Cartilage Proteoglycan Degradation Research Articles

Magnetic resonance imaging reflects cartilage proteoglycan degradation in the rabbit knee

Cartilage degeneration in osteoarthritis is initiated by a loss of proteoglycan. Intra-articular injection of papain causes a reversible loss of proteoglycan in rabbit knees. Rabbits were scanned with magnetic resonance imaging (MRI), using a 1.5T Signa superconducting magnet with 3 inch surface coil. Spin echo sequences were performed in the coronal and sagittal planes at 0, 24, 48, and 72 h after intra-articular injection of papain to obtain T1, proton density, and T2-weighted images. Cartilage proteoglycan content was measured biochemically and histochemically. Reduced articular cartilage thickness in the MR images of papain-treated knees corresponded to changes in cartilage proteoglycan content.

Elevated substance p and accelerated cartilage degradation in rabbit knees injected with interleukin‐1 and tumor necrosis factor

Cytokines, interleukin-1 (IL-1), tumor necrosis factor alpha, and the neurotransmitter, substance P, have been implicated in the pathogenesis of arthritis because they stimulate synovial cells to secrete prostaglandin E2 and collagenase in vitro. We investigated in vivo changes in intraarticular substance P and the degradation of cartilage proteoglycan in response to intraarticular cytokine injections in rabbits. Twenty-four hours after a single injection of 10 ng, 30 ng, or 100 ng of recombinant human IL-1 alpha (rHuIL-1 alpha) per joint, the mean +/- SEM levels of substance P detected in the cell-free joint lavage fluid were 250 +/- 67 fmoles, 480 +/- 60 fmoles, and 530 +/- 130 fmoles (n = 4-5), respectively. The level of substance P in the contralateral knees injected with diluent was 58 +/- 8 fmoles (n = 12). The level of substance P had increased by 2 hours after IL-1 injection and remained elevated in the joint 48 hours after injection. Cytokine-induced proteoglycan depletion was also time- and dose-dependent. Proteoglycan concentrations in articular cartilage dissected from the weight-bearing condyles were calculated as the ratio of sulfated glycosaminoglycan measured using 1,9-dimethylmethylene blue: hydroxyproline. After 48 hours, 10 ng, 30 ng, or 100 ng of rHuIL-1 alpha per joint decreased proteoglycan levels by 9 +/- 4%, 14 +/- 4%, and 21 +/- 3% (n = 8), respectively. Likewise, the injection of recombinant human tumor necrosis factor alpha induced depletion of intraarticular substance P and cartilage proteoglycan.

Retardation of articular cartilage degradation by glycosaminoglycan polysulfate, pentosan polysulfate, and DH‐40J in the rat air pouch model

The rat subcutaneous air pouch model was adapted to examine the in vivo degradation of implanted rabbit articular cartilage, both with and without induced air pouch inflammation, over a 7-day period. The effects of 3 drugs, glycosaminoglycan polysulfate (Arteparon), pentosan polysulfate (SP-54), and zinc-chelated pentosan polysulfate (DH-40J), on inflammation-induced cartilage degradation were also examined. Implanted articular cartilage from noninflamed air pouches showed a reduction in total proteoglycan (PG) content (as hexuronic acid), but not in PG extractability or aggregation, compared with cartilage maintained in tissue culture. The injection of peptone into the air pouch as an inflammogen caused an influx of leukocytes and plasma exudate and a reduction in implanted articular cartilage PG content, extractability, and aggregation, which was significantly greater than that which occurred in noninflamed air pouches. In vitro experiments demonstrated that peptone did not have a direct effect on cartilage PG degradation. Daily injection of Arteparon, SP-54, or DH-40J (10 mg/kg) into peptone-inflamed air pouches significantly increased the PG content, extractability, and aggregation in implanted articular cartilage, compared with that in cartilage from non-drug-treated control animals. The infiltration of leukocytes into the peptone-inflamed air pouches was significantly reduced by daily administration of Arteparon, 10 mg/kg. At an equivalent dose, DH-40J increased leukocyte numbers in the pouch fluid, whereas SP-54 had no significant effect on leukocyte accumulation.

