Depletion Of Aggrecan Research Articles

Reduced tissue osmolarity increases TRPV4 expression and pro-inflammatory cytokines in intervertebral disc cells.

The mechanical behaviour and cellular metabolism of intervertebral discs (IVDs) and articular cartilage are strongly influenced by their proteoglycan content and associated osmotic properties. This osmotic environment is a biophysical signal that changes with disease and may contribute to the elevated matrix breakdown and altered biologic response to loading observed in IVD degeneration and osteoarthritis. This study tested the hypothesis that changes in osmo-sensation by the transient receptor potential vallinoid-4 (TRPV4) ion channel occur with disease and contribute to the inflammatory environment found during degeneration. Immunohistochemistry on bovine IVDs from an inflammatory organ culture model were used to investigate if TRPV4 is expressed in the IVD and how expression changes with degeneration. Western blot, live-cell calcium imaging, and qRT-PCR were used to investigate whether osmolarity changes or tumour necrosis factor α (TNFα) regulate TRPV4 expression, and how altered TRPV4 expression influences calcium signalling and pro-inflammatory cytokine expression. TRPV4 expression correlated with TNFα expression, and was increased when cultured in reduced medium osmolarity and unaltered with TNFα-stimulation. Increased TRPV4 expression increased the calcium flux following TRPV4 activation and increased interleukin-1β (IL-1β) and IL-6 gene expression in IVD cells. TRPV4 expression was qualitatively elevated in regions of aggrecan depletion in degenerated human IVDs. Collectively, results suggest that reduced tissue osmolarity, likely following proteoglycan degradation, can increase TRPV4 signalling and enhance pro-inflammatory cytokine production, suggesting changes in TRPV4 mediated osmo-sensation may contribute to the progressive matrix breakdown in disease.

The role of aggrecan in normal and osteoarthritic cartilage.

Aggrecan is a large proteoglycan bearing numerous chondroitin sulfate and keratan sulfate chains that endow articular cartilage with its ability to withstand compressive loads. It is present in the extracellular matrix in the form of proteoglycan aggregates, in which many aggrecan molecules interact with hyaluronan and a link protein stabilizes each interaction. Aggrecan structure is not constant throughout life, but changes due to both synthetic and degradative events. Changes due to synthesis alter the structure of the chondroitin sulfate and keratan sulfate chains, whereas those due to degradation cause cleavage of all components of the aggregate. These latter changes can be viewed as being detrimental to cartilage function and are enhanced in osteoarthritic cartilage, resulting in aggrecan depletion and predisposing to cartilage erosion. Matrix metalloproteinases and aggrecanases play a major role in aggrecan degradation and their production is upregulated by mediators associated with joint inflammation and overloading. The presence of increased levels of aggrecan fragments in synovial fluid has been used as a marker of ongoing cartilage destruction in osteoarthritis. During the early stages of osteoarthritis it may be possible to retard the destructive process by enhancing the production of aggrecan and inhibiting its degradation. Aggrecan production also plays a central role in cartilage repair techniques involving stem cell or chondrocyte implantation into lesions. Thus aggrecan participates in both the demise and survival of articular cartilage.

SP0019 AGG-523: Aggrecanase-selective inhibitor for the treatment of osteoarthritis

Osteoarthritis (OA) is the most common form of arthritis affecting nearly 27 million people in the United States. OA is caused by the loss of articular cartilage due to increased...

THU0042 The development and characterisation of competition elisa for measurement of active ADAMTS-4 in serum and synovial fluid

BackgroundThe depletion of aggrecan in articular cartilage is an early event in the pathogenesis of Osteoarthritis and Rheumatoid arthritis. Both ADAMTS-4 and ADAMTS-5 are believed to play important roles in...

Regulation of aggrecanases from the ADAMTS family and aggrecan neoepitope formation during in vitro chondrogenesis of human mesenchymal stem cells

