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

Wang B, Chen P, Jensen AC, Karsdal MA, Madsen SH, Sondergaard BC, Zheng Q, Qvist P - BMC Res Notes (2009)

Bottom Line: In parallel, explants were co-cultured with protease inhibitors such as GM6001, TIMP1, TIMP2 and TIMP3.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Nordic Bioscience A/S, Zhongguancun Life Science Park, Beijing 102206, PR China.

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 (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.

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.

No MeSH data available.


Related in: MedlinePlus

Epitope-specificity of monoclonal antibodies binding to aggrecan fragments. Schematic representation of the monoclonal antibodies selected for incorporation into immunoassays for quantification of aggrecan fragments carrying neo-epitopes. MAb F78 recognises a repeated epitope in the G1 and G2 domain, MAb AF28 binds to the MMP-derived neo-epitope 342FFGFG, MAb 1H11 binds the neo-epitope NITEGE373, and MAb 6D6 binds to 374ARGSV.
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Figure 1: Epitope-specificity of monoclonal antibodies binding to aggrecan fragments. Schematic representation of the monoclonal antibodies selected for incorporation into immunoassays for quantification of aggrecan fragments carrying neo-epitopes. MAb F78 recognises a repeated epitope in the G1 and G2 domain, MAb AF28 binds to the MMP-derived neo-epitope 342FFGFG, MAb 1H11 binds the neo-epitope NITEGE373, and MAb 6D6 binds to 374ARGSV.

Mentions: MAb 1H11 and MAb 6D6 were incorporated into competitive immunoassays as well as sandwich assays with MAb F78. The latter antibody recognized a repeated sequence in both the G1 and the G2 domain [16], thereby making it useful in sandwich constructions with antibodies binding to neo-epitopes in the interglobular domain (figure 1).


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

Wang B, Chen P, Jensen AC, Karsdal MA, Madsen SH, Sondergaard BC, Zheng Q, Qvist P - BMC Res Notes (2009)

Epitope-specificity of monoclonal antibodies binding to aggrecan fragments. Schematic representation of the monoclonal antibodies selected for incorporation into immunoassays for quantification of aggrecan fragments carrying neo-epitopes. MAb F78 recognises a repeated epitope in the G1 and G2 domain, MAb AF28 binds to the MMP-derived neo-epitope 342FFGFG, MAb 1H11 binds the neo-epitope NITEGE373, and MAb 6D6 binds to 374ARGSV.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2803187&req=5

Figure 1: Epitope-specificity of monoclonal antibodies binding to aggrecan fragments. Schematic representation of the monoclonal antibodies selected for incorporation into immunoassays for quantification of aggrecan fragments carrying neo-epitopes. MAb F78 recognises a repeated epitope in the G1 and G2 domain, MAb AF28 binds to the MMP-derived neo-epitope 342FFGFG, MAb 1H11 binds the neo-epitope NITEGE373, and MAb 6D6 binds to 374ARGSV.
Mentions: MAb 1H11 and MAb 6D6 were incorporated into competitive immunoassays as well as sandwich assays with MAb F78. The latter antibody recognized a repeated sequence in both the G1 and the G2 domain [16], thereby making it useful in sandwich constructions with antibodies binding to neo-epitopes in the interglobular domain (figure 1).

Bottom Line: In parallel, explants were co-cultured with protease inhibitors such as GM6001, TIMP1, TIMP2 and TIMP3.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Nordic Bioscience A/S, Zhongguancun Life Science Park, Beijing 102206, PR China.

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 (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.

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.

No MeSH data available.


Related in: MedlinePlus