Cartilage Extracts Research Articles

Proteomic characterization of mouse cartilage degradation in vitro

To develop proteomics to analyze mouse cartilage degradation and correlate transcriptional and translational responses to catabolic stimuli. Proteomic techniques were used to analyze catabolism in mouse femoral head cartilage. Using specific methods to prepare cartilage extracts and conditioned media for 2-dimensional polyacrylamide gel electrophoresis and subsequent tandem mass spectrometry, we identified novel proteins and fragments released into the media of control, interleukin-1alpha (IL-1alpha)-treated, and all-trans-retinoic acid (RetA)-treated explants. Fluorescence 2-dimensional difference gel electrophoresis was used to quantify protein expression changes. We also measured changes in messenger RNA (mRNA) expression to distinguish transcriptional and posttranslational regulation of released proteins. Differentially abundant proteins in the media of control and treated explants included fragments of thrombospondin 1 and connective tissue growth factor. IL-1alpha stimulated release of the cartilage degeneration marker matrix metalloproteinase 3, as well as proteins with uncharacterized roles in cartilage pathology, such as neutrophil gelatinase-associated lipocalin. RetA stimulated release of the extracellular matrix proteins cartilage oligomeric matrix protein, link protein, and matrilin-3 into the media, which was accompanied by a dramatic reduction in the corresponding mRNA transcript levels. Gelsolin, which has been implicated in cytoskeletal reorganization in arthritis synovial fibroblasts but has not been previously associated with cartilage pathology, was regulated by IL-1alpha and RetA. In this first analysis of mouse cartilage degradation and protein release using proteomics, we identified proteins and fragments, some of which represent novel candidate biomarkers for cartilage degradation. Applying these proteomic techniques to wild-type and genetically modified mouse cartilage will provide insights into the mechanisms of cartilage degeneration.

202 DYSREGULATION OF GLUCOSE TRANSPORT AND GLUT-1 PROTEIN IN OSTEOARTHRITIC CHONDROCYTES IN RESPONSE TO HIGH EXTRACELLULAR GLUCOSE

Methods: Opticin was extracted from normal and OA human cartilage using guanidinium chloride (4M) extraction and examined by Western blotting. Human MMP 1, -2, -3, -7, -8, -9 and ADAMTS-4, -5 proteolytic activities on opticin were analyzed on normal and OA cartilage extracts incubated for 0−24 hours Results: One of the emerging observations was that all MMPs and ADAMTS tested, except MMP-9, cleaved opticin in both normal and OA specimens. Interestingly, MMP-7 and -8 were the most effective, and degradation occurred as early as 15 minutes of incubation. At which time, degradation was almost complete for MMP-7, suggesting that this molecule is a preferential substrate for this enzyme. In contrast, MMP-1 was the least effective and degradation was noted after an incubation of 16 hours. The others enzymes showed degradation starting at about 2 hours. The degradation pattern, except for MMP-7 in which degradation appeared independent of the specimen types, was different between normal and OA. In general, the degradation pattern was slower in normal than in OA specimens, reflecting an increased sensitivity of opticin to degradation when the cartilage was damaged. Fragments were also detected, but the number and size differed depending on the protease. This indicates different digestion sites among the proteases. Surprisingly, MMP-1 and -2 generated different opticin degradation fragments when normal were compared to OA specimens. The different opticin protease cleavage sites are currently under study. Conclusions: We showed, for the first time, the ability of different MMP and ADAMTS to degrade opticin. As opticin may contribute to the structural stability of cartilage matrix, its cleavage may predispose cartilage to degradation. In vivo identification of the cleavage products may assist in an early detection of OA cartilage matrix degeneration.

