Role of tenascin-C in articular cartilage

Tenascin-C (TN-C) is a glycoprotein component of the extracellular matrix (ECM). TN-C consists of four distinct domains, including the tenascin assembly domain, epidermal growth factor-like repeats, fibronectin type III-like repeats, and the fibrinogen-like globe (FBG) domain. This review summarizes the role of TN-C in articular cartilage. Expression of TN-C is associated with the development of articular cartilage but markedly decreases during maturation of chondrocytes and disappears almost completely in adult articular cartilage. Increased expression of TN-C has been found at diseased cartilage and synovial sites in osteoarthritis (OA) and rheumatoid arthritis (RA). TN-C is increased in the synovial fluid in patients with OA and RA. In addition, serum TN-C is elevated in RA patients. TN-C could be a useful biochemical marker for joint disease. The addition of TN-C results in different effects among TN-C domains. TN-C fragments might be endogenous inducers of cartilage matrix degradation; however, full-length TN-C could promote cartilage repair and prevent cartilage degeneration. The deficiency of TN-C enhanced cartilage degeneration in the spontaneous OA in aged joints and surgical OA model. The clinical significance of TN-C effects on cartilage is not straightforward.     

Calcium pentosan polysulfate inhibits the catabolism of aggrecan in articular cartilage explant cultures

OBJECTIVE: The catabolism of aggrecan and loss of aggrecan fragments from articular cartilage is a key event in the pathogenesis of arthritic diseases such as osteoarthritis. The catabolism of aggrecan is mediated by the specific proteolytic activity termed aggrecanase. The aim of this study was to investigate the effect of the chondroprotective agent calcium pentosan polysulfate (CaPPS) on the aggrecanase-mediated catabolism of aggrecan. METHODS: The catabolism of 35S-labeled aggrecan and loss of tissue glycosaminoglycans (GAGs) were investigated using bovine articular cartilage explant cultures maintained in medium containing varying concentrations of CaPPS (1-100 microg/ml) in the presence or absence of 10(-6)M retinoic acid or 7 ng/ml recombinant human interleukin-1alpha (rHuIL-1alpha). In addition, the effect of CaPPS on the degradation of aggrecan monomers by aggrecanase activity present in conditioned medium from joint capsule explant cultures was investigated. RESULTS: CaPPS inhibited the catabolism of 35S-labeled aggrecan in a dose-dependent manner, particularly when retinoic acid or rHuIL-1alpha was used to stimulate aggrecan catabolism. These effects were reflected in the tissue levels of GAG remaining in these cultures at the end of the experiment. CaPPS inhibited the degradation of aggrecan monomers by soluble aggrecanase activity. CONCLUSION: CaPPS inhibits the catabolism of aggrecan by articular cartilage in a dose-dependent manner, particularly when the processes responsible for aggrecan loss are stimulated. This effect occurs, at least in part, through direct inhibition of aggrecanase activity. CaPPS did not adversely affect overall chondrocyte metabolism, as shown by the incorporation of 35S-sulfate and 3H-leucine into macromolecules and by lactate production in cartilage explant cultures.     

The pathophysiology of osteoarthritis

Osteoarthritis (OA) is a complex disease whose pathogenesis includes the contribution of biomechanical and metabolic factors which, altering the tissue homeostasis of articular cartilage and subchondral bone, determine the predominance of destructive over productive processes. A key role in the pathophysiology of articular cartilage is played by cell/extra-cellular matrix (ECM) interactions, which are mediated by cell surface integrins. In a physiologic setting, integrins modulate cell/ECM signaling, essential for regulating growth and differentiation and maintaining cartilage homeostasis. During OA, abnormal integrin expression alters cell/ECM signaling and modifies chondrocyte synthesis, with the following imbalance of destructive cytokines over regulatory factors. IL-1, TNF-alpha and other pro-catabolic cytokines activate the enzymatic degradation of cartilage matrix and are not counterbalanced by adequate synthesis of inhibitors. The main enzymes involved in ECM breakdown are metalloproteinases (MMPs), which are sequentially activated by an amplifying cascade. MMP activity is partially inhibited by the tissue inhibitors of MMPs (TIMPs), whose synthesis is low compared with MMP production in OA cartilage. Intriguing is the role of growth factors such as TGF-beta, IFG, BMP, NGF, and others, which do not simply repair the tissue damage induced by catabolic factors, but play an important role in OA pathogenesis.     