Transforming growth factor-β is a potent inhibitor of IL-1 induced protease activity and cartilage proteoglycan degradation

Treatment of chondrocytes in culture with interleukin-1 results in the production of neutral proteases that cause the degradation of the large aggregating proteoglycan. TGF-beta is a pleiotropic growth factor that has been shown to induce differentiation of cartilage and, in some cases, was able to inhibit the IL-1-dependent processes. In this report, we examined whether TGF-beta could block the IL-1 induced catabolic effects on chondrocytes. After treatment with IL-1 beta (30 ng/ml), rabbit articular chondrocytes produced approximately 2 units of neutral protease activity. Under identical conditions, TGF-beta 1 alone did not induce any protease activity. However, a combination of IL-1 and TGF-beta resulted in a dramatic reduction in the level of protease activity. The inhibitory effect of TGF-beta was also observed at the level of proteoglycan incorporation into the extracellular matrix. The IL-1 treated chondrocytes failed to incorporate proteoglycans into their extracellular matrix. However, addition of TGF-beta in the presence of IL-1 resulted in partial reversal towards a normal extracellular matrix. These studies indicate that TGF-beta can block and at least partially inhibit the catabolic effects of IL-1 on chondrocytes.

Increase in levels of serum keratan sulfate following cartilage proteoglycan degradation in the rabbit knee joint.

Following injection of chymopapain into a single knee joint in rabbits, serum keratan sulfate levels rose sharply and remained elevated for at least 48 hours before returning to preinjection levels. These changes were accompanied by depletion of proteoglycans from articular cartilage in the injected joint. We conclude that serum keratan sulfate levels rise predictably following acute loss of proteoglycan from a single joint.

Degradation of cartilage proteoglycans by elastase is dependent on charge-mediated interactions.

We investigated the mechanism of cartilage degradation by pancreatic elastase as a model system for the action of cationic proteases such as leukocyte elastase and cathepsin-G. It is shown that the cationic properties of elastase contribute to its proteolytic potential with respect to cartilage degradation. Elastase preparations with neutral or negative charge, obtained by chemical modification were far less effective in cartilage degradation than the cationic, native enzyme. Modification of elastase did not affect the active site of the enzyme as shown by kinetic studies, nor did it alter the complexing with alpha-1-proteinase inhibitor. Quantitative and autoradiographic studies indicate that the positive charge of the enzyme favors the penetration in cartilage and interaction with the polyanionic proteoglycan substrate. It is concluded that the potent cartilage-degrading activity of elastase is dependent on charge-mediated interactions with its substrate.

Human Arthritic Synovial Fluid Influences Proteoglycan Biosynthesis and Degradation in Organ Culture of Bovine Nasal Cartilage

The influence of synovial fluid and serum from patients with inflammatory joint disease on proteoglycan metabolism was studied in organ culture of bovine nasal cartilage. Proteoglycan biosynthesis, i.e. incorporation of [35S] -sulphate, was reduced after addition of synovial fluid from rheumatoid arthritis and reactive arthritis patients. Also some rheumatoid arthritis sera but no reactive arthritis serum reduced the biosynthesis compared to control sera. Proteoglycan degradation, i.e. release of proteoglycans prelabelled with [35S] -sulphate, as well as release of proteoglycans determined by.chemical methods, was highest under the influence of rheumatoid arthritis synovial fluid. This effect appears to represent an activity truly stimulating degradation, since added control serum did not prevent the effect. The lowest proteoglycan degradation was observed in culture medium only. Addition of synovial fluid compared to addition of control serum did not increase proteoglycan degradation in freeze-killed cartilage indicating that the effect requires living cells.The findings are consistent with the presence in synovial fluid of mediators stimulating the chondrocytes both to activate proteoglycan degradation and to reduce proteoglycan biosynthesis.