475 PROTEOMIC CHARACTERIZATION OF MOUSE CARTILAGE DEGRADATION IN VITRO

Purpose: To develop proteomics to analyze mouse cartilage degradation and correlate transcriptional and translational responses to catabolic stimuli. Methods: Proteomic techniques were used to analyze catabolism in mouse femoral head cartilage. Using specific methods to prepare cartilage extracts and conditioned media for two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and subsequent tandem mass spectrometry (MS), we identified novel proteins and fragments released into the media of control, interleukin-1α (IL-1) and all-trans-retinoic acid (RetA)-treated explants. Fluorescent difference gel electrophoresis (2-D DIGE) was used to quantify protein expression changes. We also measured changes in mRNA expression to distinguish transcriptional and post-translational regulation of released proteins. Results: We identified 20 differentially-abundant proteins in media from control and treated explants, including fragments of thrombospondin-1 and connective tissue growth factor. IL-1 stimulated release of the cartilage degeneration marker MMP-3, as well as proteins with uncharacterized roles in cartilage pathology, such as neutrophil gelatinaseassociated lipocalin. RetA stimulated release of extracellular matrix proteins COMP, link protein and matrilin-3 into the media, accompanied by dramatic reduction in corresponding mRNA transcripts levels. Gelsolin, implicated in cytoskeletal reorganization in arthritic synovial fibroblasts but not previously associated with cartilage pathology, was regulated by IL-1 and RetA. Conclusions: This first analysis of mouse cartilage degradation and protein release using proteomics has identified proteins and fragments, some of which represent novel candidate biomarkers for cartilage degradation. Applying these proteomic techniques to wild-type and genetically-modified mouse cartilage will provide insights into mechanisms of cartilage degeneration.

Immunolocalisation and expression of keratocan in tendon

Short Communication Immunolocalisation and expression of keratocan in tendon S. G. Rees Ph.D.y*, A. D. Waggett Ph.D.z, B. C. Kerr Ph.D.z, J. Probert B.Sc.z, E. C. Gealy Ph.D.z, C. M. Dent M.D.x, B. Caterson Ph.D.z and C. E. Hughes Ph.D.z ySchool of Medicine, Swansea University, Singleton Park, Swansea, SA2 8PP, UK zConnective Tissue Biology Laboratories, Cardiff School of Biosciences, Cardiff University, Museum Avenue, Cardiff, CF10 3US, UK xSchool of Medicine, Department of Orthopaedic and Trauma Surgery, Cardiff University, Llandough Hospital, Penlan Road, Penarth, Vale of Glamorgan, CF64 2XX, UK

Effects of triamcinolone acetonide, sodium hyaluronate, amikacin sulfate, and mepivacaine hydrochloride, alone and in combination, on morphology and matrix composition of lipopolysaccharide-challenged and unchallenged equine articular cartilage explants

To evaluate the effects of triamcinolone acetonide (TA), sodium hyaluronate (HA), amikacin sulfate (AS), and mepivacaine hydrochloride (MC) on articular cartilage morphology and matrix composition in lipopolysaccharide (LPS)-challenged and unchallenged equine articular cartilage explants. 96 articular cartilage explants from 4 femoropatellar joints of 2 adult horses. Articular cartilage explants were challenged with LPS (100 ng/mL) or unchallenged for 48 hours, then treated with TA, HA, AS, and MC alone or in combination for 96 hours or left untreated. Cartilage extracts were analyzed for glycosaminoglycan (GAG) content by dimethyl-methylene blue assay (ng/mg of dry wt). Histomorphometric quantification of total lacunae, empty lacunae, and lacunae with pyknotic nuclei was recorded for superficial, middle, and deep cartilage zones. LPS induced a significant increase in pyknotic nuclei and empty lacunae. Treatment with TA or HA significantly decreased empty lacunae (TA and HA), compared with groups without TA or HA, and significantly decreased empty lacunae of LPS-challenged explants, compared with untreated explants. Treatment with AS or MC significantly increased empty lacunae in unchallenged explants, and these effects were attenuated by TA. Treatment with MC significantly increased empty lacunae and pyknotic nuclei and, in combination with LPS, could not be attenuated by TA. Content of GAG did not differ between unchallenged and LPS-challenged explants or among treatments. Treatment with TA or HA supported chondrocyte morphology in culture and protected chondrocytes from toxic effects exerted by LPS, AS, and MC.