The serine protease inhibitor trappin-2 is present in cartilage and synovial fluid in osteoarthritis

OBJECTIVE: Trappins are small serine protease inhibitors bound to extracellular matrix (ECM) through the actions of transglutaminase (TGase) enzymes. Trappin-2 is present in many tissues and is upregulated at sites of injury. In osteoarthritis (OA), serine proteases contribute to articular cartilage destruction, and TGase activity is increased. Yet little is known about matrix-bound serine protease inhibitors or TGase substrates in articular cartilage. Our purpose was to determine if trappin-2 was present in OA cartilage and synovial fluid (SF). METHODS: OA knee articular cartilage and SF were assayed for trappin-2 protein by Western blotting, ELISA, and immunohistochemistry. Trappin-2 mRNA was detected with RT-PCR. The ECM components bound to trappin-2 were identified by 2-D gel electrophoresis and peptide fingerprinting. RESULTS: Trappin-2 was detectable in OA articular cartilage extracts, cultured chondrocytes, conditioned media, and SF by Western blotting. OA cartilage protein extracts contained significantly higher quantities of trappin-2 than normal cartilage protein extracts (22.98 +/- 1.28 ng/mg wet weight vs 14.97 +/- 1.92 ng/mg wet weight; p < 0.01). RT-PCR confirmed the presence of trappin-2 mRNA in OA chondrocytes. Immunohistochemical studies of OA cartilage revealed trappin-2 protein in chondrocytes. Peptide mapping of trappin-2 binding partners showed that fibromodulin was bound to trappin-2 in cartilage. CONCLUSION: We confirmed the presence of trappin-2 in OA cartilage and SF. Elevated levels of TGase activity in OA cartilage may increase levels of this serine protease inhibitor in response to injury.     

Matrix metalloproteinases and aggrecanases cleave aggrecan in different zones of normal cartilage but colocalize in the development of osteoarthritic lesions in STR/ort mice.

OBJECTIVE: To map aggrecan cleavage by matrix metalloproteinases (MMPs) and aggrecanases in normal murine tibial articular cartilage (CBA strain) and in the development of spontaneous osteoarthritis (OA) in the STR/ort mouse and to assess the influence of sex hormone status on these conditions in gonadectomized STR/ort mice. METHODS: The distributions of neoepitopes of aggrecan generated by MMP (VDIPEN) and aggrecanase (NITEGE) cleavage were investigated by immunohistochemistry. RESULTS: VDIPEN neoepitope was detected mainly in the pericellular matrix of deep-zone chondrocytes in normal tibial cartilage from STR/ort and CBA mice. In early OA, VDIPEN immunostaining also localized to the pericellular matrix of chondrocytes at the site of the lesion. With increasing severity of OA lesions, VDIPEN immunostaining was also detected in the interterritorial matrix, close to the site of the lesion. In contrast, NITEGE mapped most strongly to the pericellular matrix of upper-zone chondrocytes in normal tibial cartilage. As with VDIPEN, NITEGE was strongly expressed in the pericellular matrix at the site of early OA lesions. With advancing OA, NITEGE colocalized with VDIPEN in both the pericellular and interterritorial matrices of chondrocytes adjacent to OA lesions and in those of the deep zones. Hormone status did not appear to influence the development of OA or the distribution of aggrecan neoepitopes in STR/ort mice. CONCLUSION: MMP- and aggrecanase-generated neoepitopes map predominantly to different regions in normal murine tibial cartilage. However, both groups of enzymes generate increased amounts of neoepitopes in pericellular and interterritorial matrix adjacent to histopathologic lesions of OA. Aggrecan degradation and the development of OA appear to be independent of sex hormone status in this model.     

