Aggrecanase Activity and Proteoglycan Release from Human Articular Cartilage as modulated by Tetracyclines

  • Published 2009


INTRODUCTION: The degradation of aggrecan during osteoarthritis (OA) is catalyzed by increased activities of matrix metalloproteinases and members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motif) family. ADAMTS4 and ADAMTS5, also called aggrecanase-1 and aggrecanase-2, have been identified in cartilage and were shown to cleave aggrecan at five different sites both in vitro and in vivo. Four cleavage sites are located between the aggrecan globular domains G2 and G3 while one cleavage occurs between the globular domain G1 and G2 (Glu373 – Ala374). Identification of agents able to reduce the activity of proteoglycanases has long been a therapeutic goal for inhibiting or slowing down the degradation of cartilage. Several studies have implicated that tetracyclines, in addition to their antimicrobial properties, can slow down the progression of cartilage damage both in animal models of OA as well as in humans (1,2). In search for the underlying mechanisms we previously reported that minocycline and doxycycline can not only inhibit the activity of MMP-1 but also the expression of MMP-1, MMP3 and iNOS by bovine articular chondrocytes (3,4). In this line, the present in vitro-study was designed to examine for the first time whether tetracyclines 1. also possess an inhibitory potential on the activities of ADAMTS4 and ADAMTS5 and, 2. can thus prevent interleukin-1 (IL-1) inducible proteoglycan loss from human articular cartilage. METHODS: 1. Determination of aggrecanase activity: The activity of recombinant human (rh) ADAMTS4 and rhADAMTS5 were determined using rh aggrecan interglobular domain (rhAggrecan-IGD; mdbioscience) as a substrate. Briefly, after proteolytic cleavage of the rhAggrecan-IGD by 30 nM rhADAMTS4 (F213-A579 from mdbioscience or F213-R695 from R&D) or rhADAMTS5 (S262-F632 from R&D), an aggrecan peptide with the N-terminal sequence ARGSVIL was released, which was then quantified using two monoclonal anti-peptide antibodies. The %inhibition was calculated from a standard curve established with untreated enzymes (N=3). 2. Proteoglycan loss and viability of treated cartilage explants: Fullthickness cartilage explants of the lateral compartment of the femoral condyles were taken from OA patients undergoing knee replacement surgery. Before starting the experiments, approval by the ethical board of our university and the written informed consent of the patients were obtained. 4-mm-diameter articular cartilage discs were obtained using a biopsy punch. The degree of OA changes of the femoral condyles was determined according to Collins. Explants from mild (Collins grade 01.5) or moderately (Collins grade >1.5-3) affected human OA condyles were cultured separately in supplemented and serum-free Ham ́s F12 media together with the serum substitute CR-ITS+®. Cultures were maintained for 11 days at 37C, 5% CO2 and 95% humidity. Media were changed every 3-4 days and stored frozen at -20C in the presence of 10% (v/v) proteinase inhibitor mixture. Fresh media, rhIL-1 and drugs were added to the cartilage explants. Explants were treated with 1, 10, 50 or 100 μM minocycline, doxycycline or tetracycline in the presence or absence of 5ng/ml rhIL-1ß. Papain-digested cartilage explants and culture media were assayed for sulfated GAGs by reaction with the 1,9-dimethylmethylene blue (DMMB) dye solution in 96-well plates and spectrophotometry at 523 nm using an ELISA plate reader. Proteoglycan loss was then calculated from the ratio of GAGs found in the media to total GAGs as found in media plus explants. The viability of chondrocytes within the superficial, intermeidate and deep layer of cartilage explants was determined in separately cultured explants treated with or without tetracyclines as already described above. Cell viability was assessed microscopically using fluorescein diacetate and propidium iodide. Results were compared to untreated explants removed from the same condyle. Each experimental condition was repeated five times using explants always obtained from 6 different patients (N=6). 3. Statistical evaluation: Data presented are means + SD. Groups of data were evaluated using one-way analysis of variance (ANOVA) with Tukey ́s method to compare means. Significance was set to p < 0.05. RESULTS: Doxycycline, minocycline and tetracycline dose-dependently inhibitied the activity of rhADAMTS4 (Fig. 1) and rhADAMTS5 (data not shown). Fig. 1: Activity of rhADAMTS4 (F213-A579) in the presence of tetracyclines 0 25 50 75 100 0 20 40 60 80 100 Tetracycline

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@inproceedings{2009AggrecanaseAA, title={Aggrecanase Activity and Proteoglycan Release from Human Articular Cartilage as modulated by Tetracyclines}, author={}, year={2009} }