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Cartilage degradation is fully reversible in the presence of aggrecanase but not matrix metalloproteinase activity.

Karsdal MA, Madsen SH, Christiansen C, Henriksen K, Fosang AJ, Sondergaard BC - Arthritis Res. Ther. (2008)

Bottom Line: Highly distinct protease activities were found with aggrecanase-mediated aggrecan degradation at early stages, whereas MMP-mediated aggrecan and collagen degradation occurred during later stages.By histology, we found a complete replenishment of glycosaminoglycan at early time points and pericellular localization at an intermediate time point.In contrast, only significantly increased collagen type II formation (200%; P < 0.01) was observed at early time points.

View Article: PubMed Central - HTML - PubMed

Affiliation: Nordic Bioscience A/S, Herlev Hovedgade 207, DK-2730 Herlev, Denmark. mk@nordicbioscience.com

ABSTRACT

Introduction: Physiological and pathophysiological cartilage turnover may coexist in articular cartilage. The distinct enzymatic processes leading to irreversible cartilage damage, compared with those needed for continuous self-repair and regeneration, remain to be identified. We investigated the capacity of repair of chondrocytes by analyzing their ability to initiate an anabolic response subsequent to three different levels of catabolic stimulation.

Methods: Cartilage degradation was induced by oncostatin M and tumour necrosis factor in articular cartilage explants for 7, 11, or 17 days. The catabolic period was followed by 2 weeks of anabolic stimulation (insulin growth factor-I). Cartilage formation was assessed by collagen type II formation (PIINP). Cartilage degradation was measured by matrix metalloproteinase (MMP) mediated type II collagen degradation (CTX-II), and MMP and aggrecanase mediated aggrecan degradation by detecting the 342FFGVG and 374ARGSV neoepitopes. Proteoglycan turnover, content, and localization were assessed by Alcian blue.

Results: Catabolic stimulation resulted in increased levels of cartilage degradation, with maximal levels of 374ARGSV (20-fold induction), CTX-II (150-fold induction), and 342FFGVG (30-fold induction) (P < 0.01). Highly distinct protease activities were found with aggrecanase-mediated aggrecan degradation at early stages, whereas MMP-mediated aggrecan and collagen degradation occurred during later stages. Anabolic treatment increased proteoglycan content at all time points (maximally, 250%; P < 0.001). By histology, we found a complete replenishment of glycosaminoglycan at early time points and pericellular localization at an intermediate time point. In contrast, only significantly increased collagen type II formation (200%; P < 0.01) was observed at early time points.

Conclusion: Cartilage degradation was completely reversible in the presence of high levels of aggrecanase-mediated aggrecan degradation. After induction of MMP-mediated aggrecan and collagen type II degradation, the chondrocytes had impaired repair capacity.

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Quantification of aggrecan and collagen degradation products at days 7, 11, and 17. Articular cartilage explants were cultured in the presence or absence of oncostatin M plus tumour necrosis factor (OSM + TNF). Conditioned medium was collected at days 7, 11, and 17. (a) Aggrecanase-mediated aggrecan degradation was measured by the 374ARGSV-G2 enzyme-linked immunosorbent assay (ELISA), (b) matrix metalloproteinase (MMP)-mediated aggrecan degradation was quantified by the 342FFGVG-G2 ELISA, and (c) MMP-mediated collagen type II degradation was quantified in the CTX-II ELISA. **P < 0.01, ***P < 0.001. CTX-II, crosslinked C-terminal neo-epitopes of type II collagen.
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Figure 3: Quantification of aggrecan and collagen degradation products at days 7, 11, and 17. Articular cartilage explants were cultured in the presence or absence of oncostatin M plus tumour necrosis factor (OSM + TNF). Conditioned medium was collected at days 7, 11, and 17. (a) Aggrecanase-mediated aggrecan degradation was measured by the 374ARGSV-G2 enzyme-linked immunosorbent assay (ELISA), (b) matrix metalloproteinase (MMP)-mediated aggrecan degradation was quantified by the 342FFGVG-G2 ELISA, and (c) MMP-mediated collagen type II degradation was quantified in the CTX-II ELISA. **P < 0.01, ***P < 0.001. CTX-II, crosslinked C-terminal neo-epitopes of type II collagen.