Bradykinin, a new potential mediator of inflammation-induced bone resorption. Studies of the effects on mouse calvarial bones and articular cartilage in vitro.

The effect of bradykinin and desArg9-bradykinin on bone was studied in cultures of calvarial bones taken from 6-7-day-old mice. Bradykinin, at and above a 3-nM concentration, caused a dose-dependent stimulation of bone mineral mobilization and matrix degradation. Bradykinin-stimulated resorption was inhibited by calcitonin, an increased concentration of phosphate in the culture medium, hydrocortisone, dexamethasone, indomethacin, meclofenamic acid, naproxen, and 5, 8, 11, 14-eicosatetraenoic acid. The results suggest that bradykinin stimulates osteoclast-mediated bone resorption by a process that is dependent on endogenous prostaglandin production. The stimulatory effect of bradykinin, but not of parathyroid hormone and prostaglandin E2, was potentiated by the angiotensin-converting enzyme inhibitor, BPP5a. Treatment with carboxypeptidase B did not affect the capacity of the peptide to stimulate 45Ca release. DesArg9-bradykinin (1 mumole/liter) stimulated 45Ca release to the same degree as did bradykinin. Bradykinin (3 microM) did not affect the degradation of cartilage proteoglycans, as assessed by the release of 35S-sulfate from prelabeled calf articular cartilage in organ culture. These findings suggest that generation of bradykinin in inflammatory lesions of rheumatoid arthritis and periodontitis may contribute to the bone resorptive process seen in the joints and alveolar bone; however, bradykinin does not directly activate chondrocytes into a catabolic state.

The immunologic detection and characterization of cartilage proteoglycan degradation products in synovial fluids of patients with arthritis

Antibodies were used in radioimmunoassays with gel chromatography to detect the hyaluronic acid-binding region, core protein, and keratan sulfate of human cartilage proteoglycan in the synovial fluids of patients with rheumatoid arthritis, juvenile rheumatoid arthritis, and osteoarthritis. All fluids contained proteoglycan that was mainly included on Sepharose CL-4B; this result indicates cleavage of proteoglycan (which is normally excluded). The hyaluronic acid-binding region was the smallest and most commonly detected fragment. It was relatively free of keratan sulfate and core protein, and it could sometimes bind to hyaluronic acid. Other larger fragments containing core protein and/or keratan sulfate were detected in every fluid.

Electron microscopic analysis of articular cartilage proteoglycan degradation by growth plate enzymes.

To assess the effect of intracellular growth plate chondrocyte enzymes on proteoglycan structure, we examined enzyme-treated articular cartilage proteoglycans and untreated articular cartilage proteoglycans with the electron microscopic monolayer technique. The untreated proteoglycan monomers ranged in length from less than 20 nm to more than 700 nm, with a mean length of 224.5 +/- 101.6 nm in one experiment and 224.6 +/- 95.7 nm in a second experiment. Incubation with growth plate enzymes reduced proteoglycan monomers to fragments with lengths that varied from less than 5 nm to 143 nm, increased the variability in monomer length, and destroyed proteoglycan aggregates. The enzyme treated monomers had an average length of 29.5 +/- 17.9 nm in one experiment and 35.2 +/- 17.0 nm in a second experiment. The smallest common fragments were 15 nm long and would be expected to contain about 15 glycosaminoglycan chains. This experiment demonstrates that enzymes extracted from growth plate chondrocytes can degrade the chondroitin sulfate-rich region of proteoglycan monomer core proteins, produce a range of monomer fragment sizes with less than 2% of the fragments shorter than 5 nm or longer than 100 nm, increase the variability in monomer length, and degrade proteoglycan aggregates.

Papain-induced changes in the guinea pig knee joint with special reference to cartilage healing.

