USOS DEL SUELO RURAL Y AREAS DE PRODUCCIÓN AGROPECUARIA Y FORESTAL

a. Collagen degradation

Fibrillar collagen in its native state is resistant to breakdown by proteolytic enzymes. However, it may be damaged by helical cleavage resulting in denaturation or by telopeptide cleavage, which can lead to the removal of cross-links and depolymerisation of the fibrillar network (Barrett, 1978). At neutral pH the breakdown of the helices may occur as a consequence of MMP-13 and to a lesser extent MMP-1 and MMP-8 (Knauper et al., 1996) at a specific site Gly-Leu/Ileu bond generating the characteristic ¾ and ¼ fragments. These fragments may then be degraded further by MMP-1 or by enzymes with gelanotolytic activity such as MMP-2, MMP-9, neutrophil elastase and plasmin at neutral pH and at acidic pH by cathepsin B, S, L (Kafienah et al., 1998). Additionally it has also been demonstrated that cathepsin K cleaves collagen II in the N-terminal region of the helical domain (Kafienah et al., 1998).

25 b. Aggrecan degradation

Under pathological conditions aggrecan degradation occurs as an early event and is mediated by proinflammatory cytokines within the joint (Hubbard et al., 2000). Aggrecan degradation products are derived predominantly from two protease families the aggrecanases and the MMPs. Although aggrecan fragments found in- vivo are predominantly the products of aggrecanase activity, the MMPs are also involved (Fosang et al., 1996b; Struglics et al., 2006). Some studies have indicated a lack of MMP involvement in aggrecan degradation in short term studies of explants with cytokines (Durigova et al., 2008) others have demonstrated that they may be involved in later stages of OA (van Meurs et al., 1999). Indeed, Durigova et al. 2011 (Durigova et al., 2011) demonstrated MMP mediated aggrecan degradation within the IGD was only evident after day 12 of bovine cartilage explant culture.

Many in-vitro cartilage studies using agents such as IL-1, retinoic acid and TNF-α have been identified as promoting cartilage degradation (Ismaiel et al., 1992; MacDonald et al., 1992). These agents have been identified as causing the up- regulation of some MMPs in chondrocytes which suggested a link between MMP expression and cartilage degradation. Additionally specific MMP inhibitors prevent loss of aggrecan from cartilage explant cultures in-vivo (Cawston et al., 1999; Mort et al., 1993).

b. Aggrecanases

The ADAMTSs family is made up of 19 proteases which are expressed in a wide range of tissues, but are more restricted in foetal tissues. The ADAMTS family are multi-domain metalloproteases that are secreted into the extracellular space as furin-active proteases (Bondeson et al., 2008). They are composed of a signal sequence, prodomain, catalytic domain, disintegrin-like domain, spacer region, thrombospondin motifs (TSP) and submotifs (Figure 1.6).

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Figure 1.6. Schematic representation of the domain structure of ADAMTS-1, -4, and -5 involved in aggrecan degradation.

The latter regulate both their activity and substrate specificity (Bondeson et al., 2008). A subgroup of the enzymes, ADAMTS-1, 4, 5, 7, 8, 9, 12 and 15 have roles in the pathogenesis of arthritis with the ability to degrade aggrecan within the cartilage ECM (Hoch et al., 2011). Expression of ADAMTS-1, -4, -5, -9, and -15 has been found in normal HAC (Bau et al., 2002; Bondeson et al., 2008; Wachsmuth et al., 2004). However studies have demonstrated conflicting information on expression changes of these potential aggrecanases in OA compared with normal human cartilage. Both increased (Bau et al., 2002; Wachsmuth et al., 2004) and decreased (Kevorkian et al., 2004) levels being reported.

ADAMTS-4 and ADAMTS-5 are the pertinent enzymes in the pathogenesis of OA as demonstrated by their high in-vitro activity for aggrecan cleavage, expression in OA cartilage and localized expression in areas of aggrecan degradation (Fosang et al., 2008). Work in human chondrocytes and explants revealed that both ADAMTS-4

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and -5 were important mediators of cytokine stimulated aggrecan loss in normal and OA cartilage. It was hypothesised that this may be due to a greater induction of ADAMTS-4 in man in the presence of catabolic stimuli (Song et al., 2007). Indeed it is still not clear if ADAMTS-4 or -5 is the major aggrecanases in man as ADAMTS-5 is constitutively expressed in human chondrocytes and synovial fibroblasts, were as previously mentioned in cytokine induced degradation ADAMTS-4 expression is predominant (Fosang et al., 2008). A recent study using equine cartilage revealed an significant increase in ADAMTS-5 in synovial tissue form OA joints whilst OA cartilage revealed a significant increase in ADAMTS-4, indicating it may be the principle aggrecanases in equine OA (Kamm et al., 2010). Furthermore the use of transgenic mice has enabled the analysis of aggrecanolysis from in- vitro cultures of mouse femoral head cartilage which has helped to determine further the role of these enzymes in aggrecan degradation (Stanton et al., 2011).

Aggrecan is the first matrix component to experience measurable loss in OA (Mankin and Lippiello, 1970). Aggrecanase-mediated aggrecan degradation is one of the significant early events in this disease (Huang and Wu, 2008). Degradation is due to increased proteolytic cleavage of the aggrecan interglobular domain (IGD) (Sandy, 2006). Its loss is attributed to its accelerated degradation within the two major proteolytic cleavage sites of the IGD; the Asn341-Phe342 and Glu373-Ala374 bonds. As discussed earlier it is known that MMPs can cleave the Asn341-Phe342 bond. The signature activity of the aggrecanases; primarily ADAMTS-4 and ADAMTS-5 is the cleavage of the Glu373-Ala374 bond (Tortorella et al., 2000b). The degradation products cleaved at the Glu373-Ala374 bond have been detected in cartilage explants and chondrocyte culture (Lark et al., 1995; Loulakis et al., 1992; MacDonald et al., 1992; Sandy et al., 1991) and in the synovial fluids of patients with joint disease (Sandy et al., 1992) An assay detecting cleavage at this site has recently been developed using an immunoaffinity based LC/MS/MS (Dufield et al., 2010a). In addition to the two major cleavage sites within the IGD, proteolysis of aggrecan in-vivo also occurs within the CS domain (Ilic et al., 1995; Loulakis et al., 1992) (Figure 1.7) and studies using recombinant ADAMTS-4 and ADAMTS-5 indicate preferential cleavage of aggrecan in the CS-2 domain (Tortorella et al.,

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2002; Tortorella et al., 2000b). In bovine aggrecan the two most favoured cleavage sites correspond in human to FKEEE1714-1715GLGSV and ASELE1545-1546GRGTI. Cleavage then occurs at the signature site in the IGD and in the corresponding human sequences at PTAQE1819-1820AGEGP and TISQE1919-1920LGQRP. Similar cleavage preferences have been identified by native aggrecanases in chondrocytes culture (Sandy and Verscharen, 2001).

The proteolysis within the CS attachment domain has been demonstrated in cartilage explant cultures treated with IL-1 or retinoic acid (Ilic et al., 1995; Loulakis et al., 1992) and in the synovial fluids from arthritic joints (Lohmander et al., 1993b). Sequencing analyses revealed that these cleavage sites are located in gap regions, which are relatively devoid of glycosaminoglycan chains, within the CS domain (Sandy et al., 1995).