ADAMTS-4 activity in synovial fluid as a biomarker of inflammation and effusion.

OBJECTIVE: To evaluate the potential of ADAMTS-4 (aggrecanase -1) activity in synovial fluid (SF) as a biomarker of knee injury and joint disease. DESIGN: We have measured ADAMTS-4 activity in the synovial fluid of 170 orthopaedic patients with different degrees of joint pathology, using a commercial ADAMTS-4 fluorescence resonance energy transfer (FRET) substrate assay. Patients were classified at arthroscopy as (i) macroscopically normal, (ii) with an injury of the meniscus, anterior cruciate ligament or chondral/osteochondral defects or (iii) with osteoarthritis, and the influence of independent factors (age, patient group, effusion and synovial inflammation) on ADAMTS-4 activity levels was assessed. RESULTS: In most patients (106/170) ADAMTS-4 activity was undetectable; ADAMTS-4 ranged from 0 to 2.8 ng/mL in synovial fluid from patients with an injury, 0-4.1 ng/mL in osteoarthritic patients and 4.0-12.3 ng/mL in patients with large effusions. Four independent variables each significantly influenced ADAMTS-4 activity in synovial fluid (all P < 0.001): age (concordance = 0.69), presence of osteoarthritis (OA) (concordance = 0.66), level of effusion (concordance = 0.78) and inflammation (concordance = 0.68). Not only did effusion influence the amount of ADAMTS-4 activity most strongly, but it also did this in an ordered manner (P < 0.001). CONCLUSIONS: The main finding of this study is that ADAMTS-4 levels in synovial fluid are most strongly correlated with inflammation and severity of effusion in the knee. Further study is required to determine if it could provide a useful tool to aid clinical diagnoses, indicate treatment, to monitor progression of joint degeneration or OA or alternatively the success of treatment.

Development of a novel clinical biomarker assay to detect and quantify aggrecanase-generated aggrecan fragments in human synovial fluid, serum and urine.

OBJECTIVE: Proteolytic degradation of aggrecan in articular cartilage is a hallmark feature of osteoarthritis (OA). The present study was aimed at developing a sensitive enzyme linked immunosorbent assay (ELISA) for the detection of aggrecanase-cleaved fragments of aggrecan in human serum and urine to facilitate the clinical development of aggrecanase inhibitors for OA. METHODS: The BC3 monoclonal antibody that detects the ARGS neoepitope sequence in aggrecanase-cleaved aggrecan was engineered and optimized using complementarity determining region (CDR)-saturation mutagenesis to improve its binding affinity to the neoepitope. A sandwich ELISA (BC3-C2 ELISA) was developed using the optimized alpha-ARGS antibody (BC3-C2) as capture antibody and a commercially available antibody directed against the hyaluronic-acid binding region (HABR) of aggrecan as detection antibody. Aggrecanase-cleaved fragments of aggrecan present in in vitro digests, human cartilage explant culture supernatants and in human synovial fluid, serum and urine were detected and quantified using this ELISA. RESULTS: The optimized antibody had a 4-log improvement in affinity for the ARGS containing peptide compared to the parental BC3 antibody, while maintaining the ability to not cross-react with a spanning peptide. The BC3-C2 ELISA demonstrated the ability to detect aggrecanase-cleaved aggrecan fragments in the native state, without the need for deglycosylation. This ELISA was able to measure aggrecanase-generated ARGS containing aggrecan fragments in human articular cartilage (HAC) explant cultures in the basal state (without cytokine stimulation). Treatment with an aggrecanase inhibitor resulted in a dose-dependent inhibition of ARGS neoepitope released into the culture supernatant. The ELISA assay also enabled the detection of ARGS containing fragments in human synovial fluid, serum and urine, suggesting its potential utility as a biomarker of aggrecanase activity. CONCLUSIONS: We have developed a novel ELISA using an optimized ARGS antibody and have demonstrated for the first time, an ELISA-based measurement of aggrecan degradation products in human serum and urine. This assay has the potential to serve as a mechanistic drug activity biomarker in the clinic and is expected to significantly impact/accelerate the clinical development of aggrecanase inhibitors and other disease modifying drugs for OA.