Aggrecanases from the ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin motifs) family are important therapeutic targets due to their essential role in aggrecan depletion in arthritic diseases. Whether their function is also important for matrix rearrangements during chondrogenesis and thus, cartilage regeneration, is however so far unknown. The aim of this study was to analyse the expression and function of ADAMTS with aggrecanase activity during chondrogenic differentiation of human mesenchymal stem cells (MSCs). Chondrogenic differentiation was induced in bone marrow-derived MSC pellets and expression of COL2A1, aggrecan, ADAMTS1, 4, 5, 9, 16 and furin was followed by quantitative RT-PCR. Formation of the NITEGE (ADAMTS-cleaved) and DIPEN (MMP-cleaved) aggrecan neoepitopes was detected by immunohistochemistry. While the expression of ADAMTS4, 9, 16 and furin was up-regulated during chondrogenesis, ADAMTS1 and 5 were down-regulated. Despite this regulation of ADAMTS, no formation of NITEGE neoepitopes occurred in MSC pellets, indicating no ADAMTS-induced cleavage of aggrecan. In contrast, MMP-induced cleavage of aggrecan appeared at 14 d after induction of chondrogenesis. Submission of differentiated MSC pellets to IL1β treatment for 3 d resulted in strong upregulation of ADAMTS1, 4 and 5, rapid proteoglycan depletion, and stimulation of ADAMTS-induced but not MMP-induced cleavage of aggrecan. Thus, there is no evidence for ADAMTS-induced aggrecan cleavage during chondrogenesis, but proteoglycan turnover is rapidly inducible under inflammatory signals. Therapeutic aggrecanase inhibition for treatment of arthritic disease may thus not impede regenerative self-healing pathways based on chondrogenesis of local progenitor cells in the joint.

Matrix metalloproteinase 13–deficient mice are resistant to osteoarthritic cartilage erosion but not chondrocyte hypertrophy or osteophyte development

To investigate the role of matrix metalloproteinase 13 (MMP-13; collagenase 3) in osteoarthritis (OA). OA was surgically induced in the knees of MMP-13-knockout mice and wild-type mice, and mice were compared. Histologic scoring of femoral and tibial cartilage aggrecan loss (0-3 scale), erosion (0-7 scale), and chondrocyte hypertrophy (0-1 scale), as well as osteophyte size (0-3 scale) and maturity (0-3 scale) was performed. Serial sections were stained for type X collagen and the MMP-generated aggrecan neoepitope DIPEN. Following surgery, aggrecan loss and cartilage erosion were more severe in the tibia than femur (P<0.01) and tibial cartilage erosion increased with time (P<0.05) in wild-type mice. Cartilaginous osteophytes were present at 4 weeks and underwent ossification, with size and maturity increasing by 8 weeks (P<0.01). There was no difference between genotypes in aggrecan loss or cartilage erosion at 4 weeks. There was less tibial cartilage erosion in knockout mice than in wild-type mice at 8 weeks (P<0.02). Cartilaginous osteophytes were larger in knockout mice at 4 weeks (P<0.01), but by 8 weeks osteophyte maturity and size were no different from those in wild-type mice. Articular chondrocyte hypertrophy with positive type X collagen and DIPEN staining occurred in both wild-type and knockout mouse joints. Our findings indicate that structural cartilage damage in a mouse model of OA is dependent on MMP-13 activity. Chondrocyte hypertrophy is not regulated by MMP-13 activity in this model and does not in itself lead to cartilage erosion. MMP-13 deficiency can inhibit cartilage erosion in the presence of aggrecan depletion, supporting the potential for therapeutic intervention in established OA with MMP-13 inhibitors.

Aggrecanase and Aggrecan Degradation in Osteoarthritis: A Review

Aggrecanase-mediated aggrecan degradation is a significant event in early-stage osteoarthritis (OA). Aggrecanases belonging to the 'A Disintegrin And Metalloproteinase with ThromboSpondin motifs' (ADAMTS) family of proteinases play a significant role in aggrecan depletion in osteoarthritic cartilage. There has been considerable interest in the possible role of these aggrecanases, especially ADAMTS-4 and ADAMTS-5, as therapeutic targets in OA. This article discusses recent data regarding ADAMTS-4 and ADAMTS-5 in OA, with emphasis on the relationship between aggrecanase and aggrecan degradation as well as the role of aggrecanase in OA.

Intra-articular injection of tumor necrosis factor-α in the rat: an acute and reversible in vivo model of cartilage proteoglycan degradation