Cleavage of Type II Collagen by Cathepsin K in Human Osteoarthritic Cartilage

Cathepsin K is a cysteine protease of the papain family that cleaves triple-helical type II collagen, the major structural component of the extracellular matrix of articular cartilage. In osteoarthritis (OA), the anabolic/catabolic balance of articular cartilage is disrupted with the excessive cleavage of collagen II by collagenases or matrix metalloproteinases. A polyclonal antibody against a C-terminal neoepitope (C2K) generated in triple-helical type II collagen by the proteolytic action of cathepsin K was prepared and used to develop an enzyme-linked immunosorbent assay to study the generation of this epitope and the effects of its presence in normal adult and osteoarthritic femoral condylar articular cartilage. The generation of the C2K epitope in explant culture and the effect of a specific cathepsin K inhibitor were studied. The neoepitope, which is not generated by the collagenase matrix metalloproteinase-13, increased with age in articular cartilage and was significantly elevated in osteoarthritic cartilage compared with adult nonarthritic cartilage. Moreover, in explants from three of eight OA patients, the generation of the neoepitope in culture was significantly reduced by a specific, nontoxic inhibitor of cathepsin K. These data suggest that cathepsin K is involved in the cleavage of type II collagen in human articular cartilage in certain OA patients and that it may play a role in both OA pathophysiology and the aging process.

The type II collagen fragments Helix-II and CTX-II reveal different enzymatic pathways of human cartilage collagen degradation

Cartilage degradation in osteoarthritis (OA) generates the type II collagen fragments, Helix-II and CTX-II that can be used as clinical biological markers. Helix-II and C-telopeptide of type II collagen (CTX-II) levels are associated independently with progression of OA suggesting that they may be generated through different collagenolytic pathways. In this study we analyzed the release of Helix-II and CTX-II from human cartilage collagen by the proteinases reported to play a role in cartilage degradation. In vitro, human articular cartilage extract was incubated with activated human recombinant cathepsins (Cats) and matrix-metalloproteases (MMPs). Next, we analyzed the spontaneous release of Helix-II and CTX-II from cartilage sections of patients with knee OA who were immediately deep frozen after joint replacement to preserve endogenous enzyme activity until assay. Cartilage sections were then incubated for up to 84 h in the presence or absence of E-64 and GM6001, inhibitors of cysteine proteases and MMPs, respectively. In vitro, Cats K, L and S generated large amount of Helix-II, but not CTX-II. Cat B generated CTX-II fragment, but destroyed Helix-II immunoreactivity. Cat D was unable to digest intact cartilage. MMPs-1, -3, -7, -9, and -13 efficiently released CTX-II, but only small amount of Helix-II. Neither CTX-II nor Helix-II alone was able to reflect accurately the collagenolytic activity of Cats and MMPs as reflected by the release of hydroxyproline. In OA cartilage explants, E-64 blunted the release of Helix-II whereas the release of CTX-II could be completely abrogated by GM6001 and only partly by E-64. These in vitro and ex vivo experiments of human cartilage suggest that Helix-II and CTX-II could be released in part by different enzymatic pathways. Helix-II and CTX-II alone reflect only partially overall cartilage collagen degradation. These findings may explain why these two biological markers could provide complementary information on disease progression in OA.

Collagen type VI content in healthy and arthritis knee joint cartilage

Since collagen type VI seems to play an important role in cartilage metabolism and is increased in osteoarthritis, the aim of this study was to investigate whether different stages of osteoarthritis can be characterised by the content of collagen type VI in human knee cartilage. Collagen type VI was investigated in 148 histologically normal and 117 osteoarthritic cartilage samples from 18 different localisations of human knee joints. It was quantified in cartilage extracts using an inhibition-ELISA. In normal cartilage the average content of collagen type VI was 0.48 per cent of total collagens. Interestingly, there was a high variability not only in osteoarthritic, but also in normal cartilage. The statistical analysis showed significant differences between normal femoral, tibial or retropatellar cartilage samples. Therefore, normal and osteoarthritic samples from different localizations had to be compared separately. A significant increase of collagen type VI was already found in early osteoarthritic lesions. Inspite of a statistically significant increase of collagen type VI in osteoarthritic cartilage, the range of concentrations found in normal and osteoarthritic samples overlap. In view of the high interindividual variability, collagen type VI is not very precise in the diagnosis of early osteoarthritic lesions if used as the only marker.

