**5. References**

Ahn, J.K.; Koh, E.M., Cha, H.S., Lee, Y.S., Kim, J., Bae, E.K. & Ahn, K.S. (2008). Role of hypoxia-inducible factor-1alpha in hypoxia-induced expression of IL-8, MMP-1 and MMP-3 in rheumatoid fibroblast-like synoviocytes. *Rheumatology (Oxford),* Vol.47, No.6, pp. 834-839, ISSN: 1462-0324

studied in the extracellular fraction. Both antibodies detected both the zymogen (upper

Nevetheless, it was interesting to find that three MMPs previously correlated to the invasive potential of rheumatoid synovial fibroblasts, were up-regulated by fibrin at the mRNA level (Tolboom et al., 2002). Due to its insolubility, fibrin might be degraded in a non-constitutive way by MMP-3 secreted by surrounding cells (Bini et al., 1999). On the balance of the evidence, we believe that fibrin-rich regions should not be considered a result of long-

In agreement with previous studies, most of the synovial fibroblast cultures that we employed in our studies did not constitutively express MMP-13 as assessed with qPCR techniques, and only in some was it induced after exposure to fibrin (Moore et al., 2000). In the production of this, as well as the other proteases tested, there seemed to be a high variability between patients. In fact, our studies suggested the existence of two subsets according to their response to fibrin. Approximately half of the cultures strongly reacted with the up-regulation of MMPs, while the other half showed mild or absent response. We believe that this is another example of the heterogeneous character of rheumatoid arthritis. Although there was no regulation of MT1-MMP at the mRNA level by fibrin, an interesting finding drawn by our experiments was the high expression of MT1-MMP by unstimulated rheumatoid synovial cells, pointing to the prominent role of the protease in the activity of

In summary, using a novel culture system for the study of fibrin interaction with synovial cells, we could show induction of proteases putatively associated to invasiveness, that were

In this chapter, we give an overview of the role of fibrin in the pathogenesis of synovitis. In doing so, we have neither solved the paradigm about what starts first nor discovered what triggers the invasive behaviour of rheumatoid synovial fibroblasts. Instead we provide some lines of thinking supporting the "inside – out pathway" as it has recently been named

The "fibrin pathway" has been little explored in rheumatoid arthritis therapeutics. The balance between clotting and dissolution of fibrin is a candidate process to target that could

We thank Conchita Rábago Foundation (FCR) and the Foundation of the Spanish Society for

Ahn, J.K.; Koh, E.M., Cha, H.S., Lee, Y.S., Kim, J., Bae, E.K. & Ahn, K.S. (2008). Role of

hypoxia-inducible factor-1alpha in hypoxia-induced expression of IL-8, MMP-1 and MMP-3 in rheumatoid fibroblast-like synoviocytes. *Rheumatology (Oxford),* Vol.47,

band) and the active protease (lower band).

standing inflammation, but a site for active destruction.

synovial fibroblasts as already suggested (Miller et al., 2009).

further localized at fibrin-rich areas in the synovial tissues.

**3. Conclusion**

(Schett & Firestein, 2010).

**4. Acknowledgements** 

**5. References** 

help to retard joint destruction.

Rheumatology (FER) for their financial support.

No.6, pp. 834-839, ISSN: 1462-0324


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**8** 

*Sweden* 

**Glycoproteomics of Lubricin-Implication** 

The dynamic milieu of synovial fluid is of particular interest for biomarker discovery of joint related diseases as it is composed not only of ultra-filtrated proteins originating in serum, but also proteins exclusively expressed and secreted by cells localized within the synovial membrane, fluid or cartilage. Lubricin (proteoglycan 4, prg4) is an abundant mucinous and secretory glycoprotein (~227 to 345 kDa) in synovial fluid (SF) and one of the factors considered responsible for boundary lubrication of diarthrodial joints (Swann et al., 1981; Swann et al., 1985; Jay, 1992). Lubricin is encoded by gene *PRG4* and synthesized in synovial fibroblasts (synoviocytes) and superficial zone chondrocytes. Different transcripts of *PRG4* have been referred to as superficial zone protein (SZP), megakaryocyte stimulating factor (MSF) precursor, camptodactyly arthropathy coxa vara pericarditis (CACP) protein, and hemangiopoietin (HAPO), which has recently been reviewed by Bao et al (Bao et al., 2011). As a primarily lubricating glycoprotein, lubricin has been found in SF, superficial layer of articular cartilage, tendons, and menisci (Schumacher et al., 1994; Schumacher et al., 1999; Rees et al., 2002; Rhee et al., 2005b; Schumacher et al., 2005; Sun et al., 2006). This tissuespecific distribution makes lubricin a potential biomarker during the exacerbation of chronic

Human synovial lubricin (1404 amino acids) has a large and central mucin-like domain characterized with 59 imperfect repeating units of EPAPTTPK which is subject to extensive *O*linked glycosylation. The abundance of negatively charged sugars in this domain contributes to the protein's boundary lubrication of the cartilage surface due to strong repulsive hydration forces (Jay, 1992). The mucin domain is flanked by a *C*-terminal hemopexin (PEX)-like domain and two somatomedin B (SMB)-like domains at its *N-*terminus (Flannery et al., 1999; Schumacher et al., 1999; Ikegawa et al., 2000). The two *N*-terminal SMB-like domains have 60% similarity to that of vitronectin, while *C*-terminal PEX-like domain also shows similarity to domains in vitronectin (40-50%) as well as to the matrix metalloproteinase (MMPs) family. Purified serum hemopexin has been showed to interact with hyaluronan, suggesting that the PEX-like domain in lubricin may also medicate the binding of lubricin to hyaluronan at or near cartilage surface (Hrkal et al., 1996). In addition to boundary lubrication, lubricin protects

cartilage surfaces from protein deposition and cell adhesion (Rhee et al., 2005b).

**1. Introduction** 

articular inflammation.

Corresponding author

 \*

**of Important Biological Glyco- and Peptide-Epitopes in Synovial Fluid** 

Liaqat Ali, Chunsheng Jin and Niclas G. Karlsson\*

*Medical Biochemistry, University of Gothenburg* 

