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Dramatic changes occur in skin as a function of age, including changes in morphology, physiology, and mechanical properties. Changes in extracellular matrix molecules also occur, and these changes likely contribute to the overall age-related changes in the physical properties of skin. The major proteoglycans detected in extracts of human skin are decorin and versican. In addition, adult human skin contains a truncated form of decorin, whereas fetal skin contains virtually undetectable levels of this truncated decorin. Analysis of this molecule, herein referred to as decorunt, indicates that it is a catabolic fragment of decorin rather than a splice variant. With antibody probes to the core protein, decorunt is found to lack the carboxyl-terminal portion of decorin. Further analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry shows that the carboxyl terminus of decorunt is at Phe(170) of decorin. This result indicates that decorunt represents the amino-terminal 43% of the mature decorin molecule. Such a structure is inconsistent with alternative splicing of decorin and suggests that decorunt is a catabolic fragment of decorin. A neoepitope antiserum, anti-VRKVTF, was generated against the carboxyl terminus of decorunt. This antiserum does not recognize intact decorin in any skin proteoglycan sample tested on immunoblots but recognizes every sample of decorunt tested. The results with anti-VRKVTF confirm the identification of the carboxyl terminus of decorunt. Analysis of collagen binding by surface plasmon resonance indicates that the affinity of decorunt for type I collagen is 100-fold less than that of decorin. This observation correlates with the structural analysis of decorunt, in that it lacks regions of decorin previously shown to be important for interaction with type I collagen. The detection of a catabolic fragment of decorin suggests the existence of a specific catabolic pathway for this proteoglycan. Because of the capacity of decorin to influence collagen fibrillogenesis, catabolism of decorin may have important functional implications with respect to the dermal collagen network.

Connective tissues have many features in common, including resisting and dissipating mechanical load and providing shape, as well as acting to provide barriers regulating water flow and diffusibility of macromolecules. In this chapter we have chosen to focus on cartilage and bone, covering many aspects of connective tissues. We give special focus to the noncollagenous proteins in their matrices.

Long term anti-tumour necrosis factor alpha monotherapy in rheumatoid arthritis: effect on radiological course and prognostic value of markers of cartilage turnover and endothelial activation.den Broeder AA, Joosten LA, Saxne T, Heinegard D, Fenner H, Miltenburg AM, Frasa WL, van Tits LJ, Buurman WA, van Riel PL, van de Putte LB, Barrera P.Department of Rheumatology, University Medical Centre Nijmegen, The Netherlands. A.denbroeder@aig.azn.nlOBJECTIVES: To investigate the effect of prolonged neutralisation of tumour necrosis factor alpha (TNFalpha) on the radiological course in rheumatoid arthritis (RA). To assess whether the radiological course can be predicted by clinical variables or biological markers of cartilage and synovium turnover and of endothelial activation. PATIENTS AND METHODS: Forty seven patients with active RA enrolled at our centre in monotherapy trials with adalimumab (D2E7), a fully human anti-TNFalpha monoclonal antibody, were studied for two years. Radiographs of hands and feet obtained at baseline and after one and two years were scored in chronological order by a single, blinded observer using the modified Sharp method. Radiological course was classified as stable or progressive using the smallest detectable difference as cut off point. The relation between radiological course and serum markers of cartilage and synovium turnover (metalloproteinases (MMP-1 and MMP-3), cartilage oligomeric matrix protein (COMP), human cartilage glycoprotein-39 (HC gp-39)), endothelial activation (soluble E-selectin and intercellular adhesion molecule (ICAM-1)), and integrated measures of disease activity were assessed using univariate and multivariate analysis. RESULTS: Radiological evaluation was performed in 36 patients with paired sets of radiographs at baseline and two years. After two years a total of 15/36 (42%) presented no radiological progression. More patients with stable radiological course were still receiving anti-TNFalpha treatment after two years (13/15 (87%) v 11/21 (52%); p=0.03) and had lower baseline COMP and sICAM-1 levels (p=0.01 and 0.04, respectively) than those in the group with progressive disease. In a logistic regression model the combination of sustained TNF neutralisation and baseline COMP and sICAM-1 levels was predictive for radiological outcome (p=0.03). C reactive protein and disease activity score area under the curve were significantly correlated with changes in radiological scores after two years (r=0.40 and 0.37, p