To develop an in vivo model for rapid assessment of cartilage aggrecan degradation and its pharmacological modulation. Tumor necrosis factor-alpha (TNFalpha) was injected intra-articularly (IA) in rat knees and aggrecan degradation was monitored at various times following challenge. Articular cartilage was assessed for aggrecan content by Safranin O staining and by immunohistochemistry for the NITEGE epitope. Synovial fluids (SFs) were analyzed for sulfated glycosaminoglycans (GAGs) using the dimethylmethylene blue dye assay and for aggrecan fragments generated by specific cleavage at aggrecanase-sensitive sites by Western blot analysis with neoepitope antibodies. Indomethacin, dexamethasone, and an aggrecanase inhibitor were evaluated for their ability to modulate TNFalpha-induced proteoglycan degradation in vivo. (1) IA injection of TNFalpha in the knee joint of rats resulted in transient aggrecan degradation and release of aggrecanase-generated aggrecan fragments from the articular cartilage into the SF; (2) a correlation was observed between histologically assessed depletion of aggrecan from the articular cartilage and the appearance of specific neoepitopes in the SF; (3) aggrecan degradation was inhibited by an aggrecanase inhibitor as well as by dexamethasone, but not by the non-steroidal anti-inflammatory drug (NSAID), indomethacin. TNFalpha injection in the knee joints of rats results in rapid transient cartilage proteoglycan degradation, mediated by cleavage at the aggrecanase sites. Biomarker read-out of specific neoepitopes in the SF enables the use of this mechanism-based model for rapid evaluation of aggrecanase-mediated aggrecan degradation in vivo.

Patients with rheumatoid arthritis have an altered circulatory aggrecan profile

BackgroundRheumatoid arthritis (RA) is a chronic auto-immune disease with extensive articular cartilage destruction. Aggrecan depletion, mediated by aggrecanases is one of the first signs of early cartilage erosion. We investigated, whether measurement of aggrecan and fragments thereof in serum, could be used as biomarkers for joint-disease in RA patients and furthermore characterized the fragments found in the circulation.MethodsThe study consisted of 38 patients, 12 males (62.2 ± 16.0 years) and 26 females (59.8 ± 20.7 years) diagnosed with RA: 41.5 ± 27.5 mm/h erythrocyte sedimentation rate (ESR), 38.4 ± 34.7 mg/ml C-reactive protein (CRP) and 4.8 ± 1.7 disease activity score (DAS) and 108 healthy age-matched controls. Aggrecan levels were measured using two immunoassays, i.e. the 374ARGSVI-G2 sandwich ELISA measuring aggrecanase-mediated aggrecan degradation and the G1/G2 sandwich assay, detecting aggrecan molecules containing G1 and/or G2 (total aggrecan) We further characterized serum samples by western blots, by using monoclonal antibodies F-78, binding to G1 and G2, or by BC-3, detecting the aggrecanase-generated N-terminal 374ARGSVI neo-epitope.ResultsTotal aggrecan levels in RA patients were significantly decreased from 824.8 ± 31 ng/ml in healthy controls to 570.5 ± 30 ng/ml (31% decrease, P < 0.0001), as measured by the G1/G2 ELISA. Western blot analysis with F-78 showed one strong band at 10 kDa, and weaker bands at 25 and 45 kDa in both healthy controls and RA patients. In contrast, staining for aggrecanase-activity revealed only one strong band in RA patients of 45 kDa.ConclusionThis is the first study, which characterizes different aggrecan fragments in human serum. The data strongly suggests that total aggrecan levels, i.e. aggrecan molecules containing G1 and/or G2 are lower in RA patients, and that RA patients have at least one specific subpopulation of aggrecan fragments, namely aggrecanse generated 374ARGSVI fragments. Further clinical studies are needed to investigate the potential of G1/G2 as a structure-related biochemical marker in destructive joint-diseases.

Aggrecan Protects Cartilage Collagen from Proteolytic Cleavage

The matrix components responsible for cartilage mechanical properties, type II collagen and aggrecan, are degraded in osteoarthritis through proteolytic cleavage by matrix metalloproteinases (MMPs) and aggrecanases, respectively. We now show that aggrecan may serve to protect cartilage collagen from degradation. Although collagen in freeze-thawed cartilage depleted of aggrecan was completely degraded following incubation with MMP-1, collagen in cartilage with intact aggrecan was not. Using interleukin-1-stimulated bovine nasal cartilage explants where aggrecan depletion occurs during the first week of culture, followed by collagen loss during the second week, we evaluated the effect of selective MMP and aggrecanase inhibitors on degradation. A selective MMP inhibitor did not block aggrecan degradation but caused complete inhibition of collagen breakdown. Similar inhibition was seen with inhibitor addition following aggrecan depletion on day 6-8, suggesting that MMPs are not causing significant collagen degradation prior to the second week of culture. Inclusion of a selective aggrecanase inhibitor blocked aggrecan degradation, and, in addition, inhibited collagen degradation. When the inhibitor was introduced following aggrecan depletion, it had no effect on collagen breakdown, ruling out a direct effect through inhibition of collagenase. These data suggest that aggrecan plays a protective role in preventing degradation of collagen fibrils, and that an aggrecanase inhibitor may impart overall cartilage protection.

