Cartilage Proteoglycan Degradation Research Articles

Cytokine-induced cartilage proteoglycan degradation is mediated by aggrecanase

Objective: To evaluate the relationship between specific cleavage of aggrecan at the Glu373–Ala374‘aggrecanase’ site and degradation and release of proteoglycan catabolites from cartilage in explant cultures.Design: The monoclonal antibody, BC-3, which specifically recognizes the new N-terminus, ARGSVIL, generated by cleavage of aggrecan at the Glu373–Ala374‘aggrecanase’ site, was used to follow the generation of fragments produced by cleavage at this site as compared to degradation of proteoglycan as assessed by glycosaminoglycan (GAG) release from cartilage in response to cytokines and the ability of inhibitors to block this cleavage.Results: (1) There was a strong correlation between specific cleavage at the Glu373–Ala374bond and the release of aggrecan catabolites in response to interleukin-1 (IL-1) or tumour necrosis factor (TNF) stimulation. (2) This cleavage in the interglobular domain of aggrecan was inhibited by the inclusion of cycloheximide, thus indicating a requirement forde novoprotein synthesis in the induction of ‘aggrecanase’ activity. (3) The inhibitors, indomethacin, naproxen, tenidap, dexamethasone and doxycycline were ineffective in blocking either specific cleavage at the ‘aggrecanase’ site or aggrecan degradation as measured by GAG release from cartilage. (4) In contrast, compounds which act through two different mechanisms to inhibit MMPs were effective in blocking both specific cleavage at the ‘aggrecanase’ site and proteoglycan degradation.Conclusions: Our data suggest that ‘aggrecanase’ is primarily responsible for proteoglycan cleavage in these experimental systems and that this protease has properties in common with metalloproteases including members of the MMP and ADAM family. Inhibition of ‘aggrecanase’ may have utility in preventing cartilage loss in arthritis.

Effect of ebselen on IL-1-induced alterations in cartilage metabolism.

To evaluate the effect of the antioxidant-like anti-inflammatory agent, ebselen, on cartilage proteoglycan degradation and to determine whether its cartilage protectant activity is related to its antioxidant activity. Cartilage in organ culture was stimulated with interleukin-1 (IL-1), and proteoglycan degradation was assessed by measuring the amount of sulfated glycosaminoglycan released into the media, proteoglycan synthesis evaluated by [35S]-sulfate incorporation, and prostaglandin E2 (PGE2) release determined by radioimmunoassay (RIA). Glutathione peroxidase (GSH-Px) activity was evaluated in a coupled test system using NADPH/GSSG reductase as an indicator and cyclooxygenase activity was evaluated using sheep seminal vesicle prostaglandin synthase. Ebselen caused a concentration-dependent inhibition of IL-1-stimulated proteoglycan degradation with an IC50 of 4.7 microM. Cartilage PGE2 release was also reduced in the presence of ebselen (IC50 = 6.2 microM). However, at concentrations up to 100 microM, ebselen had no effect on the inhibition of proteoglycan synthesis by IL-1. Induction of proteoglycan breakdown was also inhibited by a sulfur analog of ebselen. This analog was devoid of GSH-Px activity and was 50-fold less potent in cyclooxygenase inhibitory activity, but was equipotent to ebselen in inhibiting cartilage degradation. Ebselen, unlike other NSAIDs, blocks cartilage proteoglycan breakdown without inhibiting proteoglycan synthesis. This effect is independent of its GSH-Px activity and its ability to inhibit cyclooxygenase and PGE2 production. Therefore, this compound may provide a new mechanism for protecting cartilage matrix from degradative factors in arthritic joints.

