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Carprofen inhibits the release of matrix metalloproteinases 1, 3, and 13 in the secretome of an explant model of articular cartilage stimulated with interleukin 1?.

ABSTRACT: Arthritic diseases are characterized by the degradation of collagenous and noncollagenous extracellular matrix (ECM) components in articular cartilage. The increased expression and activity of matrix metalloproteinases (MMPs) is partly responsible for cartilage degradation. This study used proteomics to identify inflammatory proteins and catabolic enzymes released in a serum-free explant model of articular cartilage stimulated with the pro-inflammatory cytokine interleukin 1? (IL-1?). Western blotting was used to quantify the release of selected proteins in the presence or absence of the cyclooxygenase-2 specific nonsteroidal pro-inflammatory drug carprofen.Cartilage explant cultures were established by using metacarpophalangeal joints from horses euthanized for purposes other than research. Samples were treated as follows: no treatment (control), IL-1? (10 ng/ml), carprofen (100 ?g/ml), and carprofen (100 ?g/ml)?+?IL-1? (10 ng/ml). Explants were incubated (37°C, 5% CO2) over twelve day time courses. High-throughput nano liquid chromatography/mass spectrometry/mass spectrometry uncovered candidate proteins for quantitative western blot analysis. Proteoglycan loss was assessed by using the dimethylmethylene blue (DMMB) assay, which measures the release of sulfated glycosaminoglycans (GAGs).Mass spectrometry identified MMP-1, -3, -13, and the ECM constituents thrombospondin-1 (TSP-1) and fibronectin-1 (FN1). IL-1? stimulation increased the release of all three MMPs. IL-1? also stimulated the fragmentation of FN1 and increased chondrocyte cell death (as assessed by ?-actin release). Addition of carprofen significantly decreased MMP release and the appearance of a 60 kDa fragment of FN1 without causing any detectable cytotoxicity to chondrocytes. DMMB assays suggested that carprofen initially inhibited IL-1?-induced GAG release, but this effect was transient. Overall, during the two time courses, GAG release was 58.67%?±?10.91% (SD) for IL-1? versus 52.91%?±?9.35% (SD) with carprofen?+?IL-1?.Carprofen exhibits beneficial anti-inflammatory and anti-catabolic effects in vitro without causing any detectable cytotoxicity. Combining proteomics with this explant model provides a sensitive screening system for anti-inflammatory compounds.

SUBMITTER: Williams A

PROVIDER: S-EPMC3978949 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Project description:BackgroundOsteoarthritis (OA) is an inflammatory disease of synovial joints involving the loss and degeneration of articular cartilage. The gold standard for evaluating cartilage loss in OA is the measurement of joint space width on standard radiographs. However, in most cases the diagnosis is made well after the onset of the disease, when the symptoms are well established. Identification of early biomarkers of OA can facilitate earlier diagnosis, improve disease monitoring and predict responses to therapeutic interventions.MethodsThis study describes the bioinformatic analysis of data generated from high throughput proteomics for identification of potential biomarkers of OA. The mass spectrometry data was generated using a canine explant model of articular cartilage treated with the pro-inflammatory cytokine interleukin 1 ? (IL-1?). The bioinformatics analysis involved the application of machine learning and network analysis to the proteomic mass spectrometry data. A rule based machine learning technique, BioHEL, was used to create a model that classified the samples into their relevant treatment groups by identifying those proteins that separated samples into their respective groups. The proteins identified were considered to be potential biomarkers. Protein networks were also generated; from these networks, proteins pivotal to the classification were identified.ResultsBioHEL correctly classified eighteen out of twenty-three samples, giving a classification accuracy of 78.3% for the dataset. The dataset included the four classes of control, IL-1?, carprofen, and IL-1? and carprofen together. This exceeded the other machine learners that were used for a comparison, on the same dataset, with the exception of another rule-based method, JRip, which performed equally well. The proteins that were most frequently used in rules generated by BioHEL were found to include a number of relevant proteins including matrix metalloproteinase 3, interleukin 8 and matrix gla protein.ConclusionsUsing this protocol, combining an in vitro model of OA with bioinformatics analysis, a number of relevant extracellular matrix proteins were identified, thereby supporting the application of these bioinformatics tools for analysis of proteomic data from in vitro models of cartilage degradation.

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Carprofen inhibits the release of matrix metalloproteinases 1, 3, and 13 in the secretome of an explant model of articular cartilage stimulated with interleukin 1?.

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