Neutrophil gelatinase-associated lipocalin is expressed in osteoarthritis and forms a complex with matrix metalloproteinase 9

OBJECTIVE: Expression of matrix metalloproteinase 9 (MMP-9) is up-regulated in osteoarthritis (OA) and usually presents as multiple bands when synovial fluid (SF) from OA patients is analyzed by zymography. Among these bands is an approximately 125-130-kd band for high molecular weight (HMW) gelatinase, which has not been characterized. This study was undertaken to characterize the HMW MMP activity in OA SF. METHODS: MMP activity in OA SF was determined by gelatin zymography. Recombinant MMPs were used to identify MMP activity on the zymogram. Western immunoblotting, immunoprecipitation, and immunodepletion analyses were performed using antibodies specific for human MMP-9 and human neutrophil gelatinase-associated lipocalin (NGAL). Human cartilage matrix degradation was determined by dimethylmethylene blue assay. RESULTS: Zymographic analysis showed that the HMW gelatinase in OA SF comigrated with a purified NGAL-MMP-9 complex. Results of Western immunoblotting showed that the HMW gelatinase was also recognized by antibodies specific for human NGAL or human MMP-9. These same antibodies also immunoprecipitated the HMW gelatinase activity from OA SF. The NGAL-MMP-9 complex was reconstituted in vitro in gelatinase buffer. In the presence of NGAL, MMP-9 activity was stabilized; in the absence of NGAL, rapid loss of MMP-9 activity occurred. MMP-9-mediated release of cartilage matrix proteoglycans was significantly higher in the presence of NGAL (P < 0.05). CONCLUSION: Our findings demonstrate that the HMW gelatinase activity in OA SF represents a complex of NGAL and MMP-9. The ability of NGAL to protect MMP-9 activity is relevant to cartilage matrix degradation in OA and may represent an important mechanism by which NGAL may contribute to the loss of cartilage matrix proteins in OA.     

Effect of inhibition of matrix metalloproteinases on cartilage loss in vitro and in a guinea pig model of osteoarthritis

OBJECTIVE: To study the effects of a matrix metalloproteinase (MMP) inhibitor (S-34219) on osteoarthritis (OA) cartilage cultures and in the meniscectomized guinea pig model of OA. METHODS: The inhibitory activity of S-34219 on MMPs and aggrecanase was studied by fluorimetry and immunoassay, respectively. The effects of S-34219 on proteoglycan and collagen degradation were studied in cultures of rabbit and human cartilage. Medial meniscectomy was performed on 29 Hartley male guinea pigs, and these animals were randomly allocated to 1 of 3 groups: a control meniscectomized group (MNXc) receiving the vehicle, or a meniscectomized group receiving either 10 mg/kg or 20 mg/kg S-34219, administered twice per day by oral gavage for 12 weeks from day 1 after surgery. An additional group comprised sham-operated animals. Tibial cartilage from the operated left knee was processed for histologic assessment of OA lesions. RESULTS: The 50% inhibitory concentration (IC(50)) of S-34219 on MMPs 1, 2, 3, 8, 9, and 13 was 55, 0.1, 0.5, 0.1, 0.03, and 0.2 nM, respectively; the IC(50) on aggrecanase 1 was 190 nM. In cultured rabbit cartilage, 100 nM S-34219 strongly inhibited MMP-dependent degradation of collagen and proteoglycans. A concentration 100 times higher was needed to inhibit aggrecanase-dependent degradation. In cultures of human OA cartilage, 100 nM S-34219 inhibited spontaneous type II collagen degradation by 66% and proteoglycan degradation by only 22%. For in vivo studies, treated groups were compared with the MNXc group and the results, expressed as the percentage variation versus MNXc, were as follows: in the 10 and 20 mg/kg groups, a significant decrease (P < 0.05) in global histologic score (-12% and -14%, respectively) was observed, and this was associated with a significant increase (P < 0.05) in cartilage thickness (+19% and +18%, respectively). Neither dose level changed the proteoglycan content. CONCLUSION: In both treated animal groups, S-34219 significantly prevented the loss of cartilage thickness, probably by inhibiting collagen breakdown that normally leads to the erosion of fibrillated superficial areas. The absence of a protective effect on glycosaminoglycan loss, both in vitro and in vivo, suggests that aggrecanases may have an important role in cartilage loss. This study reinforces the relevance of these models for testing chondroprotective drugs, and the potential role of dual inhibitors of collagenase and aggrecanase as disease-modifying drugs in the management of OA.     

