MMP and non-MMP-mediated release of aggrecan and its fragments from articular cartilage: a comparative study of three different aggrecan and glycosaminoglycan.

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MMP and non-MMP-mediated release of aggrecan and its fragments from articular cartilage: a comparative study of three different aggrecan and glycosaminoglycan assays E.U. Sumer, M.Sc., B.C. Sondergaard, M.Sc., J.C. Rousseau, Ph.D., P.D. Delmas, M.D., Ph.D., A.J. Fosang, Ph.D., M.A. Karsdal, Ph.D., C. Christiansen, D.M.Sc., P. Qvist, Ph.D. Osteoarthritis and Cartilage Volume 15, Issue 2, Pages 212-221 (February 2007) DOI: 10.1016/j.joca.2006.07.009 Copyright © 2006 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 (A) To investigate the specificity of F-78, intact purified bovine aggrecan, chicken neurocan, BSA as a negative control, bovine biglycan and bovine decorin were run in the G1/G2 competition assay at a concentration of 10,000ng/ml. The values are mean+s.e.m.s, and the asterisks indicate significant differences. For the statistical analysis, unpaired t test with Welch's correction was used with one-tailed P values. (B) To investigate the specificity of F-78, purified porcine G1 and BSA as a negative control were run in the G1/G2 sandwich assay at a concentration of 1000ng/ml. The values are mean+s.e.m.s, and the asterisks indicate significant differences. For the statistical analysis, unpaired t test with Welch's correction was used with one-tailed P values. (C) To investigate the specificity of F-78, purified MMP digested porcine aggrecan was run in the 342-G2 sandwich assay at a concentration of 1000ng/ml, and as a negative control BSA was used. The values are mean+s.e.m.s, and the asterisks indicate significant differences. For the statistical analysis, unpaired t test with Welch's correction was used with one-tailed P values. Osteoarthritis and Cartilage 2007 15, 212-221DOI: (10.1016/j.joca.2006.07.009) Copyright © 2006 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 (A) The G1/G2 assay detecting intact aggrecan and aggrecan fragments carrying the G1 and/or G2 domain, (B) 342-G2 assay detecting aggrecan fragments carrying both the 342FFGVG neo-epitope and the G2 globular domain. Osteoarthritis and Cartilage 2007 15, 212-221DOI: (10.1016/j.joca.2006.07.009) Copyright © 2006 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Bovine articular cartilage explants were stimulated with the cytokines OSM/TNFα and supernatants were measured for the presence of aggrecan and aggrecan fragments by immunoassays. G1/G2 assay with (-■-) and without (-□-) the general MMP inhibitor GM6001. 342-G2 assay with (-●-) and without (-○-) GM6001. Osteoarthritis and Cartilage 2007 15, 212-221DOI: (10.1016/j.joca.2006.07.009) Copyright © 2006 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 The level of total GAGs (-□-) in explant cultures stimulated with OSM/TNFα. Osteoarthritis and Cartilage 2007 15, 212-221DOI: (10.1016/j.joca.2006.07.009) Copyright © 2006 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Gelatinase activity investigated by zymography of conditioned medium from cartilage explants treated with OSM/TNFα. Lane A: Vehicle, conditioned medium without cytokines from day 12. Lane B: Conditioned medium from MI explants from day 12. The rest of the lanes represent conditioned medium of explants stimulated with OSM/TNFα at days 2, 5, 7, 9, 12 and 14. Active and pro-enzymes were identified by comparison to the molecular markers and MMP-2 and MMP-9 standards (not shown). The molecular weight marker showed the bands corresponding to the weights of pro MMP-9 92kDa, active 86kDa and pro MMP-2 72kDa and active 66kDa. Osteoarthritis and Cartilage 2007 15, 212-221DOI: (10.1016/j.joca.2006.07.009) Copyright © 2006 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Articular cartilage explants were stimulated with OSM/TNFα for 5 days (see Fig. 3) and supernatants were subsequently treated with either buffer, MMP-2 or MMP-2 with MMP inhibitor (GM6001) and measured in the G1/G2 assay (A) or 342-G2 (C). Similar supernatants from day 18 were measured in the G1/G2 (B) and 342-G2 assays (D). The values are mean+s.e.m.s, and the asterisks indicate significant differences. For the statistical analysis, unpaired t test with Welch's correction was used with one-tailed P values. Osteoarthritis and Cartilage 2007 15, 212-221DOI: (10.1016/j.joca.2006.07.009) Copyright © 2006 Osteoarthritis Research Society International Terms and Conditions

Fig. 7 Bovine articular explants were cultured in the presence (OSM/TNFα) or absence of cytokines (W/O) for 5 days (A) or 18 days (B) and subsequently fixed in formaldehyde and paraffin embedded. Sections were stained for proteoglycans by toluidine blue, and as a negative control, MI explants were also included. Osteoarthritis and Cartilage 2007 15, 212-221DOI: (10.1016/j.joca.2006.07.009) Copyright © 2006 Osteoarthritis Research Society International Terms and Conditions

Fig. 8 Serum samples from healthy individuals (n=57) and patients with RA (n=20) were analyzed in the G1/G2 assay (A) and the 342-G2 assay (B). The aggrecan degradation index (C) was obtained by dividing the concentration of 342-G2 with the level measured in the G1/G2 assay for both healthy individuals and RA patients. For standardization of samples in the G1/G2 assay, intact, purified bovine aggrecan at concentrations 0.0625–1ng/ml was used, whereas MMP treated purified porcine aggrecan at concentrations 46–3000ng/ml was used in the 342-G2 assay. The values are mean+s.e.m.s, and the asterisks indicate significant differences. For the statistical analysis, unpaired t test with Welch's correction was used with one-tailed P values. Osteoarthritis and Cartilage 2007 15, 212-221DOI: (10.1016/j.joca.2006.07.009) Copyright © 2006 Osteoarthritis Research Society International Terms and Conditions