Activation of Transforming Growth Factor β(TGFβ)by interaction of Matrix Metalloproteinase 13 (MMP‐13) with the large latent complex

Dysregulated TGFβ activation has been indicated in the pathology of osteoarthritis. We have reported that hypertrophic chondrocytes produce large quantities of activated TGFβ and a large latent complex of TGFβ that contains MMP‐13 in non‐covalent association. Bioinformatics revealed that the hemopexin domains of MMP‐13 can non‐covalently interact with the calcium‐EGF‐like domains of Latent TGF Binding Protein (LTBP1), the extracellular matrix anchor protein for the TGFβ large latent complex. The catalytic domain of MMP‐13 is in alignment with the protease‐sensitive hinge region of LTBP1, the site of release of this molecule from the extracellular matrix, suggesting a novel mechanism of TGFβ large latent complex release. To test this proposed interaction, we designed peptides of the MMP‐13 hemopexin domain that are most likely to interact with LTBP1. Tissue extract of avian hypertrophic cartilage bound five times more MMP‐13 peptide than extracts from resting zone cartilage. In addition, rat tibial growth plate cartilage immunoprecipitated with antibody to LTBP1 demonstrated four times more binding affinity for MMP‐13 peptide than the scrambled peptide control. These data support the specific interaction between MMP‐13 and LTBP1 predicted in our bioinformatics model and our proposed model of the mechanism of release of the TGFβ large latent complex by MMP‐13. Sponsored in part by a grant from the CCDA.

Cover Picture: Proteomics – Clinical Applications 2/2008

AbstractCover Illustration: The application of proteomics to investigate cartilage function and disease. Mouse cartilage structures throughout development and in disease are depicted. The articular surface, meniscal fibrocartilage and underlying bone at 3 weeks are stained with haematoxilin/eosin (upper left panel). The high magnification confocal micrograph is stained to highlight collagen VI, a marker for the pericellular matrix (upper right panel). The neonatal shoulder joint is stained with toluidine blue to illustrate the dense proteoglycan‐rich extracellular matrix (lower left panel). Proteoglycan is lost from the articular cartilage in a mouse model of arthritis (central panels). Resolution of cartilage extracts and cellular proteins by 2‐D electrophoresis (lower right panel). For further details see Wilson, R., Bateman, J. F., Proteomics Clin. Appl. 2008, 2, 251–263 (in this issue).

Dynamic activation of bone morphogenetic protein signaling in collagen-induced arthritis supports their role in joint homeostasis and disease

IntroductionRheumatoid arthritis is a chronic systemic autoimmune disease affecting peripheral joints and leading to loss of joint function. The severity and outcome of disease are dependent on the balance between inflammatory/destructive and homeostatic or repair pathways. Increasing evidence suggests a role for bone morphogenetic protein (BMP) signaling in joint homeostasis and disease.MethodsActivation of BMP signaling in collagen-induced arthritis as a model of rheumatoid arthritis was studied by immunohistochemistry and Western blot for phosphorylated SMAD1/5 at different time points. Expression of different BMP ligands and noggin, a BMP antagonist, was determined on synovium and cartilage extracts of arthritic knees, at different time points, with quantitative polymerase chain reaction. At the protein level, BMP2 and BMP7 were studied with immunohistochemistry. Finally, the effect of anti-tumor necrosis factor-alpha (TNFα) treatment on the expression of BMP2, BMP7, and growth and differentiation factor-5 (GDF5) in synovium and cartilage of arthritic knees was investigated.ResultsA time-dependent activation of the BMP signaling pathway in collagen-induced arthritis was demonstrated with a dynamic and characteristic expression pattern of different BMP subfamily members in synovium and cartilage of arthritic knees. As severity increases, the activation of BMP signaling becomes more prominent in the invasive pannus tissue. BMP2 is present in cartilage and the hyperplastic lining layer. BMP7 is found in the sublining zone and inflammatory infiltrate. Treatment with etanercept slowed down progression of disease, but no change in expression of GDF5, BMP2, and BMP7 in synovium was found; in the cartilage, however, blocking of TNFα increased the expression of BMP7.ConclusionsBMP signaling is dynamically activated in collagen-induced arthritis and is partly TNFα-independent. TNFα blocking increased the expression of BMP7 in the articular cartilage, possibly enhancing anabolic mechanisms. Different types of source and target cells are recognized. These data further support a role for BMP signaling in arthritis.