Pathobiology of the synovium in osteoarthritis

The synovium is a vascularized soft tissue lining the capsule and intra‐articular structures of diarthrodial joints. The synovial intima consists of a discontinuous layer of synovial lining cells supported by a cell‐containing scaffolding of loose extracellular matrix (ECM) or interstitium, composed of collagen (types III, IV, V, VI), proteoglycans and other extracellular proteins such as fibronectin, tenascin, entactin and laminin. The synovium has several functions critical for maintaining normal joint homeostasis. Synovial fluid (SF) and its constituents are produced by this tissue, which also mediates blood/synovial fluid exchanges, such as some nutrition of the surface cartilage chondrocytes and absorption of foreign particles from the synovial cavity. The synovial ECM also provides hydraulic resistance, preventing rapid seepage of SF out of the joint cavity and modifying the exchange of macromolecules and other solutes between the subintimal capillaries and SF. Two main types of synovial cell have been identified. The most common synovial lining cell is the type B or fibroblast‐like synoviocyte (FLS). FLS are characterised by abundant rough endoplasmic reticulum and dendritic processes which form a network over the intimal surface but are also present in the subintima. The highly proliferative FLS are involved in the production of specialised matrix constituents, including hyaluronan, collagens and fibronectin for the intimal interstitium and high molecular weight hyaluronan for the SF, essential for the preservation of high viscosity and low coefficient of friction between the surfaces of articular cartilage of the joint. The type A lining cell or macrophage‐like synoviocytes (MLS) are round, nonfixed, nonproliferative, immunoreactive cells which express MHC II molecules and cathepsins B, D and L. MLS absorb and degrade extracellular matrix constituents, cell debris, microorganisms and antigens in the synovial fluid and intimal matrix and use a system of capillaries and lymphatics to clear the joint of particles. Activated MLS are an abundant source of pro‐inflammatory mediators which can promote resorption of articular cartilage. Changes which occur in the osteoarthritic (OA) synovium are not as visually striking as those which occur in RA joints, however, this does not mean that OA synovium can be legitimately used as a control tissue for RA studies. In early OA, the synovium can appear grossly normal. A degree of synovial villous hypertrophy, fibrosis and a significant low grade inflammation are usually present in this tissue from subjects with OA, with a mild infiltrate composed primarily of lymphocytes and mononuclear cells. The cellularity is generally much less than that seen in RA tissue, however, natural killer cell activity and mast cell levels are increased in OA synovium, often greater than in RA tissue. Advanced OA is often characterised by cartilaginous fragments embedded in the synovium, resulting in more pronounced synovitis. Corresponding increases in several molecular markers have been described, including cytokines (interleukin‐1β, IL‐1β receptor antagonist) and other molecules implicated in cartilage destruction (stromelysin, aggrecanase‐2, inducible nitric oxide synthase, urokinase‐type plasminogen activator). Most of these changes in the pathology of the synovium have been demonstrated in recent years by immunohistological studies on both human synovial tissues and synovia from animals with surgically induced OA.     

Matrix metalloproteinases and aggrecanases cleave aggrecan in different zones of normal cartilage but colocalize in the development of osteoarthritic lesions in STR/ort mice

Objective

To map aggrecan cleavage by matrix metalloproteinases (MMPs) and aggrecanases in normal murine tibial articular cartilage (CBA strain) and in the development of spontaneous osteoarthritis (OA) in the STR/ort mouse and to assess the influence of sex hormone status on these conditions in gonadectomized STR/ort mice.

Methods

The distributions of neoepitopes of aggrecan generated by MMP (VDIPEN) and aggrecanase (NITEGE) cleavage were investigated by immunohistochemistry.

Results

VDIPEN neoepitope was detected mainly in the pericellular matrix of deep‐zone chondrocytes in normal tibial cartilage from STR/ort and CBA mice. In early OA, VDIPEN immunostaining also localized to the pericellular matrix of chondrocytes at the site of the lesion. With increasing severity of OA lesions, VDIPEN immunostaining was also detected in the interterritorial matrix, close to the site of the lesion. In contrast, NITEGE mapped most strongly to the pericellular matrix of upper‐zone chondrocytes in normal tibial cartilage. As with VDIPEN, NITEGE was strongly expressed in the pericellular matrix at the site of early OA lesions. With advancing OA, NITEGE colocalized with VDIPEN in both the pericellular and interterritorial matrices of chondrocytes adjacent to OA lesions and in those of the deep zones. Hormone status did not appear to influence the development of OA or the distribution of aggrecan neoepitopes in STR/ort mice.