Hyaluronan inhibits expression of ADAMTS4 (aggrecanase-1) in human osteoarthritic chondrocytes.

BACKGROUND: Intra-articular injection of hyaluronan (HA) has been suggested to have a disease-modifying effect in osteoarthritis, but little is known about the possible mechanisms. OBJECTIVE: To investigate the effects of HA species of different molecular mass, including 800 kDa (HA800) and 2700 kDa (HA2700), on the expression of aggrecanases (ie, ADAMTS species), which play a key role in aggrecan degradation. METHODS: The effects of HA species on the expression of ADAMTS1, 4, 5, 8, 9 and 15 in interleukin 1alpha (IL1alpha)-stimulated osteoarthritic chondrocytes were studied by reverse transcription PCR and real-time PCR. Expression of ADAMTS4 protein and aggrecanase activity and signal transduction pathways of IL1, CD44 and intracellular adhesion molecule 1 (ICAM1) were examined by immunoblotting. RESULTS: IL1alpha treatment of chondrocytes induced ADAMTS4, and HA800 and HA2700 significantly decreased IL1alpha-induced expression of ADAMTS4 mRNA and protein. IL1alpha-stimulated aggrecanase activity in osteoarthritic chondrocytes was reduced by treatment with HA2700 or transfection of small interfering RNA for ADAMTS4. A similar result was obtained when HA2700 was added to explant cultures of osteoarthritic cartilage. HA2700 neither directly inhibited nor bound to ADAMTS4. Downregulation of ADAMTS4 expression by HA2700 was attenuated by treatment of IL1alpha-treated chondrocytes with antibodies to CD44 and/or ICAM1. The increased phosphorylation of IL1 receptor-associated kinase-1 and extracellular signal-regulated protein kinase1/2 induced by the IL1alpha treatment was downregulated by enhanced IRAK-M expression after HA2700 treatment. CONCLUSION: These data suggest that HA2700 suppresses aggrecan degradation by downregulating IL1alpha-induced ADAMTS4 expression through the CD44 and ICAM1 signalling pathways in osteoarthritic chondrocytes.

Natural chondroitin sulphates increase aggregation of proteoglycan complexes and decrease ADAMTS-5 expression in interleukin 1 beta-treated chondrocytes.

OBJECTIVE: To assess the effect of natural chondroitin sulphate (CS) on the ability of neosynthesized sulphated proteoglycans (PGs) to aggregate in cultured chondrocytes treated with interleukin (IL)1 beta. METHODS: Primary cultured rabbit articular chondrocytes were treated or not with IL1 beta alone or with concentrations of CS for 20 h. Neosynthesized PGs were labelled by incorporation of [35SO(4)]-sulphate and analysed by chromatography on Sepharose 2B columns. Gelatinolytic activity was measured by zymography, and matrix metalloproteinase (MMP)1 mRNA level in chondrocytes underwent real-time PCR. Expression of ADAMTS (for "a disintegrin and metalloproteinase with thrombospondin motifs") -4 and -5 was analysed by real-time PCR and western blotting. RESULTS: The production of [35SO(4)]-labelled PGs was significantly increased with 10 microg/ml CS in the cellular pool rather than in the incubation medium. The addition of CS to IL1 beta-treated cells inhibited in part the disaggregation of sulphated PGs induced by IL1 beta. This inhibitory effect of CS is associated with a significant decrease in ADAMTS-5 expression at the mRNA and protein levels. No effect of CS was observed on IL1 beta-induced gelatinolytic activity, MMP1 mRNA expression or ADAMTS-4 expression. CONCLUSION: CS increases the production of functional sulphated PGs in the direct environment of chondrocytes in vitro. This beneficial effect of CS in IL1 beta-treated cells is associated with decreased expression of ADAMTS-5.

Discordant localization of WFA reactivity and brevican/ADAMTS-derived fragment in rodent brain.