Cartilage chondrolysis by fibronectin fragments causes cleavage of aggrecan at the same site as found in osteoarthritic cartilage

Summary/Introduction Fibronectin proteolytic fragments (Fn-fs) have very potent cartilage chondrolytic activities toward cultured cartilage explants in vitro [1] and when injected into rabbit knee joints [2]. Their relevance to cartilage pathology is supported by their presence at elevated levels in synovial fluids of patients with osteoarthritis and rheumatoid arthritis [3, 4]. Their relevance is also supported by the discovery that their effects are mediated through other factors, thought to have roles in cartilage pathology, catabolic cytokines. Cytokines have been detected in conditioned media of Fn-f treated human cartilage [5] and neutralization with antibodies directed toward specific cytokines blocks cartilage damage by Fn-fs and induction of matrix metalloproteinases (MMPs) [5]. The major MMP responsible for cartilage damage appears to be MMP-3 (stromelysin-1), since antibodies to MMP-3 greatly slow cartilage damage caused by the Fn-fs [6, 7] and conditioned media of Fn-f treated cartilage contains levels of up to 800 nm of active MMP-3 [7]. Since MMPs cleave at the the Asn 360 -Phe 361 bond * in the interglobular domain of aggrecan [8], it would be expected that this cleavage would occur in Fn-f treated cartilage as well. However, this site is not cleaved in normal cartilage aggrecan turnover in vitro and in vivo [9] or in adult cartilage explants treated with interleukin-1 (IL-1) or retinoic acid [9–11]. In the latter cases, cleavage occurs exclusively at Glu 392 -Ala 393 . Further, the expected N-terminal Ala aggrecan fragments are found in human synovial fluids of patients with both osteoarthritis [11] and inflammatory joint diseases [12]. Accordingly, the enzymatic activity that may be responsible for this has been called ‘aggrecanase', but the responsible proteinase has not yet been identified or isolated. Our objective here was to determine whether the Fn-fs caused cleavage at the physiologically relevant ‘aggrecanase' site or at the MMP site in bovine cartilage aggrecan. The use of bovine cartilage was appropriate since proteoglycan synthesis suppression, cartilage proteoglycan degradation and release of active MMP-3 occurs to similar extents in both bovine and human knee cartilage explants [5–7]. We report here that cleavage was detected at the aggrecanase site, as well as other sites, but not at the MMP site. Thus, these data support a potential physiological role for Fn-fs in cartilage pathology in osteoarthritis or inflammatory joint disease. The data also support the relevance of the use of Fn-fs in animal models of knee joint damage as described by us earlier [2] as well as in in vitro explant culture models [1, 5–7].

MRI contrast enhanced study of cartilage proteoglycan degradation in the rabbit knee.

Early degeneration of cartilage is accompanied by a loss of proteoglycans and consequent changes in the content of water. Conventional magnetic resonance imaging (MRI) cannot reliably detect this change, since the relaxation properties of the cartilage are dominated by its collagen content. The applicability of a positively charged nitroxide as an MRI contrast agent in detection of the content of the negatively charged proteoglycans within the cartilage was investigated. The results from both MRI and electron paramagnetic resonance (EPR) spectroscopy indicate that the accumulation of the contrast agent reflects the amount of proteoglycans within the cartilage, presumably due to the electrostatic interactions between the negatively charged proteoglycans and the positively charged nitroxide. Such a contrast agent could be useful in the detection and study of early stages of the degeneration of joints.

Association of Proteoglycan Degradation with Catabolic Cytokine and Stromelysin Release from Cartilage Cultured with Fibronectin Fragments