Prognostic Factors of Metastatic Renal Cell Carcinoma After Failure of Immunotherapy: New Paradigm From a Large Phase III Trial With Shark Cartilage Extract AE 941

We analyzed prognostic factors, described survival and generated a prognostic model in patients with metastatic renal cell carcinoma in whom immunotherapy failed and who were potentially eligible for novel agents. An analysis of the relationship between clinical features and survival was performed in 300 patients with advanced renal cell carcinoma in whom immunotherapy had failed and who were subsequently treated as part of a single, phase III clinical trial with the anti-angiogenic agent Neovastat (shark cartilage extract AE 941). Clinical features were first examined univariately and a stepwise modeling approach based on Cox proportional hazard regression was then performed to generate a multivariate model. Median and progression-free survival (prognostic factors) for the whole cohort was 12.6 and 2 months, respectively. Prognostic features associated with shorter survival on multivariate analysis were the number of metastatic sites (greater than 1), time from nephrectomy to metastatic disease (less than 2 years), high alkaline phosphatase, abnormal corrected serum Ca and high lactate dehydrogenase (greater than 1.5 x the upper limit of normal). Four prognostic subgroups were identified by counting the number of adverse prognostic factors. Median survival in patients with zero adverse prognostic factors was 15.6 months compared to 11.7 months in patients with 1, 8.5 months in patients with 2 and 3.5 months in patients with 3 or more. We identified 4 risk groups to predict survival in previously treated patients with renal cell carcinoma. This model was based on data from what is to our knowledge the largest experience in this population. It should be used in clinical trial design, risk stratification and patient counseling.

Requirement for protein kinase R in interleukin-1α-stimulated effects in cartilage

Interleukin-1 (IL-1) has pleiotropic effects in cartilage. The interferon-induced, double stranded RNA-activated protein kinase PKR that phosphorylates eukaryotic initiation factor 2 (eIF2) α has been implicated in cytokine effects in chondrocytes. A compound was recently identified that potently suppresses PKR autophosphorylation (IC50 approximately 200 ηM) and partially restores PKR-inhibited translation in a cell-free system with significant effect in the nanomolar range. The objectives of this study were to exploit this potent PKR inhibitor to assess whether PKR kinase activity is required for catabolic and proinflammatory effects of IL-1α in cartilage and to determine whether IL-1α causes an increase in eIF2α phosphorylation that is antagonized by the PKR inhibitor. Cartilage explants were incubated with the PKR inhibitor and IL-1α. Culture media were assessed for sulfated glycosaminoglycan as an indicator of proteoglycan degradation and for prostaglandin E 2. Cartilage extracts were analyzed by Western blot for cyclooxygenase-2 and phosphorylated signaling molecules. Nanomolar concentrations of the PKR inhibitor suppressed proteoglycan degradation and cyclooxygenase-2 accumulation in IL-1α-activated cartilage. The PKR inhibitor stimulated or inhibited PGE 2 production with a biphasic dose response relationship. IL-1α increased the phosphorylation of both PKR and eIF2α, and nanomolar concentrations of PKR inhibitor suppressed the IL-1α-induced changes in phosphorylation. The results strongly support PKR involvement in pathways activated by IL-1α in chondrocytes.