Mentions: To further characterize the molecular mechanism underlying the loss of repair capacity, we measured levels of the catabolic biomarkers 374ARGSV, 342FFGVG, and CTX-II after the individual catabolic treatments. We found that OSM + TNF-stimulated degradation, mediated by aggrecanases and measured using the 374ARGSV-G2 assay, was high at day 7, intermediate at day 10, and almost absent at day 17 (Figure 3a). This is consistent with the S-GAG release data showing that the majority of S-GAGs are released at the early stages of catabolic stimulation. The levels of the MMP-generated fragment 342FFGVG-G2 showed that MMP-mediated aggrecan was undetectable at days 7 and 10 and high at day 17 (Figure 3b). The high aggrecanase activity at the early stages of culture may mask the MMP-mediated aggrecan epitope (342FFGVG-G2) by further processing in generating the aggrecanase (374ARGSV) site; however, Fosang and colleagues [32] have found that further processing of 342FFGVG to generate 374ARGSV cannot occur, at least not in vitro. High levels of MMP activity should have generated CTX-II fragments that are not further processed by other proteases, suggesting that MMP activity is present only at a lower level at early culture time points. This was verified by the use of a fluorescence substrate technique, in which MMP levels were detectable only in the presence of catabolic stimulation and only at late time points (data not shown), which correlate well with previous findings, documenting extensive MMP activities at later stages of catabolic induction but not at early stages [6,9,32]. Interestingly, most S-GAG is released at earlier time points than the 342FFGVG release, indicating that aggrecan loss is due primarily to aggrecanase activity, but later, aggrecanolysis shifts to an MMP-mediated degradation mode. Finally, we found that the release of the collagen type II degradation fragment CTX-II (Figure 3c) occurred with a pattern similar to that of 342FFGVG (Figure 3b), consistent with the fact that the CTX-II fragment is MMP-generated [33].


Cartilage degradation is fully reversible in the presence of aggrecanase but not matrix metalloproteinase activity.

Karsdal MA, Madsen SH, Christiansen C, Henriksen K, Fosang AJ, Sondergaard BC - Arthritis Res. Ther. (2008)

Quantification of aggrecan and collagen degradation products at days 7, 11, and 17. Articular cartilage explants were cultured in the presence or absence of oncostatin M plus tumour necrosis factor (OSM + TNF). Conditioned medium was collected at days 7, 11, and 17. (a) Aggrecanase-mediated aggrecan degradation was measured by the 374ARGSV-G2 enzyme-linked immunosorbent assay (ELISA), (b) matrix metalloproteinase (MMP)-mediated aggrecan degradation was quantified by the 342FFGVG-G2 ELISA, and (c) MMP-mediated collagen type II degradation was quantified in the CTX-II ELISA. **P < 0.01, ***P < 0.001. CTX-II, crosslinked C-terminal neo-epitopes of type II collagen.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Quantification of aggrecan and collagen degradation products at days 7, 11, and 17. Articular cartilage explants were cultured in the presence or absence of oncostatin M plus tumour necrosis factor (OSM + TNF). Conditioned medium was collected at days 7, 11, and 17. (a) Aggrecanase-mediated aggrecan degradation was measured by the 374ARGSV-G2 enzyme-linked immunosorbent assay (ELISA), (b) matrix metalloproteinase (MMP)-mediated aggrecan degradation was quantified by the 342FFGVG-G2 ELISA, and (c) MMP-mediated collagen type II degradation was quantified in the CTX-II ELISA. **P < 0.01, ***P < 0.001. CTX-II, crosslinked C-terminal neo-epitopes of type II collagen.
Mentions: To further characterize the molecular mechanism underlying the loss of repair capacity, we measured levels of the catabolic biomarkers 374ARGSV, 342FFGVG, and CTX-II after the individual catabolic treatments. We found that OSM + TNF-stimulated degradation, mediated by aggrecanases and measured using the 374ARGSV-G2 assay, was high at day 7, intermediate at day 10, and almost absent at day 17 (Figure 3a). This is consistent with the S-GAG release data showing that the majority of S-GAGs are released at the early stages of catabolic stimulation. The levels of the MMP-generated fragment 342FFGVG-G2 showed that MMP-mediated aggrecan was undetectable at days 7 and 10 and high at day 17 (Figure 3b). The high aggrecanase activity at the early stages of culture may mask the MMP-mediated aggrecan epitope (342FFGVG-G2) by further processing in generating the aggrecanase (374ARGSV) site; however, Fosang and colleagues [32] have found that further processing of 342FFGVG to generate 374ARGSV cannot occur, at least not in vitro. High levels of MMP activity should have generated CTX-II fragments that are not further processed by other proteases, suggesting that MMP activity is present only at a lower level at early culture time points. This was verified by the use of a fluorescence substrate technique, in which MMP levels were detectable only in the presence of catabolic stimulation and only at late time points (data not shown), which correlate well with previous findings, documenting extensive MMP activities at later stages of catabolic induction but not at early stages [6,9,32]. Interestingly, most S-GAG is released at earlier time points than the 342FFGVG release, indicating that aggrecan loss is due primarily to aggrecanase activity, but later, aggrecanolysis shifts to an MMP-mediated degradation mode. Finally, we found that the release of the collagen type II degradation fragment CTX-II (Figure 3c) occurred with a pattern similar to that of 342FFGVG (Figure 3b), consistent with the fact that the CTX-II fragment is MMP-generated [33].

