Identification of important interactions between subchondral bone ...

CHAPTER 7: PAPER IV
The MMP inhibitor, GM6001, was able to attenuate the levels of AGNx-II in normal bovine
cartilage, whereas the pre-cultured bovine cartilage was not affected by the inhibitor (Table 2).
The bovine cartilage pre-cultured with cytokines for 4 and 11 days before metabolic inactivation
and incubation with cysteine buffer, decreased the levels of AGNx-II compared to normal
bovine cartilage (Table 2), which was the opposite effects from the one seen in the MMP buffer.
Furthermore, the human OA cartilage had levels of AGNx-II as the normal bovine cartilage
(Table 2). The cysteine protease inhibitor, E64, did not affect the normal or pre-cultured bovine
samples, whereas E64 increased the levels significantly in the human OA samples (Table 2).
Discussion
The aim of our study was to understand the molecular foundation for the progression of articular
cartilage degradation that leads to OA. We analyzed the release of cartilage degradation
biomarkers from human OA cartilage by the proteinases reported to play a role in cartilage
degradation 5,10 . Furthermore, we investigated how diseased cartilage is altered from normal
cartilage with regards to the endogenous proteases, and we were observant to possible masking
effects among the proteases.
We showed that the cartilage degradation markers, CTX-II, NBC2, and AGNx-II,
are released in part by different proteolytic pathways in human OA cartilage, as different
proteases generated different biomarkers (fig. 1). The biomarkers were only released by specific
metalloproteinases, and none of the supplied cathepsins (fig. 1). Moreover, Cathepsin-B (and a
tendency for Cathepsin-K and Cathepsin-L) inhibited the release of CTX-II in cysteine buffer,
suggesting either a protective effect by the cathepsins on cartilage degradation or more likely a
masking effect (fig. 2) from post-cleavage of CTX-II fragments by the cathepsins. The aggrecan
degradation increased in the presence of both MMPs and aggrecanases, although AGNx-II is
MMP-specific (fig. 1G), suggesting pre- or post-cleavages by the ADAMTS’ may aid the
degradation.
In human OA cartilage explants, the collagen type II and aggrecan may already be
severely degraded by the increased production of proteases during the disease 10 , so further
protease-cleavage ex vivo may be difficult to detect, as specific targets measurable by our
biomarkers have already been cleaved and further processed, thereby masking the effect from the
MMPs and cysteine proteases we tested. This possible masking effect could be investigated by a
follow up study, testing the library of proteases in normal cartilage and comparing the results
with those from the present study in human OA cartilage.
The endogenous proteases in human OA samples activated in the MMP buffer
(Table 1) failed to increase the levels of CTX-II and NBC2. One suggestion could be that the
damaged OA cartilage had less chondrocytes and ECM proteins, and thus fewer enzymes
present, compared to normal cartilage. This is supported by the increased levels of CTX-II and
NBC2, when supplying MMP-7 and MMP-9 (fig.1). However, as we were able to decrease MMP-
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