The repair of articular cartilage following papain injection into the knee joint of the guinea pig was studied by light and electron microscopy, as well as by autoradiography using tritiated thymidine. Papain injection rapidly produced complete degradation of cartilage proteoglycan. Although a number of chondrocytes were also destroyed, the remaining chondrocytes showed mitotic cell division with resultant formation of cell clusters. Such chondrocytic regeneration, however, did not contribute significantly to the repair of cartilage tissue. On the other hand, mesenchymal cells proliferated from the transition zone and extended over the surface of the damaged cartilage. At the peripheral portion of the articular surface, they migrated and differentiated into chondrocytes with the formation of abundant intercellular matrix to produce hyaline cartilage. From these findings, it was apparent that mesenchymal cells in the transition zone were actively engaged in the repair of articular cartilage.

Interleukin 1 induces leukocyte infiltration and cartilage proteoglycan degradation in the synovial joint.

Interleukin 1 (IL-1) is a polypeptide released by activated macrophages and is thought to be a key mediator of host responses to infection and inflammation. The availability of highly purified and recombinant material has now permitted the evaluation of IL-1 as a mediator of chronic inflammatory processes in vivo. We have demonstrated that intraarticular injection of IL-1 into rabbit knee joints induces the accumulation of polymorphonuclear and mononuclear leukocytes in the joint space and the loss of proteoglycan from the articular cartilage. The effects on cartilage could not be explained solely by the presence of leukocytes, since injections of endotoxin also stimulated leukocyte accumulation in the joint but had no effect on proteoglycan loss. Responses to IL-1 were not associated with increased production of the icosanoids prostaglandin E2 or leukotriene B4 and were not reduced by an inhibitor of their synthesis. The pattern of leukocyte infiltration and cartilage breakdown 24 hr after IL-1 injection was similar to that seen in animals with antigen-induced arthritis of 1 week's duration. These observations support the hypothesis that IL-1 acts directly to mediate the erosive processes of chronic arthritis.

Degradation of bovine articular cartilage proteoglycans in vitro. The effect of Bacteroides gingivalis.

The black-pigmented Bacteroides gingivalis has previously been isolated from periodontal pockets and been shown capable of inflicting advanced tissue damage. Its effect on the degradation of articular cartilage proteoglycans has not previously been known. In these experiments it was demonstrated that under aerobic conditions the anaerobic microbe B. gingivalis is very potent in degrading the proteoglycans of fresh articular cartilage. It is even more potent in the presence of fetal calf serum (FCS) than in its absence. When the cartilage has been frozen/thawed there is still a slight enhancement of the degradation by B. gingivalis, but when the cartilage has been devitalized and de-enzymed by heat, the cartilage-degrading capacity of B. gingivalis is totally abolished. However, addition to the cartilage cultures of filtered conditioned medium from B. gingivalis inhibits in some degree the degradation of articular cartilage proteoglycans. It is therefore suggested that the great cartilage-degrading ability of Bacteroides gingivalis shown in this culture system could be due to its ability to degrade proteinase inhibitors.

An assay for proteoglycanase (PGase) and it's application to articular chondrocyte cultures.

