Interleukin-1alpha treatment of meniscal explants stimulates the production and release of aggrecanase-generated, GAG-substituted aggrecan products and also the release of pre-formed, aggrecanase-generated G1 and m-calpain-generated G1-G2.

Pro-inflammatory cytokines induce meniscal matrix degradation and inhibition of endogenous repair mechanisms, but the pathogenic mechanisms behind this are mostly unknown. Therefore, we investigated details of interleukin-1 (IL-1alpha)-induced aggrecan turnover in mature meniscal tissue explants. Fibro-cartilagenous disks (3 mm diameter x 1 mm thickness) were isolated from the central, weight-bearing region of menisci from 2-year-old cattle. After 3 or 6 days of IL-1alpha-treatment, GAG loss (DMMB assay), biosynthetic activity ([(35)SO(4)]-sulfate and [(3)H]-proline incorporation), gene expression (quantitative RT-PCR) and the abundance (zymography, Western blot) of matrix-degrading enzymes and specific aggrecan products were determined. Meniscal fibrocartilage had a 4-fold lower GAG content (per wet weight) than adjacent articular cartilage, and expressed MMPs-1, -2, -3 and ADAMTS4 constitutively, whereas ADAMTS5 m-RNA was essentially undetectable. Significant IL-1 effects were a decrease in biosynthetic activity, an increase in GAG release and in the expression/abundance of MMP-2, MMP-3 and ADAMTS4. Fresh tissue contained aggrecan core protein products similar to those previously described for bovine articular cartilage of this age. IL-1 induced the release of aggrecanase-generated CS-substituted products including both high (>250 kDa) and low molecular weight (about 75 kDa) species. TIMP-3 (but not TIMP-1 and -2 or a broad spectrum MMP inhibitor) inhibited IL-1-dependent GAG loss. In addition, IL-1 induced the release of preformed pools of three known G1-bearing products. We conclude that aggrecanases are responsible for IL-1-stimulated GAG release from meniscal explants, and that IL-1 also stimulates release of G1-bearing products, by a process possibly involving hyaluronan fragmentation.

Tumor necrosis factor alpha-dependent aggrecan cleavage and release of glycosaminoglycans in the meniscus is mediated by nitrous oxide-independent aggrecanase activity in vitro.

INTRODUCTION: Little is known about factors that induce meniscus damage. Since joint inflammation appears to be a causative factor for meniscal destruction, we investigated the influence of tumor necrosis factor (TNFalpha) on glycosaminoglycan (GAG) release and aggrecan cleavage in an in vitro model. METHODS: Meniscal explant disks (3 mm diameter x 1 mm thickness) were isolated from 2-year-old cattle. After 3 days of TNFalpha-treatment GAG release (DMMB assay), biosynthetic activity (sulfate incorporation), nitric oxide (NO) production (Griess assay), gene expression of matrix-degrading enzymes (quantitative RT-PCR, zymography), and immunostaining of the aggrecan fragment NITEGE were determined. RESULTS: TNFalpha induced release of GAG as well as production of NO in a dose-dependent manner, while sulfate incorporation was decreased. TNFalpha increased matrix metalloproteinase (MMP)-3 and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 mRNA expression, whereas collagen type I was decreased, and aggrecan, collagen type II as well as MMP-1, -2, -13 and ADAMTS-5 were variably affected. Zymography also showed a TNFalpha-dependent increase in MMP-3 expression, but pre-dominantly in the pro-form. TNFalpha-dependent formation of the aggrecanase-specific aggrecan neoepitope NITEGE was induced. Tissue inhibitor of metalloproteinases (TIMP)-3, but not TIMP-1 or -2 inhibited TNFalpha-dependent GAG release and NITEGE production, whereas inhibition of TNFalpha-dependent NO generation with the NO-synthetase inhibitor L-NMMA failed to inhibit GAG release and NITEGE production. CONCLUSIONS: Our study shows that aggrecanase activity (a) is responsible for early TNFalpha-dependent aggrecan cleavage and GAG release in the meniscus and (b) might be involved in meniscal degeneration. Additionally, the meniscus is a TNFalpha-dependent source for MMP-3. However, the TNFalpha-dependent NO production seems not to be involved in release of proteoglycans under the given circumstances.

The influence of biomechanical parameters on the expression of VEGF and endostatin in the bone and joint system.

Many degenerative processes in the skeletal system are induced by mechanical overload. Osteoarthritis and spontaneous tendon ruptures are two examples of mechanically influenced diseases. Incubator-housed compression apparatuses and cyclic strain chambers are adequate models to investigate the cellular processes. Recent studies have shown that growth factors are involved in the transduction pathways of mechanical overload leading to tissue degradation. Vascular endothelial growth factor (VEGF) is a dimerized, 45 kDa peptide that normally attracts endothelial cells in wound healing. VEGF can be detected in the superficial zone of the tibial plateau in osteoarthritic (OA) patients with degenerative changes but not in healthy articular cartilage. Blood vessels are only rarely observed in OA cartilage suggesting that there are other roles for VEGF in cartilage. VEGF is also detectable in ruptured but not in normal tendons. The mechanically induced expression of VEGF in avascular tissues like articular cartilage or fibrocartilage of contact areas from gliding tendons initiates degenerative processes. Chondrocytes from OA cartilage also express the VEGF receptor 2. In vitro assays have shown that VEGF binds the VEGFR-2 leading to a phosphorylation of MAP kinases (ERK1/2) with subsequent transcription factor accumulation (activator protein 1 = AP-1). One of the antagonists of VEGF is endostatin. Endostatin, a fragment of collagen type XVIII, is expressed in avascular tissues and has the potency to decrease VEGF induced effects (ERK1/2 phosphorylation). The increase in matrix metalloproteinase (MMP) production and the decrease in tissue inhibitor metalloproteinase (TIMP) synthesis is a result of the signal transduction cascade activation. MMPs participate in the degradation processes of osteoarthritis whereas TIMPs are inhibitors of the MMPs. Taken together mechanically induced VEGF is involved in the destruction and endostatin in the maintenance of avascular tissues of the bone and joint system.