Chondromodulin-I and Tenomodulin: The Negative Control of Angiogenesis in Connective Tissue

The negative regulation of angiogenesis may provide a promising therapeutic target for a number of lifestyle-related diseases, as the switch to an angiogenic phenotype in many tissues represents a critical step during the progression of such disorders. Cartilage is avascular and shows resistance to vascular invasion from the surrounding well-vascularized mesenchyme. Using guanidine extracts of fetal bovine cartilage, we have identified and purified chondromodulin-I (ChM-I) as an angiogenesis inhibitor. The cDNA sequence of this factor has revealed that the ChM-I precursor protein is a type II transmembrane glycoprotein (334 amino acids) and that mature ChM-I is encoded in the C-terminal region of the precursor. After cleavage of the ChM-I precursor at its processing site, mature ChM-I (120 amino acids) is secreted from chondrocytes into the extracellular matrix. Following on from the identification of ChM-I as an angiogenesis inhibitor in cartilage, we have also cloned both mouse and human tenomodulin (TeM), which share significant homology with ChM-I at their C-termini. Moreover, exogenous expression experiments in COS cells suggests that TeM is a type II transmembrane glycoprotein (317 amino acids). When overexpressed in HUVECs, the C-terminal domain (116 amino acids) of the TeM protein shows both anti-angiogenic and anti-tumorigenic activities at equivalent levels to mature ChM-I. In our present review, we discuss the structure, biological activities and localization of these anti-angiogenic molecules.

A phase III study of Æ-941 with induction chemotherapy (IC) and concomitant chemoradiotherapy (CRT) for stage III non- small cell lung cancer (NSCLC) (NCI T99–0046, RTOG 02–70, MDA 99–303)

7527 Background: Æ-941 is a shark cartilage extract with antiangiogenic properties. We conducted a placebo-controlled trial testing Æ-941, with IC and CRT, in unresectable stage III NSCLC. Methods: Eligibility criteria included performance status (PS) < 2, weight loss < 10%. Subjects received one of two treatment regimens depending on site of enrollment: carboplatin (C) (AUC 6) and paclitaxel (P) (200 mg/m2) × 2 cycles followed by CRT (60 Gy/30 fractions) with weekly C (AUC 2) and P (45 mg/m2) × 6 doses or cisplatin (CDDP) (75 mg/m2, d1) and vinorelbine (V) (30 mg/m2, d1 and 8) × 2 cycles followed by CRT (60 Gy/30 fractions) with CDDP (75 mg/m2, day 1) and V (15 mg/m2, d1 and 8) × 2 cycles. Subjects were randomized to receive Æ-941 (Arm A) or placebo (Arm B), 120 mL orally twice daily, at the start of IC and continuing after CRT as maintenance therapy. Randomization was stratified for stage, gender, and type of chemotherapy. The primary endpoint was overall survival (OS), with a planned sample size of 756 subjects providing 80% power to detect a 25% difference in OS, assuming a control arm median survival time (MST) of 13 months, type I error 0.05. Results: Between 6/00 and 2/06, 384 subjects were enrolled onto the trial and randomized. In 2/06 the trial was closed to new patient entry due to insufficient accrual. This final analysis is based on 379 randomized and eligible subjects (188 arm A, 191 arm B). Subject characteristics: 60% male, median age 63 years (range 37–84), 56% stage IIIB, 58% C-based chemotherapy, median follow-up 3.7 years. There was no significant difference in OS between arms A and B, with MSTs of 14.4 (95% CI 12.6–17.9) and 15.6 (95% CI 13.8–18.1) months, respectively (log-rank p=0.73). OS by pre-specified stratification factors: stage IIIB vs IIIA (MST 13.9 vs. 17.4 months, p=0.25), C vs. CDDP chemotherapy (MST 14.4 vs. 16.7 months, p=0.13), and male vs. female (MST 15.7 vs. 15.1 months, p=0.74). The study drug was well tolerated. Fewer subjects in arm A experienced grade 3 or higher adverse events (66% vs. 77%, p=0.018). Conclusions: The addition of Æ−941 to IC and CRT does not improve OS in patients with unresectable stage III NSCLC. No significant financial relationships to disclose.

