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Herlev, Denmark

He Y.,NordicBioscience | Zheng Q.,NordicBioscience China | Simonsen O.,University of Aalborg | Petersen K.K.,University of Aalborg | And 3 more authors.
Matrix Biology

ADAMTS-4 (aggrecanase1) is believed to play an important role in the degradation of aggrecan during the progression of joint diseases. ADAMTS-4 is synthesized as a latent pro-enzyme that requires the removal of the pro-domain, exposing the N-terminal neoepitope, to achieve activity. We developed a monoclonal antibody against this neoepitope of active ADAMTS-4. Furthermore, we established and characterized a competitive ELISA for measuring active ADAMTS-4 form applying the specific antibody. We used this assay to profile the presence of active ADAMTS-4 and its aggrecan degradation product (NITEGE373) in a bovine cartilage ex vivo model. We found that after stimulation with catabolic factors, the cartilage initially released high levels of aggrecanase-derived aggrecan fragments into supernatant but subsequently decreased to background levels. The level of active ADAMTS-4 released into the supernatant and retained in the cartilage matrix increased continuously throughout the 21days of the study. The activity of ADAMTS-4 on the last day of catabolic stimulation was verified in vitro by adding deglycosylated or native aggrecan to the conditioned medium. Samples of human cartilage affected by varying degrees of osteoarthritis stained strongly for active ADAMTS-4 where surface fibrillation and clustered chondrocytes were observed. This assay could be an effective tool for studying ADAMTS-4 activity and for screening drugs regulating ADAMTS-4 activation. Moreover, it could be a potential biomarker for degenerative joint disease. © 2012 International Society of Matrix Biology. Source

He Y.,NordicBioscience | Zheng Q.,NordicBioscience | Jiang M.,NordicBioscience China | Sun S.,NordicBioscience | And 4 more authors.

Objective: The specific degradation of type II collagen and aggrecan by matrix metalloproteinase (MMP)-9, -13 and ADAMTS-4 and -5 (aggrecanase-1 and -2) in the cartilage matrix is a critical step in pathology of osteoarthritis (OA). The aims of this study were: i) To investigate the relative contribution of ADAMTS-4 and ADAMTS-5 to cartilage degradation upon catabolic stimulation; ii) To investigate the effect of regulating the activities of key enzymes by mean of broad-spectrum inhibitors. Methods: Bovine full-depth cartilage explants stimulated with tumor necrosis factor alpha (TNF-α) and Oncostatin M (OSM) were cultured for 21 days with or without a number of inhibitors targeting different types of proteases. Monoclonal antibodies were raised against the active sites of ADAMTS-4, -5, MMP-9 and -13, and 4 ELISAs were developed and technically validated. In addition, the established AGNxI (ADAMTS-degraded aggrecan), AGNxII (MMP-degraded aggrecan), and CTX-II (MMP-derived type II collagen) were quantified in the explantsconditioned media. Results: We found that: i) Active ADAMTS-4, MMP-9, -13 were released in the late stage of TNF-α/ OSM stimulation, whereas no significant active ADAMTS-5 was detected in either extracts or supernatants; ii) Active ADAMTS-4 was primarily responsible for E373-374 A bond cleavage in aggrecan in this setting; and iii) The compensatory mechanism could be triggered following the blockage of the enzyme caused by inhibitors. Conclusions: ADAMTS-4 appeared to be the major protease for the generation of 374 ARGS aggrecan fragment in the TNF-α/OSM stimulated bovine cartilage explants. This study addresses the need to determine the roles of ADAMTS-4 and ADAMTS-5 in human articular degradation in OA and hence identify the attractive target for slowing down human cartilage breakdown. © 2015 He et al. Source

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