The role of PGase in the degradation of cartilage proteoglycans (PG) is still unclear. Although several assays are available, there still exists some problems in the sensitivity, specificity and simplicity of these procedures. We therefore decided to develop an improved assay for PGase in order to detect small quantities of enzyme activity in the culture medium of articular chondrocytes. As substrates of PGase, we prepared fluorescein isothiocyanate labelled core protein (FITC-PGC) and fluorescein amine labelled hyaluronic acid-proteoglycan subunit complex (FAHA-PGS) from new born calf costal cartilages. PGase activity was estimated from the change of elution profile of Sepharose CL-4B chromatography of fluorescein labelled substrates after incubation with the activated enzyme solution. For measurement of PGase in large numbers of samples, we developed the microultrafiltration method using ultrafiltration membrane (MPS-I kit, Amicon). The concentrated media of articular chondrocyte cultures was used for the investigation of the nature of PGase(s) derived from articular chondrocytes. It was found that APMA, CoCl2, and CaCl2 were needed for activation of the enzyme. Sephadex-G75 gel chromatography of the crude enzyme solution suggests that the molecular weight of PGase from rabbit articular chondrocytes was approximately 5 X 10(4) daltons. From the results of gel chromatography of the digested FITC-PGC and FAHA-PGS it was concluded that one of the sites of attack of the chondrocyte secreted PGase might be near the hyaluronic acid binding region of core protein. The effects of various agents on the production of PGase were also studied. Interleukin-I was found to stimulate PGase production in cultured articular chondrocytes.

Neutrophil enzymes, antiproteases, and oxygen radicals: interaction in cartilage proteoglycan degradation and effect of D-penicillamine.

Neutrophil enzymes, antiproteases, and oxygen radicals: interaction in cartilage proteoglycan degradation and effect of D-penicillamine.

Incorporation of [35S]sulphate and [3H]thymidine into isolated bovine articular chondrocytes in vitro. Effect of autogenous conditioned synovial medium.

The present work is a part of a study series concerned with the metabolic events taking place in the synovial joint tissues. We have previously reported that synovial products not only enhance the degradation of articular cartilage proteoglycans but also inhibit the synthesis of articular cartilage proteoglycans, and that these cellular interactions may be partly modulated by arachidonic acid metabolites. In the experiments reported here we show that the addition of conditioned synovial medium to confluent monolayer cultures of articular chondrocytes not only inhibits the synthesis of proteoglycans, measured as the incorporation of radiosulphate into cartilage proteoglycans but also inhibits the synthesis of DNA, measured as the incorporation of radiothymidine into the chondrocytes, in a dose-dependent manner. In our culture system, calf knee-joint synovial tissue produces PGE, and it has been reported that previous exposure to PGE2 affects the ability of the cell to respond to that same hormone. In these experiments, previous exposure to conditioned synovial medium affected neither the inhibition of the incorporation of radiosulphate nor the inhibition of the incorporation of radiothymidine.

The interleukin-1 family of molecules.

AbstractTwo types of interleukin 1 (IL‐1α and IL‐1β) have been defined by purifying the molecules from activated human peripheral blood cells, followed by cloning and expressing the molecules in Escherichia coli. Both types of IL‐1 stimulate proliferation and differentiation of T‐ and B‐lymphocytes and induce cartilage proteoglycan degradation but differ in other properties. For example, demineralization and appears to be a major mediator in the pathogenesis of joint erosion in rheumatoid arthritis. Synthetic adjuvants elicit the production of IL‐1 separate from endogenous pyrogen, and this property is required for the new generation of vaccines based on antigens produced by recombinant DNA technology.

Continuous mechanical pressure and joint tissue. Effect of synovial membrane products and indomethacin in vitro.

We have previously reported that a continuous mechanical pressure of approximately 30 kgfcm-2 greatly inhibited the degradation of articular proteoglycans in calves. In the present report it is shown that indomethacin (5 micrograms/ml) failed to inhibit cartilage degradation caused by mechanical pressure. It is therefore suggested that prostaglandins do not mediate the inhibition of cartilage degradation exerted by continuous mechanical pressure. We have also reported previously that bovine-conditioned synovial medium (SM) greatly enhances the degradation of calf articular cartilage proteoglycans. We now show that the catabolic activity of SM was greatly (but not entirely) inhibited by the application of a continuous mechanical pressure. Simultaneous addition of indomethacin (5 micrograms/ml) to the cartilage cultures did not affect this result. However, the addition of indo-SM (SM from synovial tissue cultured in the presence of 5 micrograms/ml indomethacin) to cartilage cultures subjected to continuous mechanical pressure did not increase cartilage degradation, compared with the addition of DMEM alone. A hypothesis is proposed that continuous mechanical pressure may cause a change in the state of synovial factor receptors on the membranes of chondrocytes.