The tissues in the joint, i.e. cartilage and bone, continuously remodel in a finely tuned balance between matrix synthesis and degradation. In common diseases like osteoarthritis (OA) and rheumatoid arthritis (RA), the normal balance is disturbed and shifted towards degradation in established disease. This eventually leads to disruption of the structural and functional integrity of the joint. As a consequence of the disturbed matrix turnover, increased amounts of macromolecules or fragments thereof are released into synovial fluid and may subsequently reach the blood stream. Some fragments may also be found in the urine. This sequence of events forms the rationale for efforts to identify alterations in the tissue by quantifying matrix macromolecules, “molecular markers”, in body fluids with the purpose to define non-invasive methods to monitor pathological tissue processes [1].

Cartilage oligomeric matrix protein (COMP) belongs to the thrombospondin family and is a homopentamer primarily expressed in cartilage. Mutations in the COMP gene result in the autosomal dominant chondrodysplasias pseudoachondroplasia (PSACH) and some types of multiple epiphyseal dysplasia (MED), which are characterized by mild to severe short-limb dwarfism and early-onset osteoarthritis. We have generated COMP-null mice to study the role of COMP in vivo. These mice show no anatomical, histological, or ultrastructural abnormalities and show none of the clinical signs of PSACH or MED. Northern blot analysis and immunohistochemical analysis of cartilage indicate that the lack of COMP is not compensated for by any other member of the thrombospondin family. The results also show that the phenotype in PSACH/MED cartilage disorders is not caused by the reduced amount of COMP.

Back pain affects a large portion of the population across all ages and intervertebral disc (IVD) degeneration is its most implicated common cause. Presently, there are no established treatments to prevent, stop, or even retard disc degeneration, and surgery is often the offered option. Previous studies have shown that Link N can act as a growth factor and stimulate the synthesis of proteoglycans and collagens in bovine IVD in vitro and in intact human IVDs ex vivo, as well as increase disc height in a rabbit model of disc degeneration. However, the sequences in Link N involved in modulating cellular activity are not well understood. To determine if disc cells can proteolytically process Link N, human disc cells in monolayer were exposed to native Link N over a 48-hour period and mass spectrometric analysis revealed that a peptide spanning residues 1 to 8 was generated in the presence of AF cells but not NP cells. Link N 1-8 significantly induced proteoglycan production in the presence of IL-1β in both NP and AF cells, confirming that the biological effect is maintained in the first eight amino acids of the peptide and indicating that the effect is sustained in an inflammatory environment. Thus Link N 1-8 could be a promising candidate for biologically induced disc repair, and the identification of such a stable specific peptide may facilitate the design of compounds to promote disc repair and provide alternatives to surgical intervention for early stage disc degeneration. Acknowledgments The authors thank Ms. Areej Khabut for her assistance with mass spectrometry. This work would not have been possible without funding from the Faculty of Medicine McGill University, AO Spine, North American Spine Society and Canadian Institutes of Health Research. We also thank Transplant Quebec for coordinating the retrieval of human lumbar spines through their organ donation program. Lastly, we thank The Crafoord Foundation and the Inga-Britt & Arne Lundberg Foundation for funding the mass spectrometers used in this study. Disclosure of Interest None declared

