the controls, and the core protein was substituted with the same number of glycosaminoglycan chains. The chains were the same average length and charge as normal and were sulphated to the same extent as the controls. Newly synthesized extracellular proteoglycan was not preferentially degraded. A 2-3-fold increase in glycosaminoglycan synthesis occurred in control and catabolin-treated cartilage in the presence of beta-D-xyloside (1 mM), more than 80% being secreted into the medium as free chains. Decreased incorporation of sulphate was not reversed in the presence of lysosomal-enzyme inhibitors, and there was no evidence in pulse-chase experiments of increased intracellular degradation of glycosaminoglycan chains before secretion. It is concluded that catabolin-treated cartilage synthesizes a smaller number of normal proteoglycan molecules.
Acetates↗
Chondrocyte-mediated depletion of articular cartilage proteoglycans in vitro.
The degradation of proteoglycan was examined in cultured slices of pig articular cartilage. Pig leucocyte catabolin (10 ng/ml) was used to stimulate the chondrocytes and induce a 4-fold increase in the rate of proteoglycan loss from the matrix for 4 days. Material in the medium of both control and depleted cultures was mostly a degradation product of the aggregating proteoglycan. It was recovered as a very large molecule slightly smaller than the monomers extracted with 4M-guanidinium chloride and lacked a functional hyaluronate binding region. The size and charge were consistent with a very limited cleavage or conformational change of the core protein near the hyaluronate binding region releasing the C-terminal portion of the molecule intact from the aggregate. The 'clipped' monomer diffuses very rapidly through the matrix into the medium. The amount of proteoglycan extracted with 4M-guanidinium chloride decreased during culture from both the controls and depleted cartilage, and the average size of the molecules initially remained the same. However, the proportion of molecules with a smaller average size increased with time and was predominant in explants that had lost more than 70% of their proteoglycan. All of this material was able to form aggregates when mixed with hyaluronate, and glycosaminoglycans were the same size and charge as normal, indicating either that the core protein had been cleaved in many places or that larger molecules were preferentially released. A large proportion of the easily extracted and non-extractable proteoglycan remained in the partially depleted cartilage and the molecules were the same size and charge as those found in the controls. There was no evidence of detectable glycosidase activity and only very limited sulphatase activity. A similar rate of breakdown and final distribution pattern was found for newly synthesized proteoglycan. Increased amounts of latent neutral metalloproteinases and acid proteinase activities were present in the medium of depleted cartilage. These were not thought to be involved in the breakdown of proteoglycan. Increased release of proteoglycan ceased within 24h of removal of the catabolin, indicating that the effect was reversible and persisted only while the stimulus was present.
Animals↗
The effect of cortisol on porcine articular tissues in organ culture.
(1) The effects of hydrocortisone succinate (1.0--0.01 micrograms HC/ml of medium) on porcine articular tissues in organ culture have been studied by histological and biochemical methods. (2) 1.0 and 0.1 micrograms HC/ml considerably inhibited the severe breakdown of matrix that occurs in living and dead cartilage explained in contact with synovial tissue. (3) The depletion of matrix in living cartilage cultivated in the same dish as but not in contact with synovial tissue, is much diminished when the medium contains 1.0 or 0.1 micrograms HC/ml. (4) Cartilage grown in the used medium of synovial tissue loses both proteoglycan and hydroxyproline; the addition of HC to the used medium has little (1.0 microgram HC/ml) or 0.1 microgram HC/ml) no inhibitory effect. If, however, the used medium is from synovial tissue that has been cultured in the presence of 1.0 or 0.1 micrograms HC/ml, loss of proteoglycan and collagen from the cartilage is much reduced. (5) In isolated cartilage in normal medium there is outgrowth of cells from the cut surface and some loss of proteoglycan and collagen; outgrowth is completely, and loss of matrix components partially suppressed by 1.0 micrograms HC/ml. (6) Isolated synovial tissue cultured in normal medium shrinks to about one-third of its original size, loses much of its collagen and secretes neutral metallo-proteinases into the medium; in the presence of 1.0 microgram HC/ml these changes are largely prevented. The explants also secrete an inhibitor of metallo-proteinases the production of which is not reduced by HC.
Animals↗
The activation of latent pig synovial collagenase.
Latent pig synovial collagenase (EC 3.4.24.7) can be activated by a variety of different treatments to give an active enzyme form of lower molecular weight which rapidly degrades collagen. Trypsin and plasmin effectively activated the latent collagenase whilst elastase and cathepsin G degraded most of the latent enzyme before it was activated. A number of mercurials were compared and maximum activation was achieved using 4-aminophenylmercuric acetate and phenylmercuric chloride. The latent collagenase bound to a mercurial-Sepharose column and was eluted in the active form with NaCl. The latent collagenase also activated spontaneously and the conditions which encouraged and prevented this activation were studied. High NaCl concentration, diisopropylphosphofluoridate, soybean trypsin inhibitor, low Zn2+ concentration and high and low pH all prevented the spontaneous activation of latent pig synovial collagenase.
Animals↗
Properties of pig synovial collagenase.
1. Properties of a purified chemically activated form of pig synovial collagenase were examined and compared with a spontaneously active form of the enzyme. 2. The active enzyme has a specific activity of 53 000 units (microgram/min)/mg, a mol.wt. of 44 000 (by sodium dodecyl sulphate/polyarcylamide-gel electrophoresis in 2-mercaptoethanol) and pI 5.2 (by isoelectric focusing in polyacrylamide gels). 3. The activity has the characteristics of a metalloproteinase that degrades types I and III soluble or insoluble collagens in preference to type II, at an optimum pH of 6.5-8.5. 4. There is no detectable difference in these properties between the chemically activated and spontaneously active form of collagenase.
Animals↗
Purification of pig synovial collagenase to high specific activity.
1. Pig synovium in tissue culture secretes a specific collagenase in a latent form. 2. The latent enzyme was concentrated by (NH4)2SO4 precipitation and activated with 4-aminophenylmercuric acetate, and the active enzyme was purified by chromatography on Ultrogel AcA44, DEAE-cellulose, heparin-Sepharose and a zinc-chelate medium to a specific activity of 53 400 units/mg. of protein. 3. The enzyme was shown to be essentially homogeneous by polyacrylamide-gel electrophoresis. 4. The purified collagenase digested collagen to give the characteristic three-quarter and one-quarter pieces.
Animals↗