AB0171 Aggrecan Degradation Is Not Just Aggrecan Degradation – A Study of Neo-Epitopes (NITEGE and ARGS) of Aggrecanase Degradation

Background

Cartilage degradation is a hallmark of arthritic disease. The main constituents of cartilage is aggrecan and type II collagen. Previous studies have shown that aggrecan degradation is prior to type II collagen degradation and that only aggrecanase degradation but not matrix metalloproteinases (MMP) degradation of aggrecan was reversible¹. It is therefore essential to have biomarkers that can detect reversible and irreversible aggrecan degradation to identify the condition of the cartilage. Of the well-known aggrecanase degradation site at NITEGE^373_374ARGS, ARGS is released by only aggrecanse, but NITEGE is retained in the aggrecan molecule and in theory needs additional aggrecan degradation to be released. The numbers indicate the aggrecanase cleavage site.

Objectives

Investigate the profile of two neo-epitope biomarkers of aggrecanase degraded aggrecan at NITEGE373–374ARGS in a bovine cartilage explants model.

Methods

Catabolic stimulated (oncostatin M and TNF-a; O+T) bovine cartilage explants were treated with or without the generic MMP inhibitor, GM6001. Explants without catabolic stimulation was used as negative control. In the culture supernatant two biomarkers investigating aggrecanase degraded aggrecan was measured. The AGNxI competitive ELISA detects the NITEGE³⁷³ neo-epitope and the ARGS sandwich ELISA detects the ³⁷⁴ARGSVI neo-epitope. Statistical differences between groups were tested by two-way ANOVA.

Results

The AGNxI level was significantly increased compared to w/o from day 5 to day 17. GM6001 significantly lowered the release of AGNxI compared to O+T alone at day 7 (p<0.0001), day 10 (p=0.0007) and 12 (p=0.01). In addition, GM6001 shifted the release to 2days later in the culture period, as the AGNxI level was first significantly increased compared to w/o at day 7 to day 17, where the level was higher than O+T, but non-significant. The ARGS level was significantly increased at day 5, 10 and 12 for both O+T and O+T + GM6001 compared to w/o. At day 10 the ARGS level was higher in O+T + GM6001 than O+T (p<0.0001).

Conclusions

The prolonged release of AGNxI and the significantly lowered level of AGNxI with the MMP-inhibitor, suggest that the AGNxI release is somewhat dependent of MMP activity. In contrast, the ARGS release seems not to be MMP-dependent as there was a significant increase in O+T with the MMP-inhibitor compared to w/o and the peak was pronounced. In summary, aggrecan degradation is not just aggrecan degradation and different neo-epitopes have different importance in cartilage degradation.

References

Arthritis Res Ther. 2008;10(3):R63

Disclosure of Interest

A. S. Siebuhr Employee of: NORDIC BIOSCIENCE A/S, Y. He Employee of: NORDIC BIOSCIENCE A/S, S. Hoielt: None declared, Y. Luo: None declared, M. Karsdal Shareholder of: NORDIC BIOSCIENCE A/S, Employee of: NORDIC BIOSCIENCE A/S, A.-C. Bay-Jensen Employee of: NORDIC BIOSCIENCE A/S