BACKGROUND: Proteoglycan (PG) in the extracellular matrix (ECM) of the central nervous system (CNS) may act as a barrier for neurite elongation in a growth tract, and regulate other characteristics collectively defined as structural neural plasticity. Proteolytic cleavage of PGs appears to alter the environment to one favoring plasticity and growth. Brevican belongs to the lectican family of aggregating, chondroitin sulfate (CS)-bearing PGs, and it modulates neurite outgrowth and synaptogenesis. Several ADAMTSs (a disintegrin and metalloproteinase with thrombospondin motifs) are glutamyl-endopeptidases that proteolytically cleave brevican. The purpose of this study was to localize regions of adult CNS that contain a proteolytic-derived fragment of brevican which bears the ADAMTS-cleaved neoepitope sequence. These regions were compared to areas of Wisteria floribunda agglutin (WFA) reactivity, a common reagent used to detect "perineuronal nets" (PNNs) of intact matrix and a marker which is thought to label regions of relative neural stability. RESULTS: WFA reactivity was found primarily as PNNs, whereas brevican and the ADAMTS-cleaved fragment of brevican were more broadly distributed in neuropil, and in particular regions localized to PNNs. One example is hippocampus where the ADAMTS-cleaved brevican fragment is found surrounding pyramidal neurons, in neuropil of stratum oriens/radiatum and the lacunosum moleculare. The fragment was less abundant in the molecular layer of the dentate gyrus. Mostly PNNs of scattered interneurons along the pyramidal layer were identified by WFA. In lateral thalamus, the reticular thalamic nucleus stained abundantly with WFA whereas ventral posterior nuclei were markedly immunopositive for ADAMTS-cleaved brevican. Using Western blotting techniques, no common species were reactive for brevican and WFA. CONCLUSION: In general, a marked discordance was observed in the regional localization between WFA and brevican or the ADAMTS-derived N-terminal fragment of brevican. Functionally, this difference may correspond to regions with varied prevalence for neural stability/plasticity.

Dietary loading and aggrecanase-1/TIMP-3 expression in rat mandibular condylar cartilage.

AIMS: To examine the expression of aggrecanase-1 and a tissue inhibitor of metalloproteinases (TIMP-3) in the condylar cartilage of young rats and to determine their relationship during altered dietary loading at different time points after weaning. METHODS: One hundred Sprague-Dawley rats were randomly assigned to 1 of 2 groups: the soft-diet group, which served as the control group (n=50), or the hard-diet group, which served as the experimental group (n=50). Ten soft- and 10 hard-diet rats were killed at 6 hours, 12 hours, 24 hours, 48 hours, and 9 days after weaning (i.e., after initiation of diet change for hard-diet rats). The right-side temporomandibular joints (TMJs) were prepared for immunohistochemical staining. The cartilage from the left-side mandibular condyles of all 10 animals in each group was combined for Western blot analysis. RESULTS: Immunohistochemical analysis revealed strong staining for aggrecanase-1 localized mainly in the chondrocytes of proliferative and upper hypertrophic cartilage zones at all time points in both groups. The immunohistological expression of aggrecanase-1 was significantly higher in the hard-diet group at 12 and 24 hours than in the soft-diet group. Strong staining for TIMP-3 was mainly localized in the chondrocytes of proliferative and upper hypertrophic zones at all time points in both groups. The expression of TIMP-3 in the hard-diet group was at a significantly lower level compared to the soft-diet group at 6 hours. Western blot analysis also showed time-related differences in aggrecanase-1 and TIMP-3, but there was no significant difference between the 2 groups. CONCLUSION: The temporary change in aggrecanase-1 and TIMP-3 expression reflects the complex interaction of these enzymes in the physiologic range and cartilage response to altered dietary loading.

Dermatosparaxis in a Himalayan cat: I. Biochemical studies of dermal collagen.

Dermatosparaxis, a genetic disease, results from the deficiency of the NH2 procollagen peptidase, an enzyme which removes the NH2-terminal nontriple-helical extensions from procollagen. We have identified a Himalayan cat which has deficient amino terminal procollagen peptidase activity. The partially processed precursor chains pNalpha 1 (110,000 daltons) and pNalpha 2 (99,000 daltons) were identified by sodium dodecyl sulfate electrophoresis. In contrast to that from a normal animal, the 20,000 xg supernatant of a skin homogenate failed to convert pNcollagen to collagen. Amino acid analysis of pNalpha 1 and pNalapha 2 chains demonstrated the presence of cysteine and a lower percentage of hydroxyprolyl and glycyl residues due to the presence of the amino terminal extensions. The disorder in this animal is milder than that in sheep and cattle which is reflected in the longer survival and relatively smaller proportion of pNalpha chains in skin. The defect was also demonstrated by skin fibroblasts in culture.

