Dataset Information

The effect of protease inhibitors on the induction of osteoarthritis-related biomarkers in bovine full-depth cartilage explants.

ABSTRACT:

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-?) 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 explants-conditioned media.

Results

We found that: i) Active ADAMTS-4, MMP-9, -13 were released in the late stage of TNF-?/ OSM stimulation, whereas no significant active ADAMTS-5 was detected in either extracts or supernatants; ii) Active ADAMTS-4 was primarily responsible for E373-374A 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 374ARGS aggrecan fragment in the TNF-?/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.

SUBMITTER: He Y

PROVIDER: S-EPMC4409205 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Publications

The effect of protease inhibitors on the induction of osteoarthritis-related biomarkers in bovine full-depth cartilage explants.

He Yi Y Zheng Qinlong Q Jiang MengMeng M Sun Shu S Christiansen Thorbjørn G TG Kassem Moustapha M Karsdal Morten A MA Bay-Jensen Anne C AC

PloS one 20150424 4

<h4>Objective</h4>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 inhibitor ...[more]

PMID: 25909781

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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.

Project description:BackgroundLimited chondrocyte migration and impaired cartilage-to-cartilage healing is a barrier in cartilage regenerative therapy. Collagenase treatment and delivery of a chemotactic agent may play a positive role in chondrocyte repopulation at the site of cartilage damage. This study evaluated chondrocyte migratory activity after enzymatic treatment in cultured cartilage explant. Differential effects of platelet-derived growth factor (PDGF) dimeric isoforms on the migratory activity were investigated to define major chemotactic factors for cartilage.MethodsFull-thickness cartilage (4-mm3 blocks) were harvested from porcine femoral condyles and subjected to explant culture. After 15 min or 60 min of actinase and collagenase treatments, chondrocyte migration and infiltration into a 0.5-mm cartilage gap was investigated. Cell morphology and lubricin, keratan sulfate, and chondroitin 4 sulfate expression in superficial- and deep-zone chondrocytes were assessed. The chemotactic activities of PDGF-AA, -AB, and -BB were measured in each zone of chondrocytes, using a modified Boyden chamber assay. The protein and mRNA expression and histological localization of PDGF-β were analyzed by western blot analysis, real-time reverse transcription polymerase chain reaction (RT-PCR), and immunohistochemistry, and results in each cartilage zone were compared.ResultsSuperficial-zone chondrocytes had higher migratory activity than deep-zone chondrocytes and actively bridged the cartilage gap, while metachromatic staining by toluidine blue and immunoreactivities of keratan sulfate and chondroitin 4 sulfate were detected around the cells migrating from the superficial zone. These superficial-zone cells with weak immunoreactivity for lubricin tended to enter the cartilage gap and possessed higher migratory activity, while the deep-zone chondrocytes remained in the lacuna and exhibited less migratory activity. Among PDGF isoforms, PDGF-AB maximized the degree of chemotactic activity of superficial zone chondrocytes. Increased expression of PDGF receptor-β was associated with higher migratory activity of the superficial-zone chondrocytes.ConclusionsIn enzymatically treated cartilage explant culture, chondrocyte migration and infiltration into the cartilage gap was higher in the superficial zone than in the deep zone. Preferential expression of PDGF receptor-β combined with the PDGF-AB dimeric isoform may explain the increased migratory activity of the superficial-zone chondrocytes. Cells migrating from superficial zone may contribute to cartilage regeneration.

Project description:Anterior cruciate ligament (ACL) transection surgery in the minipig induces post-traumatic osteoarthritis (PTOA) in a pattern similar to that seen in human patients after ACL injury. Prior studies have reported the presence of cartilage matrix-degrading proteases, such as Matrix metalloproteinase-1 (MMP-1) and A disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS-4), in the synovial fluid of injured or arthritic joints; however, the tissue origin of these proteases is unknown. The objective of this study was to identify transcriptional processes activated in the synovium after surgical induction of PTOA with ACL transection, and to determine if processes associated with proteolysis were enriched in the synovium after ACL transection. Unilateral ACL transection was performed in adolescent Yucatan minipigs and synovium samples were collected at 1, 5, 9, and 14 days post-injury. Transcriptome-wide gene expression levels were determined using bulk RNA-Sequencing in the surgical animals and control animals with healthy knees. The greatest number of transcripts with significant changes was observed 1 day after injury. These changes were primarily associated with cellular proliferation, consistent with measurements of increased cellularity of the synovium at the two-week time point. At five to 14 days, the expression of transcripts relating to proteolysis and cartilage development was significantly enriched. While protease inhibitor-encoding transcripts (TIMP2, TIMP3) represented the largest fraction of protease-associated transcripts in the uninjured synovium, protease-encoding transcripts (including MMP1, MMP2, ADAMTS4) predominated after surgery. Cartilage development-associated transcripts that are typically not expressed by synovial cells, such as ACAN and COMP, were enriched in the synovium following ACL-transection. The upregulation in both catabolic processes (proteolysis) and anabolic processes (cartilage development) suggests that the synovium plays a complex, balancing role in the early response to PTOA induction.

Dataset Information

The effect of protease inhibitors on the induction of osteoarthritis-related biomarkers in bovine full-depth cartilage explants.

Objective

Methods

Results

Conclusions

Publications

The effect of protease inhibitors on the induction of osteoarthritis-related biomarkers in bovine full-depth cartilage explants.

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