Bottom Line: Highly distinct protease activities were found with aggrecanase-mediated aggrecan degradation at early stages, whereas MMP-mediated aggrecan and collagen degradation occurred during later stages.By histology, we found a complete replenishment of glycosaminoglycan at early time points and pericellular localization at an intermediate time point.In contrast, only significantly increased collagen type II formation (200%; P < 0.01) was observed at early time points.

View Article: PubMed Central - HTML - PubMed

Affiliation: Nordic Bioscience A/S, Herlev Hovedgade 207, DK-2730 Herlev, Denmark. mk@nordicbioscience.com

ABSTRACT

Introduction: Physiological and pathophysiological cartilage turnover may coexist in articular cartilage. The distinct enzymatic processes leading to irreversible cartilage damage, compared with those needed for continuous self-repair and regeneration, remain to be identified. We investigated the capacity of repair of chondrocytes by analyzing their ability to initiate an anabolic response subsequent to three different levels of catabolic stimulation.

Methods: Cartilage degradation was induced by oncostatin M and tumour necrosis factor in articular cartilage explants for 7, 11, or 17 days. The catabolic period was followed by 2 weeks of anabolic stimulation (insulin growth factor-I). Cartilage formation was assessed by collagen type II formation (PIINP). Cartilage degradation was measured by matrix metalloproteinase (MMP) mediated type II collagen degradation (CTX-II), and MMP and aggrecanase mediated aggrecan degradation by detecting the 342FFGVG and 374ARGSV neoepitopes. Proteoglycan turnover, content, and localization were assessed by Alcian blue.

Results: Catabolic stimulation resulted in increased levels of cartilage degradation, with maximal levels of 374ARGSV (20-fold induction), CTX-II (150-fold induction), and 342FFGVG (30-fold induction) (P < 0.01). Highly distinct protease activities were found with aggrecanase-mediated aggrecan degradation at early stages, whereas MMP-mediated aggrecan and collagen degradation occurred during later stages. Anabolic treatment increased proteoglycan content at all time points (maximally, 250%; P < 0.001). By histology, we found a complete replenishment of glycosaminoglycan at early time points and pericellular localization at an intermediate time point. In contrast, only significantly increased collagen type II formation (200%; P < 0.01) was observed at early time points.

Conclusion: Cartilage degradation was completely reversible in the presence of high levels of aggrecanase-mediated aggrecan degradation. After induction of MMP-mediated aggrecan and collagen type II degradation, the chondrocytes had impaired repair capacity.

Show MeSH
Related in: MedlinePlus