Introduction Back pain affects a large portion of the population across all ages, and intervertebral disc (IVD) degeneration is its most common cause. Presently, there are no established treatments to prevent, stop, or even retard disc degeneration, and surgery is often the offered option. Previous studies have shown that Link-N can act as a growth factor and stimulate the synthesis of proteoglycans and collagens in articular cartilage, in bovine IVD in vitro, and in intact human IVDs ex vivo, as well as increase disc height in a rabbit model of disc degeneration. However, the sequences in Link-N involved in modulating cellular activity and the stability of Link-N in the presence of IVD cells are not well understood. Materials and Methods Human lumbar spines were obtained through the Transplant Quebec's organ donation program. Six spinal segments were retrieved (mean age 30 years) and IVD cells were isolated from the nucleus pulposus (NP) and inner annulus fibrosus (iAF) regions by sequential enzymatic digestion. IVD cells were cultured in monolayer in the presence of Link-N to assess its stability in culture. Media was collected at designated time points from the cultures and was analyzed by MALDI-TOF and MS-MS mass spectrophotometry to evaluate the stability of Link-N peptide. IVD cells embedded in 1.2% alginate were exposed to the identified Link-N fragments and to the native scrambled or reversed Link-N peptides in the absence and presence of IL-1β to assess the biological activity of the peptides. SO4 incorporation was used to assess proteoglycan synthesis in response to the peptides. All peptides were used at equimolar concentrations. Results Mass spectrometric analysis revealed that a peptide spanning residues 1 to 8 was generated in the presence of human AF, while the original 16 amino acids peptide remained intact in the presence of NP cells. Link-N 1-8 significantly induced proteoglycan production in the absence and presence of IL-1β in both human NP and AF cells, confirming that the biological effect is maintained within the first 8 amino acids of the peptide. A reversed or scrambled Link-N peptide, as well as residues 9-16 of Link-N, had no biological effect. Conclusion In conclusion, Link-N 1-8 peptide is a promising therapeutic alternative for treating early disc degeneration before major collagen degradation has occurred. One advantage in using this shorter 8 amino acid peptide rather than the original 16 amino acid Link-N in therapy is the production cost. More important, this small size should be amenable to medicinal chemists allowing them to optimize the structure for maximal biological activity and stability. Thus Link-N 1-8 could be a promising candidate for biologically induced disc repair, and the identification of such a stable specific peptide may facilitate the design of compounds to promote disc repair and provide alternatives to surgical intervention for early stage disc degeneration. Disclosure of Interest None declared

An early step in the biosynthesis of dermatan sulfate is polymerization to chondroitin, which then is modified by the d -glucuronyl C5-epimerase and mainly 4-O-sulfotransferase. The final structure of the dermatan sulfate side chains varies and our aim was to identify, which of the two enzymes that are crucial to generate dermatan sulfate copolymeric structures in tissues. Dermatan sulfate side chains of biglycan and decorin were prepared from fibroblasts and nasal and articular chondrocytes and characterized regarding detailed structure. Microsomes were prepared from these cells and the activities of d -glucuronyl C5-epimerase and 4-O-sulfotransferase were determined. Chondrocytes from nasal cartilage synthesized biglycan and decorin containing 10%, articular chondrocytes 20–30%, and fibroblast 80% of the uronosyl residues in the l -iduronyl configuration. All three tissues contained high amount of 4-O-sulfotransferase activity. The activity of d -glucuronyl C5-epimerase showed different relationships. Fibroblasts contained a high level of the epimerase activity, articular chondrocytes intermediary activity, and in nasal cartilage it was barely detectable. The data indicate that the activity of the d -glucuronyl C5-epimerase is the main factor for formation of dermatan sulfate in tissues.