Assessment of cartilage degradation effects of matrix metalloproteinase-13 in equine cartilage cocultured with synoviocytes

To determine the effects of matrix metalloproteinase (MMP)-13, compared with interleukin (IL)-1alpha, on cartilage matrix molecule gene expression in a coculture system of equine cartilage explants and synoviocytes. Articular cartilage and synovium specimens harvested from femoropatellar joints of 4 horses, aged 3 to 5 years. Synoviocytes were isolated and cocultured with cartilage explants. Cultures were treated with human recombinant MMP-13 (1, 25, or 100 ng/mL) or IL-1alpha (0.01, 0.1, 1.0, or 10 ng/mL) for 96 hours, with medium exchange at 48 hours. Cartilage extracts and media were analyzed for glycosaminoglycan (GAG) content, and results were adjusted to cartilage DNA content. Quantitative PCR was performed on mRNA from cartilage (MMP-3, MMP-13, aggrecan, and collagen type IIB [COL2A1]) and synoviocytes (MMP-3 and MMP-13), and results were adjusted to 18S ribosomal subunit mRNA expression. Treatments were performed in triplicate, and the experiment was repeated 4 times. Cultures treated with MMP-13 or IL-1alpha had increased media GAG concentration at 48 and 96 hours. Aggrecan and COL2A1 mRNA expression were increased by application of MMP-13 or IL-1alpha. Gene expression of the catabolic mediator, MMP-3, in cartilage and synoviocytes was increased in cultures treated with MMP-13 or IL-1alpha. Expression of MMP-13 mRNA in cartilage was increased by IL-1alpha, but decreased in synoviocytes by MMP-13 treatment. Results support the use of recombinant MMP-13 in a coculture system of synoviocytes and cartilage explants for the study of osteoarthritis.

Aggrecanolysis in human osteoarthritis: confocal localization and biochemical characterization of ADAMTS5–hyaluronan complexes in articular cartilages

Human osteoarthritis (OA) is characterized by aggrecanase-mediated depletion of cartilage aggrecan. We have examined the abundance, location and some biochemical properties of the six known aggrecanases (A disintegrin and metalloproteinase with thrombospondin-like motifs 1 (ADAMTS1) 4, 5, 8, 9 and 15) in normal and OA human cartilages. Formalin-fixed, ethylenediamine tetraacetic acid (EDTA)-decalcified sections of full-depth cartilage from human OA tibial plateaus and normal control samples were studied by confocal imaging. Probes included specific antibodies to aggrecanases and two aggrecan epitopes, as well as biotinylated hyaluronan binding protein (HABP) for hyaluronan (HA) visualization. Cartilage extracts were analyzed by Western blot for the individual proteinases and aggrecan fragments. ADAMTS5 was present in association with cells throughout normal cartilage and was markedly increased in OA, particularly in clonal groups in the superficial and transitional zones, where it was predominantly co-localized with HA. Consistent with the confocal analysis, a high molecular weight complex of ADAMTS5 and HA was isolated from human OA cartilage by isotonic salt extraction and chromatography on Superose 6. The complex eluted with an apparent molecular size of about 2x10(6) and contained major ADAMTS5 forms of 150, 60, 40 and 30kDa. The yield of most forms on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was markedly enhanced by prior digestion of the complex with either Streptomyces hyaluronidase or chondroitinase ABC. ADAMTS5 abundance and distribution in human OA cartilages is consistent with a central role for this enzyme in destructive aggrecanolysis. HA-dependent sequestration of ADAMTS5 in the pericellular matrix may be a mechanism for regulating the activity of this proteinase in human OA cartilage.

A poster of this work was presented at the 23rd IFSCC Congress 2004, Orlando, Florida, USA. Supplementation with nutritional cartilage extract positively influences skin hydration, skin barrier and skin structure: a double blind, randomized, placebo‐controlled study

The aim of the study was to evaluate the efficacy of polysaccharides from fish cartilage with regard to their skin aging properties. An application test was carried out during the intake of cartilage tablets as a nutritional supplement. The study was designed as a monocentric, double‐blind, randomized, placebo‐controlled application test. 28 healthy female volunteers (35–60 years) with dry skin were included in the study. They were divided into two groups. Group 1 received tablets containing placebo and group 2 the verum treatment (cartilage hydrolysate combined with vitamin C). The duration of the study was 12 weeks. The focus of interest was to find out about the hydration properties, and to see whether the skin barrier and structure were influenced by the test formulation compared with placebo. Hydration measurements were made before and during the study, and the transepidermal water loss (barrierer function of the skin) was measured. The thickness and density of the skin of all volunteers were determined by means of ultrasound measurements during the study. Statistical analysis was based on Wilcoxon signed rank test. The following results were obtained in this study: there was a significant improvement in the hydration properties, a significant decrease in transepidermal water loss and a significant increase in the skin density in the verum group (cartilage extract). No or minor improvements could be detected in the placebo group.