Degradation of cartilage proteoglycan and collagen by synovial cells: Stimulation by macrophages under activation by phagocytosis, lymphocyte factors, bacterial products or other inflammatory stimuli

When cultured together with dead 35S-labelled cartilage discs or at the surface of [ 3 H]proteoglycan [ 14 C]collagen-coated plates, synovial cells from either arthritic or normal rabbit joints digested both the proteoglycan and the collagen of the substrates after a lag-period of 1–2 days. These digestions were inversely related to the age (number of subculture passages) of the synovial cells and they could be modulated by serum components that were either inhibitory or stimulatory. They were dependent on a protein synthesis by the cells and were paralleled, in young cultures, by the release of collagenase and of a proteoglycan-degrading neutral proteinase. The co-culture of synovial cells with macrophages or their culture with macrophage-conditioned culture media caused a more rapid and more extensive degradation of collagen and proteoglycan due to the stimulation of the synovial cells by a nondialysable macrophage factor. The production of this synovial cell-activating ‘matrix regulatory monokine’ by the macrophage was enhanced by several immunological or inflammatory stimuli such as lymphocyte factors, phagocytosis, asbestos fibres, endotoxin, adjuvant muramyl dipeptide or chemotactic formyl-methionyl peptide, as well as by other membrane-active agents (phorbol myristate acetate, concanavalin A). It is presumed that these interactions are of importance in the development of cartilage destruction in rheumatoid and other chronic inflammatory arthritis.

In vitro inhibition of Pseudomonas aeruginosa elastase by metal-chelating peptide derivatives.

Pseudomonas aeruginosa elastase is a zinc metalloendopeptidase, probably responsible for the tissue destruction observed during infections with this organism. The elastase of a virulent Pseudomonas aeruginosa strain (Habs serotype 1) was isolated and found to have a molecular weight of 35,000; it readily degraded elastin and cartilage proteoglycans. A series of amino acid and peptide derivatives containing the metal-chelating moieties hydroxamate, phosphoryl, or thiol were synthesized and tested as potential inhibitors of the enzyme. Inhibition constants (K(i)s) for the compounds were determined with the chromophoric substrate furylacryloyl-glycyl-l-leucyl-l-alanine. The hydroxamic acid derivatives of benzyloxycarbonyl-glycine, benzyloxycarbonyl-l-leucine and benzyloxycarbonyl-l-phenylalanine had inhibition constants in the range of 11 to 28 muM. The 2-mercaptoacetyl derivatives of l-leucyl-d-phenylalanine and l-leucyl-l-phenylalanine had K(i) values of 34 and 1.5 muM, respectively, demonstrating the stereospecificity of the inhibition. The most potent inhibitors tested were 2- mercaptoacetyl-l-phenylalanyl-l-leucine and phosphoryl-l-leucyl-l-phenylala-nine (K(i) = 0.2 muM). Similar compounds lacking the metal-chelating moiety were about 3 orders of magnitude poorer inhibitors. When the inhibition of the enzyme activity towards azocasein, elastin, or cartilage was examined, inhibitor concentrations approximately 50-fold higher than the respective K(i)s were required to obtain 60 to 90% inhibition. Virtually complete inhibition was achieved with these substrates at inhibitor concentrations 500-fold higher than the respective K(i)s (0.1 to 14 mM). Although, 2-mercaptoacetyl-l-phenylalanyl-l-leucine and phosphoryl-l-leucyl-l-phenylalanine exhibited the same affinity to the enzyme, the latter was inferior in inhibiting cartilage proteoglycan degradation. 2-Mercaptoacetyl-l-phenylalanyl-l-leucine represents a class of potent elastase inhibitors that might prove useful in the management of P. aeruginosa infections.