1H magnetic resonance spectroscopy of nanomelic chicken cartilage: effect of aggrecan depletion on cartilage T 2

Objective: To determine the effect of proteoglycan depletion on cartilage proton magnetic resonance (MR) spectroscopy T 2using nanomelic chicken cartilage, a genetic mutant that completely lacks aggrecan. Design: Proton MR spectroscopic T 2measurements of normal embryonic and nanomelic femoral epiphyseal cartilage were obtained using a 96-echo pulse sequence with inter-echo delay times increased logarithmically over the TE period of 60 μs to 1.7 s. The relative intensity and distribution of cartilage T 2components were determined by fitting signal decay curves to a multi-exponential function. The number of T 2components in the signal decay curves was determined by the degree of freedom limited r 2of the fit. Results: For normal fetal chicken cartilage, 97.6±0.2% (mean±95% confidence interval) of the total signal comprises a long T 2component (179.1±1.3 ms) with a relatively small short T 2component (0.5±0.4 ms). The T 2distribution for nanomelic cartilage is more heterogeneous with four components identified: two short T 2components (0.5±0.02 and 7.3±0.6 ms), a large intermediate component (56.4±5.6 ms), and a broadly distributed long component (137.5±16.6 ms). In nanomelic cartilage there is greater heterogeneity of cartilage T 2indicating greater variation in water proton mobility and exchange of water with the extracellular matrix. Conclusion: Absence of aggrecan in the extracellular cartilage matrix produces greater heterogeneity in cartilage T 2, but will not increase T 2as has been previously reported with degenerative change of the collagen matrix.

Stimulation of hyaluronan metabolism by interleukin‐1α in human articular cartilage

To determine the effects of interleukin-1alpha (IL-1alpha) on the expression of hyaluronan synthase (HAS), CD44, and aggrecan in human articular chondrocytes, and to assess the net result of these metabolic changes on the accumulation of hyaluronan within articular cartilage.Normal human articular cartilage slices, as well as isolated chondrocytes, were treated with IL-1alpha. Changes in the relative expression of messenger RNA (mRNA) for HAS-2, CD44, and aggrecan were determined by competitive, quantitative reverse transcriptase-polymerase chain reaction. Hyaluronan accumulation was characterized by staining with a hyaluronan-specific binding protein and by fluorophore-assisted carbohydrate electrophoresis, while proteoglycan content was determined by alcian blue and Safranin O staining, CD44 protein expression by immunohistochemistry, and aggrecan biosynthesis by 35S-sulfate incorporation. Changes in cell-associated matrix sizes were visualized by a particle exclusion assay.IL-1alpha stimulated the expression of HAS-2 and CD44 mRNA (3.5-fold and 3-fold, respectively), but inhibited the expression of aggrecan mRNA. In IL-1-treated chondrocytes, extracellular hyaluronan decreased, while intracellular accumulation of hyaluronan was enhanced. Together with the decrease in expression of aggrecan, a dramatic reduction in cell-associated matrix was observed. IL-1-treated cartilage slices displayed a prominent depletion of aggrecan as well as hyaluronan within the upper layers of the tissue. The regional loss of hyaluronan coincided with a regional up-regulation of CD44.These data demonstrate that IL-1alpha stimulates HAS-2 at the same time as it inhibits the expression of aggrecan. Although hyaluronan biosynthesis is up-regulated, so too is the expression of CD44 and the internalization/catabolism of hyaluronan. The net result is a loss of hyaluronan in areas of the articular cartilage where increases in CD44 expression are most prominent. This depletion of hyaluronan in the upper layers of the tissue likely facilitates the prominent loss of aggrecan from the tissue.

Stimulation of hyaluronan metabolism by interleukin-1alpha in human articular cartilage.