ADAMTS-5 Deficiency Does Not Block Aggrecanolysis at Preferred Cleavage Sites in the Chondroitin Sulfate-rich Region of Aggrecan

In the mouse, proteolysis in the aggrecan interglobular domain is driven by ADAMTS-5, and mice deficient in ADAMTS-5 catalytic activity are protected against aggrecan loss and cartilage damage in experimental models of arthritis. Here we show that despite ablation of ADAMTS-5 activity, aggrecanolysis can still occur at two preferred sites in the chondroitin sulfate-rich region. Retinoic acid was more effective than interleukin-1alpha (IL) in promoting cleavage at these sites in ADAMTS-5-deficient cartilage. These results suggest that cleavage at preferred sites in the chondroitin sulfate-rich region is mediated by ADAMTS-4 or an aggrecanase other than ADAMTS-5. Following retinoic acid or IL-1alpha stimulation of cartilage explants, aggrecan fragments in medium and extracts contained SELE(1279) or FREEE(1467) C-terminal sequences. Some SELE(1279) and FREEE(1467) fragments were retained in the cartilage, with intact G1 domains. Other SELE(1279) fragments were released into the medium and co-migrated with the (374)ALGS neoepitope, indicating they were aggrecanase-derived fragments. In contrast none of the FREEE(1467) fragments released into the medium co-migrated with the (374)ALGS neoepitope, suggesting that, despite their size, these fragments were not products of aggrecanase cleavage in the interglobular domain. ADAMTS-5, but not ADAMTS-1, -4, or -9, was up-regulated 8-fold by retinoic acid and 17-fold by IL-1alpha treatment. The data show that whereas ADAMTS-5 is entirely responsible for cleavage in the interglobular domain, cleavage in the chondroitin sulfate-rich region is driven either by ADAMTS-4, which compensates for loss of ADAMTS-5 in this experimental system, or possibly by another aggrecanase. The data show that there are differential aggrecanase activities with preferences for separate regions of the core protein.

G1-G2 Aggrecan Product that can be Generated by M-calpain on Truncation at Ala709-Ala710 is Present Abundantly in Human Articular Cartilage

To elucidate the specific function of m-calpain in the metabolism of aggrecan in human articular cartilage, the prevalence and localization of a large glycosaminoglycan-bearing aggrecan product generated by m-calpain in human osteoarthritis (OA) cartilage were investigated. Extracts of human OA articular cartilage were analysed by immunostaining using new polyclonal anti-VPGVA antiserum that detects the COOH terminal neoepitope IVTQVVPGVA(709) generated by m-calpain-related cleavage within the keratan sulphate rich region of human aggrecan. Immunoblotting analyses of aggrecan populations in guanidine hydrochloride-extracts showed that OA cartilages contained anti-VPGVA positive aggrecan products with the COOH terminal neoepitope ... VPGVA(709), resulting from truncation between the Ala(709)-Ala(710) m-calpain-related cleavage site. This aggrecan product consisted of two NH(2) terminal globular domain (G1 and G2) and KS side chains. Immunohistochemical staining showed that anti-VPGVA positive staining was localized within chondrocytes and spread to the surrounding interterritorial matrix. Confocal microscopic analysis showed subcellular colocalization of anti-VPGVA and anti m-calpain. These results indicate that the aggrecan product with the COOH terminal neoepitope VPGVA(709) is synthesized regularly by intracellular processing in chondrocytes, and is present abundantly as a limited form of aggrecan. M-calpain is the major candidate of the proteinase to generate this aggrecan product during the intracellular aggrecan processing.