Effects of chemically induced hypoxia on in vitro expression of hypoxia inducible factor-lα, vascular endothelial growth factor, aggrecanase-1, and tissue inhibitor of metalloproteinase-3 in rat mandibular condylar chondrocytes.

AIMS: To examine the expression of hypoxia inducible factor-lα (HIF-1α), vascular endothelial growth factor (VEGF), aggrecanase-1 (ADAMTS-4), and tissue inhibitor of metalloproteinase-3 (TIMP-3) in rat mandibular condylar chondrocytes under hypoxic conditions and examine the relationship of these expressed factors in early condylar cartilage growth. METHODS: CoCl2 was used to mimic a hypoxic microenvironment by chemically inducing hypoxia. Chondrocytes, which were obtained from the mandibular condyles of 3-week-old female Sprague-Dawley rats, were treated with 125 µmol/L CoCl2 for 48 hours. The HIF-1α, VEGF, ADAMTS-4, and TIMP-3 mRNA levels in chondrocytes were detected using a semiquantitativepolymerase chain reaction (PCR) at 12, 24, and 48 hours after the initiation of hypoxia and normoxia conditions. A univariate variance analysis (pairwise least significant difference t test) using a SPSS 13.0 software package was performed to test for differences between different groups. RESULTS: PCR analysis of the chondrocytes showed that the expression of HIF-1α and VEGF mRNA increased at 12, 24, and 48 hours after induction of hypoxia. HIF-1α expression gradually increased throughout the study duration, while VEGF expression peaked at 12 hours. Compared to normoxia conditions, hypoxia resulted in constantly elevated expression levels of both. On the other hand, there was no significant difference in the ADAMTS-4 and TIMP-3 mRNA expression between hypoxic and normoxic conditions throughout the study (P > .05). CONCLUSION: An upregulated HIF-1α and VEGF mRNA expression indicates their important roles in cartilage vascularization and development in newly hypoxic microenvironments. Additionally, chemically induced hypoxia has little effect on the expressions of ADAMTS-4 and TIMP-3.

Detection of ADAMTS-4 activity using a fluorogenic peptide-conjugated Au nanoparticle probe in human knee synovial fluid.

A disintegrin and metalloproteinase with thrombospondin motif-4 (ADAMTS-4) plays a pivotal role in degrading aggrecan, which is an early event in cartilage degrading joint diseases such as osteoarthritis (OA). Detection of ADAMTS-4 activity could provide useful clinical information for early diagnosis of such diseases and disease-modifying therapy. Therefore, we developed a ADAMTS-4 detective fluorescent turn-on AuNP probe (ADAMTS-4-D-Au probe) by conjugating gold nanoparticles with a FITC-modified ADAMTS-4-specific peptide (DVQEFRGVTAVIR). When the ADAMTS-4-D-Au probe was incubated with ADAMTS-4, the fluorescence recovered and fluorescence intensity markedly increased in proportion to concentrations of ADAMTS-4 and the probe. A nearly 3-fold increase in fluorescent intensity in response to only 3.9 pM of ADAMTS-4 was detected, whereas almost no fluorescence recovery was observed when the probe was incubated with matrix metalloproteinase (MMP)-1, -3, and -13. These results indicate a relative high sensitivity and specificity of the probe. Moreover, ADAMTS-4-D-Au probe was used to detect ADAMTS-4 activity in synovial fluid from 11 knee surgery patients. A substantial increase in fluorescent intensity was observed in the acute joint injury group as compared to the chronic joint injury and end-stage OA groups, indicating that this simple and low-cost sensing system might serve as a new detection method for ADAMTS-4 activity in biological samples and in screens for inhibitors for ADAMTS-4-related joint diseases. Additionally, this probe could be a potential biomarker for early diagnosis of cartilage-degrading joint diseases.