Pathomechanisms of cartilage destruction by mechanical injury.

Mechanical injury is considered to be a major inductor of articular cartilage destruction and therefore a risk factor for the development of secondary osteoarthritis. Mechanical injury induces damage to the tissue matrix directly or mediated by chondrocytes via expression of matrix-degrading enzymes and reduction of biosynthetic activity. As a consequence the mechanical properties of cartilage change. Some of the pathomechanisms of mechanical injury have already been uncovered by the use of a broad range of in vitro-models. They demonstrate that mechanical injury induces tissue swelling and decrease in both the compressive and shear stiffness of articular cartilage, probably due to disruption of the collagen network. Injurious compression induces chondrocyte death by necrosis and apoptosis and the remaining cells decrease their biosynthetic activity. The tissue content of proteoglycans also decreases with time in injured cartilage, and the tissue loses its ability to respond to physiological levels of mechanical stimulation with an increase in biosynthesis. Immature cartilage seems to be more vulnerable to injurious compression than more mature tissue. The expression of several matrix-degrading enzymes like ADAM-TS5 and matrix-metalloproteinases (MMP-1, MMP-2, MMP-3, MMP-9, MMP-13) is increased after injury and may in part be regulated by an autocrine vascular endothelial growth factor (VEGF)-dependent signalling pathway. Apoptosis seems to be mediated by caspase activity and reactive oxygen species. For that reason activation of antioxidative defense mechanisms as well as the inhibition of angiogenetic factors and MMPs might be key regulators in the mechanically induced destruction of cartilage and might be suggested as potential therapeutic interventions. This review summarizes some of the most important data from in vitro injury studies dealing with the pathomechanisms of cartilage destruction.

Influence of tissue maturation and antioxidants on the apoptotic response of articular cartilage after injurious compression.

OBJECTIVE: To study the influence of tissue maturation and antioxidants on apoptosis in bovine articular cartilage induced by injurious compression. METHODS: Bovine articular cartilage disks were obtained from the femoropatellar groove of animals ages 0.5-23 months and placed in culture. Cartilage disks were preincubated overnight with the cell-permeable superoxide dismutase (SOD) mimetic Mn(III) porphyrin (0-12.5 microM) or alpha-tocopherol (0-50 microM) and then injured by a single unconfined compression to a final strain of 50% at a velocity of 1 mm/second. After 4 days of additional incubation, the disks were fixed and embedded for light and electron microscopy. Apoptotic cells were quantified morphologically by the appearance of nuclear blebbing on light microscopy. Biosynthetic activity was demonstrated by incorporation of radiolabeled proline. The antioxidative action of the SOD mimetic was confirmed by histologic examination of cartilage after incubation with nitroblue tetrazolium. RESULTS: Injurious compression induced significantly more apoptosis in cartilage disks from newborn calves (22% of cells) than in cartilage from more mature cows (2-6%). In cartilage from 22-month-old animals, the SOD mimetic reduced the percentage of apoptotic cells induced by injury in a dose-dependent manner (complete inhibition with 2.5 microM), while alpha-tocopherol had no effect. Neither antioxidant altered protein biosynthesis or cellular ultrastructure. CONCLUSION: Our data suggest that the apoptotic response of articular cartilage to mechanical injury is affected by maturation and is mediated in part by reactive oxygen species. The antioxidative status of the tissue might be important for the prevention of mechanically induced cell death in articular cartilage.

Mechanical overload induces VEGF in cartilage discs via hypoxia-inducible factor.

VEGF (vascular endothelial growth factor) is not only one of the most important angiogenesis factors, but is involved also in inflammatory processes. Recent studies have shown that VEGF as well as its receptor VEGFR-2 are expressed on osteoarthritic chondrocytes, but not on normal adult chondrocytes. Since mechanical overload is one of the causative factors for osteoarthritis, we studied its effect on VEGF expression on bovine cartilage disks that were compressed once with a strain of 50% and a strain rate of 1/second. Under these conditions, control disks (without pressure) were completely negative for VEGF expression as evidenced by immunocytochemical stainings as well as by enzyme-linked immunosorbent assay (ELISA) measurements. In contrast, 4 days after mechanical overload, the cartilage disks were positive in both detection methods. In addition, after mechanical overload chondrocytes were strongly immunopositive for hypoxia-inducible factor-1alpha (HIF-1alpha), the limiting protein of the dimeric transcription factor HIF-1 that is known to induce VEGF expression. Furthermore, the matrix metalloproteases MMP-1, MMP-3, and MMP-13, could be easily detected in pressure-treated disks by immunohistochemistry whereas staining in controls was low or undetectable. The tissue inhibitors of metalloproteinases (TIMP-1 and -2) could be detected in controls but not in samples treated with mechanical overload. To prove that increased MMP or decreased TIMP expression could be a result of the autocrine action of VEGF on chondrocytes, we repeated the experiments in the presence of a specific inhibitor for the kinase activity of the VEGFR-2. This inhibitor was effective to reduce mechanically induced MMP-1, -3, and -13 immunostaining and to restore TIMP expression. Taking together, these findings indicate that VEGF is induced in chondrocytes by mechanical overload and mediates destructive processes in osteoarthritis as an autocrine factor.