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Coculture of bovine cartilage with synovium and fibrous joint capsule increases aggrecanase and matrix metalloproteinase activity.

ABSTRACT: A hallmark of osteoarthritis is increased proteolytic cleavage of aggrecan. Cross talk between cartilage and the synovium + joint capsule (SJC) can drive cartilage degradation by activating proteases in both tissues. We investigated aggrecan proteolysis patterns in cartilage explants using a physiologically relevant explant model of joint injury combining cartilage mechanical compression and coincubation with SJC.Bovine cartilage explants were untreated; coincubated with SJC; or subjected to mechanical injury and coincubated with SJC, mechanical injury alone, or mechanical injury and incubated with tumor necrosis factor-? (TNF-?). To compare the patterns of aggrecan proteolysis between 6 h and 16 days, release of sulfated glycosaminoglycans and specific proteolytic aggrecan fragments into medium or remaining in cartilage explants was measured by dimethylmethylene blue and Western blot analysis.Aggrecanase activity toward aggrecan was observed in all conditions, but it was directed toward the TEGE?ARGS interglobular domain (IGD) site only when cartilage was coincubated with SJC or TNF-?. Matrix metalloproteinase (MMP) activity at the aggrecan IGD site (IPES?FFGV) was not detected when cartilage was exposed to TNF-? (up to 6 days), but it was in all other conditions. Compared with when bovine cartilage was left untreated or subjected to mechanical injury alone, additional aggrecan fragment types were released into medium and proteolysis of aggrecan started at an earlier time when SJC was present.Indicative of different proteolytic pathways for aggrecan degradation, the SJC increases both aggrecanase and MMP activity toward aggrecan, whereas TNF-? inhibits MMP activity against the IGD of aggrecan.

SUBMITTER: Sward P

PROVIDER: S-EPMC5498889 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Coculture of bovine cartilage with synovium and fibrous joint capsule increases aggrecanase and matrix metalloproteinase activity.

Swärd Per P Wang Yang Y Hansson Maria M Lohmander L Stefan LS Grodzinsky Alan J AJ Struglics André A

Arthritis research & therapy 20170705 1

<h4>Background</h4>A hallmark of osteoarthritis is increased proteolytic cleavage of aggrecan. Cross talk between cartilage and the synovium + joint capsule (SJC) can drive cartilage degradation by activating proteases in both tissues. We investigated aggrecan proteolysis patterns in cartilage explants using a physiologically relevant explant model of joint injury combining cartilage mechanical compression and coincubation with SJC.<h4>Methods</h4>Bovine cartilage explants were untreated; coincu ...[more]

PMID: 28679445

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Project description:BackgroundProgressive 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.FindingsBovine cartilage explants were cultured in the presence or absence of the catabolic cytokines oncostatin M (OSM) and tumor necrosis factor alpha (TNFalpha). 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 NITEGE(373)ELISA (3) sandwich G1-NITEGE(373 )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.ConclusionOur 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.

Dataset Information

Coculture of bovine cartilage with synovium and fibrous joint capsule increases aggrecanase and matrix metalloproteinase activity.

Publications

Coculture of bovine cartilage with synovium and fibrous joint capsule increases aggrecanase and matrix metalloproteinase activity.

Similar Datasets

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