To determine the effects of interleukin-1alpha (IL-1alpha) on the expression of hyaluronan synthase (HAS), CD44, and aggrecan in human articular chondrocytes, and to assess the net result of these metabolic changes on the accumulation of hyaluronan within articular cartilage. Normal human articular cartilage slices, as well as isolated chondrocytes, were treated with IL-1alpha. Changes in the relative expression of messenger RNA (mRNA) for HAS-2, CD44, and aggrecan were determined by competitive, quantitative reverse transcriptase-polymerase chain reaction. Hyaluronan accumulation was characterized by staining with a hyaluronan-specific binding protein and by fluorophore-assisted carbohydrate electrophoresis, while proteoglycan content was determined by alcian blue and Safranin O staining, CD44 protein expression by immunohistochemistry, and aggrecan biosynthesis by 35S-sulfate incorporation. Changes in cell-associated matrix sizes were visualized by a particle exclusion assay. IL-1alpha stimulated the expression of HAS-2 and CD44 mRNA (3.5-fold and 3-fold, respectively), but inhibited the expression of aggrecan mRNA. In IL-1-treated chondrocytes, extracellular hyaluronan decreased, while intracellular accumulation of hyaluronan was enhanced. Together with the decrease in expression of aggrecan, a dramatic reduction in cell-associated matrix was observed. IL-1-treated cartilage slices displayed a prominent depletion of aggrecan as well as hyaluronan within the upper layers of the tissue. The regional loss of hyaluronan coincided with a regional up-regulation of CD44. These data demonstrate that IL-1alpha stimulates HAS-2 at the same time as it inhibits the expression of aggrecan. Although hyaluronan biosynthesis is up-regulated, so too is the expression of CD44 and the internalization/catabolism of hyaluronan. The net result is a loss of hyaluronan in areas of the articular cartilage where increases in CD44 expression are most prominent. This depletion of hyaluronan in the upper layers of the tissue likely facilitates the prominent loss of aggrecan from the tissue.

Topographical Variation in the Catabolism of Aggrecan in an Ovine Annular Lesion Model of Experimental Disc Degeneration

An established model of experimental disc degeneration (Osti et al., Spine 15:762, 1990; Melrose et al., J Orthop Res 10:655, 1992) was used in this study. Four 2-year-old sheep received anterolateral incisions (4 x 10 mm) in the outer one-third of the annulus fibrosus of their L2-L3 and L4-L5 discs (lesion group). The annulus was not incised in another four sham-operated animals. After 6 months the sheep were killed, lumbar discs were dissected into lateral halves of the annulus fibrosus and the nucleus pulposus. Cells were isolated from disc tissues enzymatically and were grown in alginate bead culture to examine the proteoglycan metabolism of cells from lesion and control zones. The media of lesion zone cultures contained relatively high levels (compared with sham cultures) of catabolic fragments of the large, high-buoyant-density proteoglycans as demonstrated by Western blotting using monoclonal antibodies (5-D-4, 3-B-3, 1-C-6) and biotinylated hyaluronan and also by gel chromatography. Furthermore, cells from the vicinity of the lesion site also synthesized significantly lower levels (compared with sham cultures) of aggrecan that was retained within the alginate beads. Collectively, these data indicated that focal depletion of large, high-buoyant-density proteoglycans was evident within lesion sites in this model of experimental disc degeneration. The introduction of an annular lesion therefore significantly affected the proteoglycan metabolism of endogenous disc cell populations. The unique hydrodynamic and viscoelastic properties of the intervertebral disc are dependent to a large degree on the tissue levels of aggrecan. The focal depletion of aggrecan by annular lesions therefore may represent an important predisposing factor to the subsequent degeneration of these intervertebral discs.

Transforming Growth Factor-β Stimulates Retinoic Acid-Induced Proteoglycan Depletion in Intact Articular Cartilage

Cartilage-bearing sesamoid bones from the metacarpophalangeal joints of adult cows were cultured with retinoic acid for 1 week and allowed to recover in control medium for another 2 weeks. Retinoic acid decreased the proteoglycan synthesis of the cartilage to 33% of control values, and induced 26% loss of proteoglycans from the matrix. During recovery, the synthesis of proteoglycans returned to the control level but their content remained reduced. Transforming growth factor-β (TGF-β1, 5 ng/ml) was added to the culture medium to stimulate the recovery. However, TGF-β depressed the synthesis of proteoglycans and increased their loss to 61%. Only the large aggregating species, aggrecan, was lost from the matrix. The half-life of proteoglycans synthesized during recovery in control medium was 12.7 days, which was reduced to 8.7 days by TGF-β. The proteoglycan half-life in control cartilage cultured without retinoic acid or TGF-β was 33.8 days. Neither retinoic acid nor TGF-β-induced changes in the hyaluronate content of the tissue. Aggrecans and small proteoglycans synthesized in the presence of TGF-β were larger than those in controls. The synthesis of the small proteoglycans was stimulated 4.5-fold by TGF-β, and their content was increased. The results show that TGF-β can stimulate depletion of aggrecan in retinoic acid-treated cartilage. This indicates a catabolic function of TGF-β in cartilage remodeling.