Down-regulated genes in mouse dental papillae and pulp.

Important factors involved in odontogenesis in mouse dental papillae disappear between the pre- and post-natal stages of development. Therefore, we hypothesized that certain genes involved in odontogenesis in dental papillae were subject to pre-/post-natal down-regulation. Our goal was to identify, by microarray analysis, which genes were down-regulated. Dental papillae were isolated from embryonic 16-day-, 18-day- (E16, E18), and post-natal 3-day-old (P3) murine first mandibular molar germs and analyzed by microarray. The number of down-regulated genes was 2269 between E16 and E18, and 3130 between E18 and P3. Drastic down-regulation (fold change > 10.0) of Adamts4, Aldha1a2, and Lef1 was observed at both E16 and E18, and quantitative RT-PCR revealed a post-natal reduction in their expression (Adamts4, 1/3; Aldh1a2, 1/13; and Lef1, 1/37). These results suggest that down-regulation of these three genes is an important factor in normal odontogenesis in dental papillae.

Fibroblast growth factor 2 inhibits induction of aggrecanase activity in human articular cartilage.

OBJECTIVE: Articular chondrocytes are surrounded by an extracellular pool of fibroblast growth factor 2 (FGF-2). We undertook this study to investigate the possible role of FGF-2 in aggrecan catabolism by aggrecanase in human articular cartilage. METHODS: Aggrecan catabolism was induced by interleukin-1alpha (IL-1alpha) in normal human articular cartilage and assessed by measuring the release of glycosaminoglycan (GAG) and aggrecanase-dependent fragments by Western blotting with antibodies against neoepitopes. ADAMTS-4 and ADAMTS-5 messenger RNA (mRNA) expression was measured by quantitative real-time reverse transcriptase-polymerase chain reaction. Production of matrix metalloproteinases (MMPs) 1, 3, and 13 and tissue inhibitors of metalloproteinases (TIMPs) 1 and 3 was measured by Western blotting. IL-6 and IL-8 were measured by enzyme-linked immunosorbent assay. Proteoglycan synthesis was monitored by 35S-sulfate incorporation. RESULTS: IL-1alpha caused cleavage of aggrecan in cultured human articular cartilage explants, with release of GAG and aggrecan fragments containing ARGS and AGEG neoepitopes. This was inhibited by FGF-2 (1-100 ng/ml). Tumor necrosis factor alpha and retinoic acid also stimulated release of neoepitope, and this was also suppressed by FGF-2. IL-1alpha induced ADAMTS-4 and ADAMTS-5 mRNA in primary human chondrocytes, and this was inhibited by FGF-2. IL-1alpha-induced aggrecan breakdown was inhibited by TIMP-1 or by the N-terminal portion of TIMP-3, although FGF-2 did not affect production of the inhibitors TIMP-1 and TIMP-3 when IL-1alpha was present. FGF-2 did not prevent IL-1alpha suppression of proteoglycan synthesis and did not negate its ability to stimulate the production of IL-6, IL-8, and MMPs 1, 3, and 13. CONCLUSION: Our findings suggest that FGF-2 may play a chondroprotective role in human articular cartilage by controlling the expression and activity of the aggrecanases ADAMTS-4 and ADAMTS-5.

Low molecular weight isoforms of the aggrecanases are responsible for the cytokine-induced proteolysis of aggrecan in a porcine chondrocyte culture system.