Addition of fibronectin fragments to bovine articular cartilage explant cultures results in enhanced release of metalloproteinases and rapid cartilage proteoglycan (PG) degradation and loss. The chondrolysis begins with rapid PG degradation which markedly slows after 1 week. Preliminary observations suggest that catabolic cytokines mediate chondrolytic activities of the fibronectin fragments. The objectives of this work were to investigate the correlations between: (a) release of specific cytokines; (b) release of the metalloproteinase (MMP), stromelysin-1 (MMP-3); (c) release of the tissue inhibitor of MMPs, TIMP-1, and; (d) degradation and release of PG from cultured cartilage. We report that human articular cartilage cultured with an amino-terminal 29-kDa fragment (Fn-f), at 0.1 μM, released enhanced levels of TNF-α, IL-1β, and IL-1α with peaks at Days 2, 3, and 9, respectively. MMP-3 release was elevated with a peak at Day 6 and a profile similar to that for the Fn-f-induced cartilage PG depletion. IL-6 release was enhanced within 2 days and continued at the same level throughout the culture period but this did not lead to enhanced release of TIMP-1, a known activity of IL-6. These data suggest that in the early chondrolytic events induced in cultured cartilage by Fn-f, enhanced MMP-3 release and maximal degradation and release of PG from cultured cartilage are kinetically associated with elevated release of the catabolic cytokines, TNF-α, IL-1β, and IL-1α. Further, a later period of slowing PG loss and slowing MMP-3 release is associated with greatly slowed release of these cytokines, but prolonged release of IL-6. This model of cartilage damage may be useful for studies of the interplay between cytokines and the effects of combinations of cytokines on cartilage homeostasis.

Isothiazolones interfere with normal matrix metalloproteinase activation and inhibit cartilage proteoglycan degradation.

A series of isothiazolones that inhibit pro-(matrix metallo-proteinase) (proMMP) activation but do not inhibit the active enzyme are effective as cartilage protectants in bovine nasal cartilage organ culture, preventing interleukin-1 (IL-1)-induced proteoglycan (aggrecan) degradation without affecting its synthesis. These compounds were found to bind to prostromelysin (proMMP-3) in a non-dialysable and stoichiometric manner. Preincubation with cartilage-protectant isothiazolones prevented the binding of [14C]iodoacetamide to Cys75 of the MMP-3 propeptide, suggesting that the activity of these compounds involves their binding to the Cys75 of the MMP zymogen. Studies following chymotrypsin activation of proMMP-3 by SDS/PAGE indicated that altered processing of the 57 kDa zymogen to the active form occurred in the presence of compound. The 53 kDa intermediate seen on normal activation was not formed; instead a different intermediate appeared with a molecular mass of approx. 46 kDa. N-terminal sequence analysis indicated that this intermediate was formed by cleavage at the putative 4-aminophenylmercuric acid cleavage site. Importantly the 45 kDa active MMP-3 species formed in the presence of compound was one amino acid residue shorter than the native MMP-3. These results suggest that the inhibition of cartilage proteoglycan degradation by isothiazolones might be due to their ability to bind to the Cys75 in the propeptide region of the MMP zymogen and interfere with its normal activation process.

Leukemia Inhibitory Factor Induces Leukocyte Infiltration and Cartilage Proteoglycan Degradation in Goat Joints

Recent studies have implicated leukemia inhibitory factor (LIF) in human joint disease. LIF is produced by cultured synovial cells and articular chondrocytes, stimulates cartilage and bone resorption, and has been detected in inflammatory exudates from arthritic joints. The aim of this study was to evaluate the effect of intraarticular injections of human recombinant LIF in the goat. Endotoxin-free, sterile normal saline containing 1 micrograms recombinant human LIF (rhLIF) was injected into the right radiocarpal joints (RCJs) of eight angora goats. The left RCJs were injected with an equivalent volume of vehicle alone (n = 6) or vehicle containing 1 micrograms human albumin (n = 2). Goat joints were examined for clinical features of inflammation, and synovial fluid (SF) was aspirated on days 0, 2, and 6 postinjection. Leukocyte counts and concentrations of keratan sulfate, IL-1 beta, and TNF-alpha were determined in the SF. Proteoglycan synthesis was determined ex vivo in cartilage explants obtained on day 6 postinjection. A statistically significant increase in joint swelling and effusion volume was observed in LIF-injected joints but not in control joints. In the LIF-injected RCJs, the leukocyte count increased from 82 +/- 9 cells/microliters before injection to 10,300 +/- 3357 cells/microliters at day 2 postinjection (p < 0.005) and declined to 678 +/- 113 cells/microliters at day 6 postinjection. Polymorphonuclear leukocytes and monocyte/macrophages predominated in the infiltrate. No appreciable change in leukocyte counts was observed in control joints.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of interleukin-1 receptor antagonist and soluble tumor necrosis factor receptor in animal models of infection.