Conclusion

MMP‐ and aggrecanase‐generated neoepitopes map predominantly to different regions in normal murine tibial cartilage. However, both groups of enzymes generate increased amounts of neoepitopes in pericellular and interterritorial matrix adjacent to histopathologic lesions of OA. Aggrecan degradation and the development of OA appear to be independent of sex hormone status in this model.

     

Identification of fibronectin neoepitopes present in human osteoarthritic cartilage

OBJECTIVE: Fibronectin fragments are present at high concentrations in the cartilage of patients with rheumatoid arthritis and patients with osteoarthritis (OA) and have been shown to promote cartilage catabolism in human cartilage cultures, suggesting that fibronectin fragments participate in the initiation and progression of arthritic disease. This study was undertaken to 1) identify the major fibronectin fragments in human OA cartilage and confirm their ability to elicit cartilage catabolism, 2) identify the cleavage sites in fibronectin and generate the corresponding neoepitope antibodies, and 3) explore the utility of fibronectin neoepitopes as biomarkers. METHODS: Fibronectin fragments were purified from human OA cartilage using affinity chromatography; their N-termini were then identified by sequencing. Bovine nasal cartilage was treated with affinity-purified fibronectin fragments and assayed for aggrecan breakdown by monitoring the release of glycosaminoglycans and the aggrecan neoepitope 1771AGEG. Fibronectin neoepitopes were detected by Western blotting in cytokine-treated media of human cartilage explants, and by immunohistochemical analyses of human OA cartilage. RESULTS: Multiple fibronectin fragments were isolated from human OA cartilage, and all contained the N-terminus 272VYQP. These fragments induced aggrecanase-mediated cartilage catabolism in bovine cartilage explants. Fibronectin fragments with the N-terminus 272VYQP and fragments with the C-terminus VRAA271 were detected following cytokine treatment of human cartilage extracts. These neoepitopes localized with areas of aggrecan loss in OA cartilage. CONCLUSION: Human OA cartilage contains fibronectin fragments with catabolic activity and a major cleavage site within fibronectin. This study is the first to characterize fibronectin neoepitopes in OA cartilage, suggesting that they may represent a novel biomarker of arthritis.     

Induction of aggrecanase 1 (ADAM-TS4) by interleukin-1 occurs through activation of constitutively produced protein

OBJECTIVE: To study the production of aggrecanase 1 (ADAM-TS4) in monolayer chondrocytes, capsular fibroblasts, and cartilage. METHODS: Bovine nasal and articular cartilage, monolayer chondrocytes, and capsular fibroblasts were incubated in the absence and presence of interleukin-1 (IL-1). ADAM-TS4 production was evaluated by immunofluorescence or by Western blot analysis. Aggrecanase activity was measured in cells grown on an immobilized peptide substrate, and peptide cleavage was monitored by enzyme-linked immunosorbent assay. RESULTS: There was constitutive production of ADAM-TS4 in both cells and tissue. The protein was associated with the extracellular matrix based on the observation that the staining could be reduced following treatment of chondrocytes with heparin or exposure to chondroitinase ABC. Interestingly, there was no detectable change in the abundance of ADAM-TS4 in response to IL-1. Western blot analysis of cell lysates from IL-1-stimulated chondrocytes showed no evidence of increased ADAM-TS4 production, but resulted in activation of ADAM-TS4. The activation was associated with an increased generation in the aggrecanase neoepitope NITEGE in nasal cartilage in response to IL-1. These data suggest that induction of aggrecanase activity both in cells and in cartilage by IL-1 may involve the stimulation of an activator of ADAM-TS4. Consistent with this observation, culture of chondrocytes on a solid support containing a peptide substrate resulted in the generation of aggrecanase-mediated cleavage that could be blocked by selective inhibitors of ADAM-TS4. CONCLUSION: These data support the hypothesis that ADAM-TS4 is constitutively produced in these cells and tissue, and that stimulation by IL-1 results in aggrecanase activation. Thus, the activator could be a potential target by which to control aggrecanase-mediated degradation in arthritic diseases.     