OBJECTIVE: The major proteases responsible for aggrecan turnover in articular cartilage are the aggrecanases (ADAMTS-4 and ADAMTS-5). Although several studies have demonstrated C-terminal truncation of these aggrecanases, the mechanism and importance of this processing are poorly understood. The objective of this study was to further investigate ADAMTS-4 and ADAMTS-5 C-terminal truncation in a porcine model in vitro culture system. METHODS: Chondrocyte-agarose cultures with well-established extracellular matrices were treated with or without interleukin-1 (IL-1), for a variety of different culture time periods. Cultures were analyzed for release of sulfated glycosaminoglycan, aggrecanase-generated interglobular domain (IGD)-aggrecan cleavage, and the presence of ADAMTS-4 and ADAMTS-5 isoforms. Inhibition of aggrecanase activity with monoclonal antibodies, tissue inhibitor of metalloproteinases 3 (TIMP-3), and cycloheximide pretreatment were used to identify ADAMTS isoforms involved in IGD-aggrecan catabolism. RESULTS: Multiple isoforms, including possible zymogens, of ADAMTS-4 and ADAMTS-5 were sequestered within the extracellular matrix formed by 3-week chondrocyte-agarose cultures. IL-1 exposure induced production of a low molecular weight (37 kd) isoform of ADAMTS-4. This isoform was capable of degrading exogenous aggrecan at the IGD-aggrecanase site, was inhibited by TIMP-3, was blocked after preincubation with an antibody to a sequence in the catalytic domain of ADAMTS-4, and required de novo synthesis in the presence of IL-1 for its generation. CONCLUSION: In porcine chondrocyte-agarose cultures, a 37-kd ADAMTS-4 isoform appears to be the major matrix protease responsible for the IGD-aggrecanase activity detected in response to exposure to IL-1. This conclusion contradicts that of recent studies of transgenic knockout mice and highlights the need to determine the roles of the different aggrecanase(s) in human disease.

Aggrecanases and aggrecanase-generated fragments in the human intervertebral disc at early and advanced stages of disc degeneration.

STUDY DESIGN: A comparative study of aggrecanases and aggrecan fragmentation profile in the human intervertebral disc at early and advanced stages of disc degeneration. OBJECTIVE: To determine differences in the content of the aggrecanases and the profile of aggrecan fragmentation in early and advanced stages of disc degeneration using cadaveric human intervertebral discs. SUMMARY OF BACKGROUND DATA: Aggrecanases and aggrecanase-generated aggrecan fragments have been found in human degenerated discs. However, the association between the grade of disc degeneration and the content of the aggrecanases and the profile of aggrecan fragments has not been well studied. METHODS: A total of 108 cadaveric donor spines were assessed by MRI T2 imaging and graded based on the Thompson scale. Twelve donor spines (average age, 63 years), each specifically exhibiting 2 different stages (Grade 2 and Grade 4) of disc degeneration at different disc levels, were included in this study. After harvesting the preselected discs, tissue samples were obtained from the center of the nucleus pulposus (NP) and the middle zone of the anulus fibrosus (AF). The amount of the aggrecanases, specifically ADAMTS-4 and ADAMTS-5, and the pattern of aggrecan fragmentation in the isolated tissues were assessed by western blot using specific antibodies. RESULTS: In both NP and the AF tissues, the amount of ADAMTS-4 detected was higher in disc tissues with a higher level of degeneration (Grade 4) than in Grade 2 disc tissues with a lower level of degeneration. However, the amount of ADAMTS-5 detected did not differ between the 2 disc tissue grades. The aggrecan fragmentation analysis of these samples demonstrated the presence of aggrecanase-mediated fragmentation in both groups; however, there was no apparent difference in the aggrecan fragmentation profile between discs at early and advanced stages of disc degeneration. CONCLUSION: Aggrecanases are involved in aggrecanolysis at both the early and advanced stages of disc degeneration. The aggrecan fragmentation profile analysis demonstrates the involvement of aggrecanases, as well as that of matrix metalloproteinases and/or cathepsins, during disc degeneration.

Changes in immunolocalisation of beta-dystroglycan and specific degradative enzymes in the osteoarthritic synovium.