Intracisternal or intraarticular inoculation of rabbit recombinant interleukin (IL)-1 beta and rabbit tumor necrosis factor-alpha combined with IL-1 receptor antagonist (IL-1RA) and soluble tumor necrosis factor receptor (sTNFR), respectively, produced significantly less inflammation in rabbits than after inoculation of these cytokines alone. In contrast, when Haemophilus influenzae type b (Hib) or Hib lipooligosaccharide (LOS) was given intraarticularly with IL-1RA, sTNFR, or the combination, there was no significant or consistent modulation of synovial inflammation and cartilage proteoglycan degradation. In the experimental meningitis model, IL-1RA and sTNFR did not significantly reduce the meningeal inflammatory response associated with intracisternal inoculation of Hib LOS. These data indicate that specific cytokine inhibitors (sTNFR and IL-1RA) may not be effective in modulating inflammation induced by a broad inflammatory stimulus such as gram-negative bacteria or their products and suggest caution in using them to treat these infectious conditions in humans.

Proteoglycan degradation in hemarthrosis. Intraarticular, autologous blood injection in rat knees.

We determined the degradation of articular cartilage proteoglycans in a single episode of experimental hemarthrosis in rat knees. The right knee joints of rats were injected once with autologous whole blood. Both knee joints were examined histologically. Biochemical studies of cartilage proteoglycans extracted from the knees were also conducted. Histological examination revealed an accumulation of mononuclear cells in intraarticular fibrin clots and subsynovial layers 8 hours after the injection of blood. Accordingly, initiation of proteoglycan degradation occurred 8 hours after injection of blood, lasting from 1 day of limited degradation to 3 days; recovery then occurred within 7 days. The proteoglycan degradation could be inhibited by 1 mM phenylmethylsulfonyl fluoride, a general serine proteinase inhibitor, 0.1 M 6-aminohexanoic acid, a specific inhibitor of plasminogen activators, 10 mM EDTA, and 10(-6) to 10(-8) M dexamethasone, indicating that the accumulation of mononuclear cells in intraarticular fibrin clots and subsynovial layers may play an important role in cartilage damage.

Inhibition of human neutrophil elastase and cathepsin G by a diphenyl disulfonic acid copolymer

The sulfated polymer MDL 101,028 was found to be a potent inhibitor of both human neutrophil elastase (HNE) and human neutrophil cathepsin G (CatG). Cleavage of synthetic substrate by HNE was inhibited by MDL 101,028 with an IC 50 of 40 nM, while CatG was inhibited with an IC 50 of 80 nM. Degradation of a macromolecular connective tissue substrate (cartilage proteoglycan) by HNE or CatG was inhibited by MDL 101,028 with an IC 50 of approximately 10 μM. MDL 101,028 at concentrations of 4, 10 and 25 μM inhibited degradation of cartilage proteoglycan by human neutrophil lysate or stimulated human neutrophils by 54%, 70% and 79%, and 31%, 47% and 73%, respectively. Acute pulmonary injury resulting from the intratracheal (i.t.) instillation of HNE in rats was inhibited by 48%, 90% and 90% at concentrations of MDL 101,028 of 1.1 mg/kg, 2.8 mg/kg and 11 mg/kg. The duration of action of the compound after i.t. instillation was between 2 and 4 h. These results suggest that sulfated polymers such as MDL 101,146 may be useful as inhibitors of HNE-mediated lung injury.