Matrix-degrading metalloproteinases and their roles in joint destruction

Progressive degradation of the extracellular matrix (ECM) of articular cartilage and bone by enhanced activities of proteinases is an essential step for joint destruction in rheumatoid arthritis (RA) and osteoarthritis (OA). Among the proteinases, matrix-degrading metalloproteinases play a key role in joint destruction. Recent studies have indicated that these metalloproteinases comprise members of the matrix metalloproteinase (MMP) and a disintegrin and metalloproteinase (ADAM) gene families. The MMP family is composed of 19 different members and classified into five subgroups of collagenases, gelatinases, stromelysins, membrane-type MMPs, and other MMPs. They have the ability to digest almost all ECM components in human tissues when they act in concert. Their prospective roles in RA and OA joint destruction have been well established. On the other hand, the ADAM family members are classified into ADAM metalloproteinases and catalytically inactive nonproteolytic homologues. The ADAM metalloproteinases contain ADAM with a transmembrane domain (membrane-type ADAM) and ADAM with thrombospondin motifs (ADAMTS). Although members in both groups are known to degrade ECM components, ADAMTS species may be especially important for the aggrecan (cartilage proteoglycan) degradation of articular cartilage in RA and OA, since aggrecanases-1 and -2 are included in this group. This review outlines the characters of the MMP and ADAM gene family members and their roles in joint destruction in RA and OA. Received: March 14, 2000     

Matrix-degrading metalloproteinases and their roles in joint destruction

Abstract

Progressive degradation of the extracellular matrix (ECM) of articular cartilage and bone by enhanced activities of proteinases is an essential step for joint destruction in rheumatoid arthritis (RA) and osteoarthritis (OA). Among the proteinases, matrix-degrading metalloproteinases play a key role in joint destruction. Recent studies have indicated that these metalloproteinases comprise members of the matrix metalloproteinase (MMP) and a disintegrin and metalloproteinase (ADAM) gene families. The MMP family is composed of 19 different members and classified into five subgroups of collagenases, gelatinases, stromelysins, membrane-type MMPs, and other MMPs. They have the ability to digest almost all ECM components in human tissues when they act in concert. Their prospective roles in RA and OA joint destruction have been well established. On the other hand, the ADAM family members are classified into ADAM metalloproteinases and catalytically inactive nonproteolytic homologues. The ADAM metalloproteinases contain ADAM with a transmembrane domain (membrane-type ADAM) and ADAM with thrombospondin motifs (ADAMTS). Although members in both groups are known to degrade ECM components, ADAMTS species may be especially important for the aggrecan (cartilage proteoglycan) degradation of articular cartilage in RA and OA, since aggrecanases-1 and -2 are included in this group. This review outlines the characters of the MMP and ADAM gene family members and their roles in joint destruction in RA and OA.     

Host metalloproteinases in Lyme arthritis.

OBJECTIVE: To assess the role of matrix metalloproteinases (MMPs) in cartilage and bone erosions in Lyme arthritis METHODS: We examined synovial fluid from 10 patients with Lyme arthritis for the presence of MMP-2, MMP-3, MMP-9, and "aggrecanase" activity using gelatinolytic zymography and immunoblot analysis. We developed an in vitro model of Lyme arthritis using cartilage explants and observed changes in cartilage degradation in the presence of Borrelia burgdorferi and/or various protease inhibitors. RESULTS: Synovial fluid from patients with Lyme arthritis was found to contain at least 3 MMPs: gelatinase A (MMP-2), stromelysin (MMP-3), and gelatinase B (MMP-9). In addition, there was evidence in 2 patients of "aggrecanase" activity not accounted for by the above enzymes. Infection of cartilage explants with B. burgdorferi resulted in induction of MMP-3, MMP-9, and "aggrecanase" activity. Increased induction of these enzymes by B. burgdorferi alone was not sufficient to cause cartilage destruction in the explants as measured by glycosaminoglycan (GAG) and hydroxyproline release. However, addition of plasminogen, which can act as an MMP activator, to cultures resulted in significant GAG and hydroxyproline release in the presence of B. burgdorferi. The MMP inhibitor batimastat significantly reduced the GAG release and completely inhibited the collagen degradation. CONCLUSION: MMPs are found in synovial fluids from patients with Lyme arthritis and are induced from cartilage tissue by the presence of B. burgdorferi. Inhibition of MMP activity prevents B. burgdorferi-induced cartilage degradation in vitro.     

