Identification of important interactions between subchondral bone ...

Introduction
CHAPTER 7: PAPER IV
Cartilage turnover is a complex process in which proteases play a prominent role in both health
and disease. The extracellular matrix (ECM) in healthy articular cartilage is expressed and
regulated by the chondrocytes, which include expression of matrix proteins, proteases, and
protease inhibitors 1 . One of the major mediators of cartilage destruction in osteoarthritis (OA) is
the metalloproteinases; matrix metalloproteinases (MMPs) and A Disintegrin And
Metalloproteinase with Thrombospondin Motifs (ADAMTS’) 2 . In few words, the destruction of
ECM in OA results from initial aggrecan degradation by ADAMTS’ and subsequently
degradation by diverse MMPs that continues to degrade the major components of the ECM, e.g.
collagen type II and aggrecan 2,3,4 . However, cysteine proteases are also essential players in the
proteolytic cleavage of ECM in OA, which are synthesized both by the chondrocytes and
synovial cells in response to cytokines and growth factors 5 .
Currently, treatment of cartilage degenerative diseases is either analgesic or joint
replacement. Thus, the discovery and development of new treatments are needed. Since proteases
- such as MMPs and cathepsins - play a major role in the net degradation of cartilage, they are a
natural target for drug development. Early treatment with protease inhibitors to either reduce
inflammation and/or reduce aggrecan/collagen loss may have the potential to reduce the onset
of future idiopathic or posttraumatic osteoarthritis. However, to target the right protease at the
right time during the progression of OA, a better understanding of the proteolytic processes is
needed. ECM-neoepitope biomarkers are great tools for investigating the proteolytic processes 6 .
The collagen type II degradation marker, C-terminal telopeptide degradation fragment (CTX-II) 7
and the aggrecan degradation marker, AGNx-II 8 , are both MMP-specific. The novel collagen
type II intra-helical degradation markers, NBC2, are supposed to represent cathepsin-specificity.
However, the biomarker will be evaluated in the present study.
In this study, we identified which MMPs, ADAMTS’ and cathepsins were able to
generate CTX-II, NBC2, and AGNx-II fragments using an in vitro and ex vivo model of human
OA cartilage. Such data could provide information on the specificity of the different proteases
for cleavage of collagen and aggrecan, and also inform us about the proteolytic pathways for each
biomarker release. Furthermore, the endogenous proteases in normal bovine cartilage were
compared with cytokine-stimulated bovine and human OA cartilage. The cytokines, oncostatin M
(OSM) and tumor necrosis factor-α (TNF-α), are inducers of cartilage degradation in vivo and in
vitro 9 , and would allow us to induce a controlled catabolic system towards the one seen in human
OA cartilage. I.e. induction of aggrecanases after 4 days with cytokine stimulation and induction
of MMPs after 11 days with cytokine stimulation 3 . By inhibiting one type of protease, we aimed
to find the proteases responsible for collagen type II and aggrecan degradation and possible
masking effects by other proteases.
91