ARG-Gly-AsP-Ser Peptide Analogs Suppress Cartilage Chondrolytic Activities of Integrin-Binding and Nonbinding Fibronectin Fragments

We have reported that Fn fragments (Fn-f), which have been detected in synovial fluids of osteoarthritis and rheumatoid arthritis patients, can potently cause cartilage chondrolysis and depress proteoglycan (PG) synthesis in cartilage tissue cultured as explants. Amino-terminal 29-kDa, gelatin-binding 50-kDa, and integrin-binding 140-kDa Fn-f are active. In order to investigate the mode of action and devise means of blocking the damage mediated by all Fn-f, we have tested the effects of various analogs resembling the integrin binding sequence, Arg-Gly-Asp-Ser, on blocking Fn-f-mediated chondrolysis. The analog peptides, Gly-Arg-Ala-Asp-Ser-Pro-Lys and Arg-Phe-Asp-Ser, at concentrations as low as 1 μM, blocked the effects of all three Fn-f on cartilage degradation, while the native sequence peptide, Arg-Gly-Asp-Ser, had very low Fn-f-blocking activity and by itself caused cartilage damage. Random sequence peptides dissimilar to the analog sequences were inactive as inhibitors as well as was a sequence analog, Phe-Asp-Arg-Ser, related to the Arg-Phe-Asp-Ser inhibitor. The analog inhibitory peptides decreased rates of Fn-f-mediated PG degradation and release from cartilage and decreased Fn-f-mediated PG synthesis depression. The analog inhibitory peptides alone had no detectable effect on cartilage PG degradation or PG synthesis rates. These data show that the chondrolytic activities of integrin-binding and nonbinding Fn-f can be blocked by synthetic peptide analogs of the Arg-Gly-Asp-Ser sequence and suggest that these peptides may be useful for blocking other activities of Fn-f.

Effects of Retinoic acid on cartilage differentiation in a chondrogenic cell line

Previously we have isolated the monopotential chondrogenic cell line RCJ 3.1 C5.18 from the multipotential mesenchymal cell line RCJ 3.1 [Grigoriadis et al.: Endocrinology, 125:2103-2110, 1989]. When cultured for approximately 20 days under appropriate conditions, these cells from cartilage nodules. In the present investigation, we have used this cell line to study the effects of all-trans retinoic acid (RA) on chondroblast differentiation, cartilage formation, and cartilage degradation. Continuous exposure of cultures to RA (0.01-100 nM) inhibited chondroblast differentiation and glycosaminoglycan (GAG) accumulation in a dose-dependent manner, without comparable effects on cell growth. Pulse treatment with RA for various 4 day periods during a 17-24 day culture period established that RA inhibited differentiation of chondroprogenitors at all periods tested. These effects were reversible, except for part of the effect on early chondroprogenitors. Treatment with RA on days 13-17 in 17 day cultures not only resulted in cessation of cartilage formation, but also in disappearance of pre-existing cartilage nodules. We demonstrated that this was associated with RA-induced downregulation of GAG synthesis and increased degradation of cartilage proteoglycans. Hence, the inhibitory effects of RA on cartilage formation consist of inhibition of chondroblast differentiation, inhibition of GAG synthesis by differentiated chondroblasts, and stimulation of cartilage proteoglycan degradation by differentiated chondroblasts and/or chondrocytes. These results indicate that the clonal monopotential chondrogenic cell line RCJ 3.1 C5.18 forms a good model system to study the effects of retinoids on cartilage differentiation, formation, and degradation.