Neutrophil gelatinase–associated lipocalin is expressed in osteoarthritis and forms a complex with matrix metalloproteinase 9

Objective

Expression of matrix metalloproteinase 9 (MMP‐9) is up‐regulated in osteoarthritis (OA) and usually presents as multiple bands when synovial fluid (SF) from OA patients is analyzed by zymography. Among these bands is an ∼125–130–kd band for high molecular weight (HMW) gelatinase, which has not been characterized. This study was undertaken to characterize the HMW MMP activity in OA SF.

Methods

MMP activity in OA SF was determined by gelatin zymography. Recombinant MMPs were used to identify MMP activity on the zymogram. Western immunoblotting, immunoprecipitation, and immunodepletion analyses were performed using antibodies specific for human MMP‐9 and human neutrophil gelatinase–associated lipocalin (NGAL). Human cartilage matrix degradation was determined by dimethylmethylene blue assay.

Results

Zymographic analysis showed that the HMW gelatinase in OA SF comigrated with a purified NGAL–MMP‐9 complex. Results of Western immunoblotting showed that the HMW gelatinase was also recognized by antibodies specific for human NGAL or human MMP‐9. These same antibodies also immunoprecipitated the HMW gelatinase activity from OA SF. The NGAL–MMP‐9 complex was reconstituted in vitro in gelatinase buffer. In the presence of NGAL, MMP‐9 activity was stabilized; in the absence of NGAL, rapid loss of MMP‐9 activity occurred. MMP‐9–mediated release of cartilage matrix proteoglycans was significantly higher in the presence of NGAL (P < 0.05).

Conclusion

Our findings demonstrate that the HMW gelatinase activity in OA SF represents a complex of NGAL and MMP‐9. The ability of NGAL to protect MMP‐9 activity is relevant to cartilage matrix degradation in OA and may represent an important mechanism by which NGAL may contribute to the loss of cartilage matrix proteins in OA.

     

Sites of collagenase cleavage and denaturation of type II collagen in aging and osteoarthritic articular cartilage and their relationship to the distribution of matrix metalloproteinase 1 and matrix metalloproteinase 13

OBJECTIVE: To determine the sites of cleavage and denaturation of type II collagen (CII) by collagenase(s) in healthy and osteoarthritic (OA) human articular cartilage and their relationship to the distribution of matrix metalloproteinase 1 (MMP-1) and MMP-13. METHODS: Single (per subject) full-depth specimens from femoral condylar cartilage were isolated from articulating surfaces at autopsy from 8 subjects without arthritis and during arthroplasty from 10 patients with OA. Fixed frozen sections of cartilage were examined by immunoperoxidase localization, using antibodies to the collagenase-generated cleavage site in CII, to an intrachain epitope recognized only in denatured CII, and to MMP-1 and MMP-13 (proenzyme, activated enzyme, or enzyme/inhibitor complex). RESULTS: Staining for collagen cleavage, denaturation, and both MMPs was weak to moderate and was frequently observed in pericellular sites in cartilage from younger, nonarthritic subjects. In specimens from older subjects, this staining was often more widespread and of greater intensity. Similar staining was usually, but not always, seen for all antibodies. In OA cartilage, staining was often stronger and more intense than that in normal cartilage from older subjects, and the distribution of staining was often similar for the different antibodies. Pericellular staining in the deep zone was frequently more pronounced in arthritic cartilage and extended to territorial and sometimes interterritorial sites. In very degenerate specimens, staining was distributed throughout most of the cartilage matrix. CONCLUSION: These observations provide evidence for the presence of limited cleavage and denaturation of CII restricted to mainly pericellular and superficial sites in cartilage from younger, healthy subjects, where MMP-1 and MMP-13 are also selectively localized. Collagen degradation is more extensive and often more pronounced in cartilage from older, nonarthritic subjects. Characteristic changes in early OA are similar to those seen with aging in cartilage from older, healthy subjects, with collagen damage and collagenases concentrated closer to the articular surface. There was usually a close correspondence between the cleavage and denaturation of CII and the sites at which these collagenases were detected, suggesting that both MMPs are involved in the physiology and pathology. There was no evidence that the damage to CII is ordinarily initiated in sites other than at and near the articular surface and around chondrocytes.     