The extracellular matrix of skeletal tissues contains molecules with key roles in tissue homeostasis. Particularly important are those that are part of the collagen network and maintains its integrity. Examples are members of the thrombospondin family, in cartilage particularly COMP, and members of the family of extracellular leucine rich repeat (LRR) proteins. These proteins have key roles in the assembly of the collagen II fibrillar network and have the capacity to crosslink neighbouring fibrils by containing at least two functional domains, one binding to one fibril and the other binding to another fibril. An example is decorin showing an interaction site of the core proteins on one hand, while another being the dermatan sulfate side chain. Similarly fibromodulin contains structures with potential for interactions with different collagen fibrillar structures. Current data indicate that an early event in osteoarthritis is the fragmentation of the binding proteins to separate the functional domains. This will result in an inability of the collagen network to prevent swelling of the tissue and maintain functionality. The alterations elicit a response where molecules that have roles in catalysing collagen fibril assembly are produced. Such regulating proteins include COMP. This modular protein contains five identical subunits held together close to their C-terminus and having a C-terminal end globular domain that promotes tight binding to collagen. COMP appears to bring together the collagen monomers and catalyse the formation of early fibrils in a chaperon like mode. It is also present, apparently crosslinking completed fibrils by simultaneously binding several collagen IX molecules that are covalently bound on the surface of the collagen II/XI fibres. Other matrix constituents like PRELP and chondroadherin bind to cell surface molecules, apparently involved in signalling to the cells. These two molecules also bind to collagens I and II. Cell binding proteins have key roles in bone turnover. Examples are osteopontin, a protein binding the αvβ3 as well as the α4β1 integrin. The protein is produced by the osteoblast and binds to the bone mineralized surface. It then appears to have a central role in recruiting and promoting the development of osteoclast precursors to a fully functional cell. Osteopontin is also expressed by a number of cells in the inflammatory system. It is likely that release of osteopontin in joint destruction will influence the inflammatory response. Other bone proteins with a capacity to bind αvβ3 include BSP and osteoadherin. Their role in bone turnover and interactions with other tissues remains to be elucidated, but available data indicate primary functions in bone formation.

OBJECTIVE: To elucidate whether differences in the destructive tissue process in cartilage and bone in psoriatic arthritis (PsA) and rheumatoid arthritis (RA) can be recognised by different release patterns of molecular fragments derived from joint tissue. METHODS: Aggrecan, cartilage oligomeric matrix protein (COMP), and bone sialoprotein (BSP) were quantified by immunoassays in knee joint synovial fluid samples. These were obtained early in the disease course of patients with PsA and RA. At the time of arthrocentesis radiographs of their knee and hip joints were normal. RESULTS: At follow up no destruction had developed in the knees and hips of most patients with PsA (n=18), whereas the patients with RA could be separated into one "destructive" group (n=18) and one "non-destructive" group (n=25). Patients with PsA had low synovial fluid aggrecan concentrations (p

Aggrecan, versican, neurocan, and brevican are important components of the extracellular matrix in various tissues. Their amino-terminal globular domains bind to hyaluronan, but the function of their carboxyl-terminal globular domains has long remained elusive. A picture is now emerging where the C-type lectin motif of this domain mediates binding to other extracellular matrix proteins. We here demonstrate that aggrecan, versican, and brevican lectin domains bind fibulin-2, whereas neurocan does not. As expected for a C-type lectin, the interactions are calcium-dependent, with K(D) values in the nanomolar range as measured by surface plasmon resonance. Solid phase competition assays with previously identified ligands demonstrated that fibulin-2 and tenascin-R bind the same site on the proteoglycan lectin domains. Fibulin-1 has affinity for the common site on versican but may bind to a different site on the aggrecan lectin domain. By using deletion mutants, the interaction sites for aggrecan and versican lectin domains were mapped to epidermal growth factor-like repeats in domain II of fibulin-2. Affinity chromatography and solid phase assays confirmed that also native full-length aggrecan and versican bind the lectin domain ligands. Electron microscopy confirmed the mapping and demonstrated that hyaluronan-aggrecan complexes can be cross-linked by the fibulins.

Cartilage oligomeric matrix protein (COMP) is a noncollagenous extracellular matrix protein found predominantly in cartilage, but also in tendon, ligament and meniscus. Studies in man have demonstrated that it may be used as a prognostic marker in rheumatoid arthritis and osteoarthritis. The present study investigated whether tendon injury contributes to serum and tendon sheath synovial fluid levels of COMP in horses. COMP levels, analysed by competitive ELISA, in the digital sheath synovial fluid were more than 10-fold higher than in the serum. Levels were significantly raised when tendon damage or sepsis was present within the tendon sheath but showed only mild, statistically insignificant, elevation in cases of tenosynovitis alone. COMP concentrations in serum were found to vary with age. Foals (age < or = 1 year) had significantly (P