Lysosomal cysteine endopeptidases mediate interleukin 1-stimulated cartilage proteoglycan degradation

The peptidyl diazomethane inactivator of cysteine endopeptidases, benzyloxycarbonyl-Tyr-Ala-CHN2, was tested as an inhibitor of interleukin 1 alpha-stimulated release of proteoglycan from bovine nasal septum cartilage explants. Like the previously tested epoxidyl peptide proinhibitor trans-epoxysuccinyl-leucylamido-(3-methyl)butane ethyl ester, it proved to be an effective inhibitor of proteoglycan release from cartilage, with significant inhibition at a concentration of 1 microM. The inhibition did not seem to be due to a general toxic effect. The rates of inactivation of the bovine cysteine endopeptidases by the peptidyl diazomethane, the epoxidyl peptide proinhibitor and its active form were determined. Benzyloxycarbonyl-Tyr-Ala-CHN2 proved to be a rapid inactivator of cathepsins L, S and B, but reacted much more slowly with cathepsin H and calpain. Thus it would appear that the latter two enzymes are not implicated in proteoglycan release in our test system. The peptidyl diazomethane and epoxidyl peptide proinhibitor (above) were also tested for their effects on three other interleukin 1-mediated cellular events, namely epidermal growth factor receptor transmodulation, and interleukin 6 and prostaglandin E2 production. In all cases the inactivators did not interfere with the response to interleukin 1 in human gingival fibroblasts. We conclude that one or more of the lysosomal cysteine endopeptidases cathepsins B, L and S mediate interleukin 1-stimulated cartilage proteoglycan degradation without affecting signal transduction.

Monoclonal antibodies recognizing protease-generated neoepitopes from cartilage proteoglycan degradation. Application to studies of human link protein cleavage by stromelysin.

Monoclonal antibodies were raised that specifically recognize the NH2-terminal neoepitope sequence present in link protein cleavage products derived from stromelysin-degraded proteoglycan aggregate. Competitive enzyme-linked immunosorbent assay, using synthetic peptides as inhibitors, showed that one of these antibodies (CH-3) required, for antibody recognition, the free NH2-terminal amino acid isoleucine (residue 17 of the intact protein) in the sequence NH2-IQAENG at the stromelysin cleavage site of link protein 3. Human proteoglycan aggregate was digested with recombinant human stromelysin, bovine chymotrypsin, bovine trypsin, and porcine elastase, and their respective link protein degradation products were tested for immunoreactivity with antibody CH-3. Only stromelysin- and chymotrypsin-generated link protein 3 were recognized by antibody CH-3. Both of these enzymes generate link protein NH2 termini with the sequence 17IQAENG. . .; hence these studies indicated that monoclonal antibody CH-3 recognized this neoepitope sequence in only specific proteolytically modified link protein molecules. Since the occurrence of link protein 3 increases with aging, the incidence of CH-3 epitope in proteoglycans isolated from human knee articular cartilage of individuals of different ages was investigated. The prevalence of CH-3 epitope was found to be highest in newborn and adolescent articular cartilage samples. However, little CH-3 epitope was detected in older adult cartilage, although considerably more link protein 3 was present in these samples. These results suggest that additional proteolytic agents are responsible for the increased occurrence of link protein degradation products with aging.

Platelet-activating factor inhibits proteoglycan synthesis and enhances neutrophil-mediated proteoglycan degradation in cartilage explants.

Platelet-activating factor (PAF), which stimulates the release of tissue-destructive enzymes and reactive oxygen metabolites from neutrophils, was investigated for its role in neutrophil-mediated cartilage breakdown. Bovine cartilage explants were incubated with or without human neutrophils, PAF, and other reagents. Cartilage damage was measured as either proteoglycan degradation (percent release of 35S-labeled proteoglycan from 35S-labeled cartilage) or inhibition of proteoglycan synthesis (rate of incorporation of 35S into proteoglycan). PAF increased neutrophil-mediated proteoglycan degradation in the 2-20 microM range. Three specific PAF-receptor antagonists, WEB2086, CV3988, and CV6209, reversed this effect of PAF. These antagonists also reduced the enhancement of neutrophil-mediated cartilage damage caused by granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha (TNF alpha). The results suggest that there may be a positive feedback mechanism whereby cytokine-primed neutrophils produce PAF, which amplifies the release of other tissue-damaging substances from neutrophils. In the absence of neutrophils, PAF (2-20 microM) inhibited the synthesis of proteoglycan by bovine cartilage. Neutrophils also inhibit proteoglycan synthesis, but PAF probably is not involved in this effect of neutrophils because the PAF receptor antagonists had no consistent effect. PAF increases neutrophil-mediated cartilage proteoglycan degradation in vitro. GM-CSF and TNF alpha enhancement of neutrophil damage to cartilage is partly due to PAF. PAF alone inhibits cartilage proteoglycan synthesis.

