Suppression of MMP activity in bovine cartilage explants cultures has little if any effect on the release of aggrecanase-derived aggrecan fragments

Abstract

Abstract

Background: Progressive loss of articular cartilage is a central hallmark in many joint disease, however, the relative importance of individual proteolytic pathways leading to cartilage erosion is at present unknown. We therefore investigated the time-dependant release ex vivo of MMP- and aggrecanase-derived fragments of aggrecan and type II collagen into the supernatant of bovine cartilage explants cultures using neo-epitope specific immunoassays, and to associate the release of these fragments with the activity of proteolytic enzymes using inhibitors.

Findings: Bovine cartilage explants were cultured in the presence or absence of the catabolic cytokines oncostatin M (OSM) and tumor necrosis factor alpha (TNFα). In parallel, explants were co-cultured with protease inhibitors such as GM6001, TIMP1, TIMP2 and TIMP3. Fragments released into the supernatant were determined using a range of neo-epitope specific immunoassays; (1) sandwich ³⁴² FFGVG-G2 ELISA, (2) competition NITEGE³⁷³ ELISA (3) sandwich G1-NITEGE³⁷³ ELISA (4) competition ³⁷⁴ ARGSV ELISA, and (5) sandwich ³⁷⁴ ARGSV-G2 ELISA all detecting aggrecan fragments, and (6) sandwich CTX-II ELISA, detecting C-telopeptides of type II collagen. We found that (1) aggrecanase-derived aggrecan fragments are released in the early (day 2-7) and mid phase (day 9-14) into the supernatant from bovine explants cultures stimulated with catabolic cytokines, (2) the release of NITEGE ³⁷³ neo-epitopes are delayed compared to the corresponding ³⁷⁴ ARGSV fragments, (3) the MMP inhibitor GM6001 did not reduce the release of aggrecanase-derived fragment, but induced a further delay in the release of these fragments, and finally (4) the MMP-derived aggrecan and type II collagen fragments were released in the late phase (day 16-21) only.

Conclusion: Our data support the model, that aggrecanases and MMPs act independently in the processing of the aggrecan molecules, and furthermore that suppression of MMP-activity had little if any effect on the quantity of aggrecanase-derived fragments released from explants cultures.