Tyrosine sulfate residues were identified in fibromodulin produced by tracheal chondrocytes, by tendon and sclera fibroblasts in primary culture, as well as in Chinese hamster ovary cells transfected with a construct containing fibromodulin cDNA. The tyrosine sulfate residues were located in the N-terminal part of fibromodulin. Thus, Chinese hamster ovary cells expressing a deleted variant of fibromodulin lacking the N-terminal 52 amino acids following the predicted signal peptide did not contain any tyrosine sulfate residues. The substitution with keratan sulfate chains was not restricted to chondrocytes, but was also identified in fibromodulin synthesized by bovine tendon fibroblasts and sclera fibroblasts, as well as in fibromodulin isolated from tendon. Digestion of fibromodulin with N-glycosidase F reduced the apparent size of fibromodulin to that of the core protein, as predicted from sequence analysis (Oldberg, A., Antonsson, P., Lindblom, K., and Heinegård, D. (1989) EMBO J.8, 2601-2604). Thus fibromodulin from cartilage, tendon, and sclera contains N-glycosidically linked oligosaccharides, some of which are extended to keratan sulfate chains.

The human cDNA for cartilage intermediate layer protein (CILP) codes for a larger precursor protein that consists of CILP and a homologue to porcine Nucleotide pyrophosphohydrolase (NTPPHase) [Lorenzo et al. 1998a. J. Biol. Chem. 273, 23469–23475]. The human gene has now been isolated and characterized. Southern blot analysis indicated a single copy of the CILP gene in the human genome. The gene spans approximately 15.3 kbp of genomic DNA, and is organized in nine exons. The 5′ flanking region contains a putative promoter region with a TATA-like box localized from −29 to −23 bp upstream of the transcription start site. Analysis of the putative promoter region revealed potentially cis-regulatory eukaryotic elements such as GATA-1, MyoD, MZF1, and CdxA. The protein coding region begins in exon 2 with the putative signal peptide. CILP is encoded from exon 3 to exon 9. In addition, exon 9 also codes for the entire NTPPHase homologue and contains the 3′ untranslated region of the gene. All the introns follow the ‘gt–ag’ rule, except the last intron, intron 8, that belongs to the minor class of pre-mRNA introns that contain ‘at–ac’ at their 5′ and 3′ ends, respectively. The CILP gene was mapped to human chromosome 15q22.

The cellular distribution of osteoclast integrin subunits alpha(v) and beta(3), the tissue distribution, and level of the apparent ligand osteopontin (OPN) as well as of the putative regulatory enzyme tartrate-resistant acid phosphatase (TRAP) were studied along with the intracellular distribution of the activation marker c-src in osteopetrotic ia/ia (incisors-absent) mutant rats and their normal littermates. In ia/ia rats, the osteoclasts are incapable of bone matrix resorption. Ultrastructurally the cells exhibit extended clear zones at the expense of ordinary ruffled borders. A secretory dysfunction in the mutant is strongly suggested by the absence of detectable extracellular TRAP, concomitant with an accumulation of the enzyme in abundant small cytoplasmic vesicles. Moreover, TRAP mRNA, protein content, as well as enzymatic activity were elevated. Furthermore, increased levels of integrin subunits alpha(v) and beta(3) were detected at the clear zone of mutant osteoclasts. OPN mRNA levels were elevated in long bones from mutants. In ia/ia rats, immunolabeling for OPN was homogeneously distributed at the surface facing osteoclasts, while in normal littermates it was concentrated at the clear zone area and barely detectable at ruffled borders. The absence of OPN labeling in the abundant, putative intracellular secretory vesicles in mutant osteoclasts suggests that these cells do not produce OPN. The osteoclasts of ia/ia rats appeared to produce and translocate the c-src protein to the cell membrane. In ia/ia a defect ruffled border-formation is observed along with extensive clear zone formation and decreased secretory function. The lesion may be due to a signaling defect, but in that case the defect seems to be located downstream to or not involving the c-src pathway. Our results illustrate the close relationship between secretory function and ruffled border formation in osteoclasts, a relationship that appears to be necessary for proper resorptive function.