OBJECTIVE: To investigate the immunolocalisation of beta-dystroglycan (beta-DG) and specific matrix metalloproteinases (MMPs)-3, -9, -13 and a disintegrin like and metalloproteinase thrombospondin type 1 motif 4 (ADAMTS-4) within the joint tissues of patients with osteoarthritis (OA) and unaffected controls. DESIGN: Cartilage, synovium and synovial fluid were obtained from the hip joints of five osteoarthritic (patients undergoing total hip replacement) and five control hip joints (patients undergoing hemiarthroplasty for femoral neck fracture). The samples were analysed for beta-DG protein using Western blot technique and by immunohistochemistry for tissue distribution of beta-DG, MMP-3, -9, -13, and ADAMTS-4. RESULTS: beta-DG was detected in the smooth muscle of both normal and osteoarthritic synovial blood vessels. Importantly, beta-DG was detected in endothelium of blood vessels of OA synovium, but not in the control endothelium. In the endothelium of osteoarthritic synovial blood vessels, beta-DG co-localised with MMP-3 and -9. MMP-13 and ADAMTS-4 showed no endothelial staining, and only weak staining of the vascular smooth muscle was found. In contrast, we did not detect beta-DG protein in cartilage or synovial fluid. CONCLUSIONS: beta-DG has been shown to have a role in angiogenesis, and our results demonstrate for the first time that there are clear differences in beta-DG staining between OA and control synovial blood vessels. The specific immunolocalisation of beta-DG within endothelium of inflamed OA blood vessels and its co-localisation with MMP-3 and -9, reported to have pro-angiogenic roles and believed to be involved in beta-DG cleavage, may also suggest that beta-DG plays a role in angiogenesis accompanying OA.

Analysis of ADAMTS4 and MT4-MMP indicates that both are involved in aggrecanolysis in interleukin-1-treated bovine cartilage.

OBJECTIVE: To investigate the mechanism of aggrecanolysis in interleukin-1 (IL-1)-treated cartilage tissue by examining the time course of aggrecan cleavages and the tissue and medium content of membrane type 4-matrix metalloproteinases (MT4-MMP) and a disintegrin and metalloproteinase with thrombospondin type I motifs (ADAMTS)4. METHODS: Articular cartilage explants were harvested from newborn bovine femoropatellar groove. The effects of IL-1 treatment with or without aggrecanase blockade were investigated by Western analysis of aggrecan fragment generation, ADAMTS4 species (p68 and p53), and MT4-MMP, as well as by realtime PCR (polymerase chain reaction) for ADAMTS4 and 5. Aggrecanase was blocked with mannosamine (ManN), an inhibitor of glycosylphosphatidylinositol anchor synthesis, and esculetin (EST), an inhibitor of MMP-1, MMP-3, and MMP-13 gene expression. RESULTS: IL-1 treatment caused a major increase in MT4-MMP abundance in the tissue and medium. ADAMTS4 (p68) was abundant in fresh cartilage and this was retained in the tissue in untreated cartilage. IL-1 treatment for 6 days caused a marked loss of p68 from the cartilage and the appearance of p53 in the medium. Addition of either 1.35 mM ManN or 31-500 microM EST blocked IL-1-mediated aggrecanolysis and this was accompanied by nearly complete inhibition of the MT4-MMP increase, the p68 loss and the formation of p53. IL-1 treatment increased mRNA abundance for ADAMTS4 ( approximately 3-fold) and ADAMTS5 ( approximately 10-fold) but this was not accompanied by a marked change in enzyme protein abundance. CONCLUSION: These studies support a central role for MT4-MMP in IL-1-induced cartilage aggrecanolysis and are consistent with the identification of p68 as the aggrecanase that cleaves within the CS2 domain, and of p53 as the aggrecanase that generates G1-NITEGE. Since the induction by IL-1 was not accompanied by marked changes in total ADAMTS4 protein, but rather in partial conversion of p68 to p53 and release of both from the tissue, we conclude that aggrecanolysis in this model system results from MT4-MMP-mediated processing of a resident pool of ADAMTS4 and release of the p68 and p53 from their normal association with the cell surface.

Peptídios séricos do procolágeno tipo III: novo teste para o estudo da hepatite alcoólica? / Serum type III procolagen peptide: a new test for the study of alcoholic hepatitis?

A hepatite alcoólica é uma das lesöes que compöem o espectro morfológico da doença hepática alcoólica. O se estudo é particularmente importante pois está demonstrado que é lesäo pré-cirrótica. Os dados clínico e laboratoriais näo detectam por vezes a presença dessa lesäo. A determinaçäo dos peptídios séricos do procolágeno tipo III parece constitutir grande avanço para o diagnóstico de hepatite alcoólica, em estilistas sem ou com cirrose. A presença de níveis séricos significantemente elevados daqueles peptídios em paciente alcoólatra justificaria indicaçäo para biópsia hepática