Serum keratan sulfate—a marker of predisposition to polyarticular osteoarthritis

We have used an ELISA to quantify a highly sulfated epitope present on keratan sulfate, a carbohydrate chain found principally in cartilage proteoglycans. The serum level of the epitope provides an indirect measure of the rate of degradation of cartilage proteoglycans during normal turnover and can be used to diagnose specific abnormalities in keratan sulfate metabolism. Serum levels of the epitope are elevated in a high percentage of patients with osteoarthritis and correlate with the number of joints involved. The elevated rate of proteoglycan turnover in these patients appears to be systemic, affecting not only the degenerating articular surfaces but apparently normal articular cartilages as well. We have postulated that this acceleration in the rate of proteoglycan turnover precedes clinical evidence of degenerative changes; and we discuss the rationale for the contention that this elevation may predispose adult humans to polyarticular osteoarthritis.

Monoclonal antibodies recognizing protease-generated neoepitopes from cartilage proteoglycan degradation : application to studies of human link protein cleavage by stromelysin

Monoclonal antibodies recognizing protease-generated neoepitopes from cartilage proteoglycan degradation : application to studies of human link protein cleavage by stromelysin

Inhibition of interleukin 1-stimulated cartilage proteoglycan degradation by a lipophilic inactivator of cysteine endopeptidases

Inactivators of cysteine endopeptidases were tested as inhibitors of the cytokine-stimulated release of proteoglycan from cartilage. The test system consisted of bovine nasal septum cartilage maintained in organ culture, and the stimulus was provided by recombinant human interleukin 1 alpha. L-3-Carboxy-2,3-trans-epoxypropionyl-leucylamido-(4-guanidin o)butane (E64) and L-3-carboxy-2,3-trans-epoxypropionyl-leucylamido-(3-methyl)b utane (Ep475) showed no inhibition at concentrations up to 100 microM. In contrast, trans-epoxysuccinyl-leucylamido-(3-methyl)butane ethyl ester (Ep453), a 'prodrug' of Ep475, was an effective inhibitor. The LL-, LD- and DL-isomers gave significant inhibition at 10 microM, and the DD-isomer was inhibitory at 100 microM. None of the isomers had any detectable effect on protein synthesis or glycolysis, and their inhibitory effects were reversible. Iodoacetate inhibited proteoglycan release by a general toxic effect. Our results suggest that cysteine endopeptidase(s) play a part in cytokine-stimulated cartilage breakdown, but that effective inhibitors must pass through membranes.

Interleukin 1-stimulated cartilage proteoglycan degradation is inhibited by some cysteine endopeptidase inactivators

Interleukin 1-stimulated cartilage proteoglycan degradation is inhibited by some cysteine endopeptidase inactivators

Magnetic resonance imaging of the rabbit knee: Detection of cartilage proteoglycan degradation

Intra-articular (i.a.) injection of papain causes a reversible loss of proteoglycan in intact rabbit knees. Twelve rabbits were scanned with magnetic resonance imaging (MRI) at 0, 24, 48 and 72 hours after 5 units of papain i.a. in a 1.5 Tesla Signa with a three inch surface coil using spin echo sequence. Total cartilage thickness in proton density images was 1.08 +/- 0.09 mm prior to papain injection. The magnetic resonance images showed a reduction in articular cartilage thickness in papain-treated rabbit femurs at 24 hours to 0.69 +/- 0.18 mm and partial restoration by 72 hours to 0.77 +/- 0.21 mm.