Table 1.
Overview of technical performance of the ELISA assays.
Fig 1.
Characterization of active ADAMTS-4 antibody, NB421-13F7, and active MMP-13 antibody, NB469-8C11.
(A) The binding of NB421-13F7 to biotinylated peptide was displaced by a specific peptide (SISRARQVEL), but not a elongated peptide (RSISRARQVEL). (B) Displacement of signal with the recombinant human ADAMTS-5 (starting from 80ng/ml to 10ng/ml in2-fold dilution steps) (C) The binding of NB469-8C11 to biotinylated peptide was displaced by a specific peptide (YNVFPRTLKW), but neither a elongated peptide (EYNVFPRTLKW) nor a cross peptide (YNFFPRKPKW, MMP2). (D) Displacement of signal by APMA-activated MMP-13form, but not Pro-MMP13 (starting from 80ng/ml to 10 ng/ml in 2-fold dilution steps). (E) Western blot of active ADAMTS-5 in the supernatant of T+Oinduced bovine cartilage explants (Lane1) and recombinant active ADAMTS-5(Ser262-Pro622) (Lane2). (F)Western Blot of active MMP-13 in APMA-activated recombinant of MMP-13 (Lane1) and the supernatant of T+O induced bovine cartilage explants, but not, recombinant Pro-MMP13 (Lane 2).
Fig 2.
Profiling of active aggrecanases, MMPs and their specific aggrecan degradation fragments into bovine explants upon T+O stimulation.
The releases of (A)active ADAMTS-4.: (D) active MMP-13, (E) active MMP-9, (F)AGNxII and (G)CTX-II started since day 14 and increased over time, while (B) active ADAMTS-5 was unable to be detected during the 21 days. (C) Aggrecanase-mediated aggrecan fragment’s levels peaked at day 7, afterwards, declined to background levels at day 21. No any release of any biomarkers was seen in the WO group. All values were shown as mean ±95% confidence intervals (n = 8 in each group). Error bars indicated the upper limit of 95% confidence intervals.
Fig 3.
The cartilage extracts by enzyme degradation method from WO group and T+O group were tested for 4 active proteases.
Cartilage explants from WO group and T+O group at day 7, 14 and 21 were extracted by using mean of enzymes as previously described [15]. (A) active ADAMTS-4 was accumulated in the WO group. However, stimulation of T+O significantly induced the elevated level of active ADAMTS-4 retained in the matrix. None of (B) active ADAMTS-5, (C) active MMP-13, (D) active MMP-9 was detected in untreated group or treated group. All values were shown as mean±95% confidence intervals (n = 4 in each group). Error bars indicated the upper limit of 95% confidence intervals.
Fig 4.
The effect of broad-spectrum inhibitors on the profiling of active aggrecanases and their specific aggrecan degradation fragments.
(A) active ADAMTS-4. (B) active ADAMTS-5. (C) AGNxI. All values were shown as mean±95% confidence intervals (n = 8 in each group). Error bars indicated the upper limit of 95% confidence intervals.
Fig 5.
The effect of broad-spectrum inhibitors on the profiling of active MMPs and their specific aggrecan and collagen degradation fragments.
(A) active MMP-13. (B) active MMP-9. (C) AGNxI. (D) CTX-II. All values were shown as mean±95% confidence intervals (n = 8 in each group). Error bars indicated the upper limit of 95% confidence intervals.
Table 2.
Area under the biomarker level vs. time curve (AUC).
Fig 6.
The schematic diagram of the effects of inhibitors on protease activation and cartilage degradation.
The broad MMP inhibitor, GM6001 (red color), almost completely inhibited the activation of Pro-ADAMTS-4, Pro-MMPs and cartilage breakdown, since the efficient gaining of activities of both pro-enzymes is MMP-dependent process. Addition with Furin-like convertase inhibitor, PCi (green color), decreased the release of active ADAMTS-4 and AGNxI induced by T+O, but induced huge release of active ADAMTS-5, suggesting active ADAMTS-4 is the predominant aggrecanase for the generation of AGNxI in T+O treated-bovine cartilage explants. PCi promoted the activation of MMP-13 and AGNxII, but not MMP-9. Blockade of cysteine proteases by E64 increased the levels of active ADAMTS-4 and the release of CTX-II, whereas had little effect on the activation of MMP-13 and MMP-9, demonstrating that other compensatory collagenases possibly were triggered following by the inhibition of cysteine protease activity. In summary, the activation of pro-enzyme is a key point in cartilage degradation of T+O treated bovine cartilage, which is regulated in an integrated cascade process.