Mechanisms and kinetics of glycosaminoglycan release following in vitro cartilage injury

OBJECTIVE: Acute joint injury leads to increased risk for osteoarthritis (OA). Although the mechanisms underlying this progression are unclear, early structural, metabolic, and compositional indicators of OA have been reproduced using in vitro models of cartilage injury. This study was undertaken to determine whether glycosaminoglycan (GAG) loss following in vitro cartilage injury is mediated by cellular biosynthesis, activation of enzymatic activity, or mechanical disruption of the cartilage extracellular matrix. METHODS: Immature bovine cartilage was cultured for up to 10 days. After 3 days, groups of samples were subjected to injurious mechanical compression (single uniaxial unconfined compression to 50% thickness, strain rate 100% per second). GAG release to the medium was measured, and levels were compared with those in location-matched, uninjured controls. The effects of medium supplementation with inhibitors of biosynthesis (cycloheximide), of matrix metalloproteinase (MMP) activity (CGS 27023A or GM 6001), and of aggrecanase activity (SB 703704) on GAG release after injury were assessed. RESULTS: GAG release from injured cartilage was highest during the first 4 hours after injury, but remained higher than that in controls during the first 24 hours postinjury, and was not affected by inhibitors of biosynthesis or degradative enzymes. GAG release during the period 24-72 hours postinjury was similar to that in uninjured controls, but the MMP inhibitor CGS 27023A reduced cumulative GAG loss from injured samples between 1 day and 7 days postinjury. Other inhibitors of enzymatic degradation or biosynthesis had no significant effect on GAG release. CONCLUSION: Injurious compression of articular cartilage induces an initially high rate of GAG release from the tissue, which could not be inhibited, consistent with mechanical damage. However, the finding that MMP inhibition reduced GAG loss in the days following injury suggests a potential therapeutic intervention.     

Pathologic indicators of degradation and inflammation in human osteoarthritic cartilage are abrogated by exposure to n-3 fatty acids

OBJECTIVE: To determine if n-3 polyunsaturated fatty acid (PUFA) supplementation (versus treatment with n-6 polyunsaturated or other fatty acid supplements) affects the metabolism of osteoarthritic (OA) cartilage. METHODS: The metabolic profile of human OA cartilage was determined at the time of harvest and after 24-hour exposure to n-3 PUFAs or other classes of fatty acids, followed by explant culture for 4 days in the presence or absence of interleukin-1 (IL-1). Parameters measured were glycosaminoglycan release, aggrecanase and matrix metalloproteinase (MMP) activity, and the levels of expression of messenger RNA (mRNA) for mediators of inflammation, aggrecanases, MMPs, and their natural tissue inhibitors (tissue inhibitors of metalloproteinases [TIMPs]). RESULTS: Supplementation with n-3 PUFA (but not other fatty acids) reduced, in a dose-dependent manner, the endogenous and IL-1-induced release of proteoglycan metabolites from articular cartilage explants and specifically abolished endogenous aggrecanase and collagenase proteolytic activity. Similarly, expression of mRNA for ADAMTS-4, MMP-13, and MMP-3 (but not TIMP-1, -2, or -3) was also specifically abolished with n-3 PUFA supplementation. In addition, n-3 PUFA supplementation abolished the expression of mRNA for mediators of inflammation (cyclooxygenase 2, 5-lipoxygenase, 5-lipoxygenase-activating protein, tumor necrosis factor alpha, IL-1alpha, and IL-1beta) without affecting the expression of message for several other proteins involved in normal tissue homeostasis. CONCLUSION: These studies show that the pathologic indicators manifested in human OA cartilage can be significantly altered by exposure of the cartilage to n-3 PUFA, but not to other classes of fatty acids.