The aggregating proteoglycans (aggrecan, versican, neurocan, and brevican) are important components of many extracellular matrices. Their N-terminal globular domain binds to hyaluronan, but the function of their C-terminal region containing a C-type lectin domain is less clear. We now report that a 90-kDa protein copurifies with recombinant lectin domains from aggrecan and versican, but not from the brain-specific neurocan and brevican. Amino acid sequencing of tryptic peptides from this protein identified it as fibulin-1. This extracellular matrix glycoprotein is strongly expressed in tissues where versican is expressed (blood vessels, skin, and developing heart), and also expressed in developing cartilage and bone. It is thus likely to interact with these proteoglycans in vivo. Surface plasmon resonance measurements confirmed that aggrecan and versican lectin domains bind fibulin-1, whereas brevican and neurocan do not. As expected for a C-type lectin, the interactions with fibulin-1 are Ca2+-dependent, with KD values in the low nanomolar range. Using various deletion mutants, the binding site for aggrecan and versican lectin domains was mapped to the epidermal growth factor-like repeats in domain II of fibulin-1. No difference in affinity was found for deglycosylated fibulin-1, indicating that the proteoglycan C-type lectin domains bind to the protein part of fibulin-1.

Fibromodulin is a member of a family of connective tissue glycoproteins/proteoglycans containing leucine-rich repeat motifs. Several members of this gene family bind to fibrillar collagens and are believed to function in the assembly of the collagen network in connective tissues. Here we show that mice lacking a functional fibromodulin gene exhibit an altered morphological phenotype in tail tendon with fewer and abnormal collagen fiber bundles. In fibromodulin-null animals virtually all collagen fiber bundles are disorganized and have an abnormal morphology. Also 10-20% of the bundles in heterozygous mice are similar to the abnormal bundles in fibromodulin-null tail tendon. Ultrastructural analysis of Achilles tendon from fibromodulin-null mice show collagen fibrils with irregular and rough outlines in cross-section. Morphometric analysis show that fibromodulin-null mice have on the average thinner fibrils than wild type animals as a result of a larger preponderance of very thin fibrils in an overall similar range of fibril diameters. Protein and RNA analyses show an approximately 4-fold increase in the content of lumican in fibromodulin-null as compared with wild type tail tendon, despite a decrease in lumican mRNA. These results demonstrate a role for fibromodulin in collagen fibrillogenesis and suggest that the orchestrated action of several leucine-rich repeat glycoproteins/proteoglycans influence the architecture of collagen matrices.

Aggrecan, the predominant large proteoglycan of cartilage, is a multidomain macromolecule with each domain contributing specific functional properties. One of the domains contains the majority of the keratan sulfate (KS) chain substituents and a protein segment with a proline-rich hexapeptide repeat sequence. The function of this domain is unknown but the primary structure suggests a potential for binding to collagen fibrils. We have examined binding of aggrecan fragments encompassing the KS-rich region in a solid-phase assay. A moderate affinity (apparent K d = 1.1 μm) for isolated collagen II, as well as collagen I, was demonstrated. Enzymatic digestion of the KS chains did not alter the capacity of the peptide to bind to collagen, whereas cleavage of the protein core abolished the interaction. The distribution of the aggrecan KS-rich region in bovine tarsometatarsal joint cartilage was investigated using immunoelectron microscopy. Immunoreactivity was relatively low in the superficial zone and higher in the intermediate and deep zones of the uncalcified cartilage. Within the pericellular and territorial matrix compartments the epitopes representing the aggrecan KS-rich region were detected preferentially near or at collagen fibrils. Along the fibrils, epitope reactivity was non-randomly distributed, showing preference for the gap region within the d-period. Our data suggest that collagen fibrils interact with the KS-rich regions of several aggrecan monomers aligned within a proteoglycan aggregate. The fibril could therefore serve as a backbone in at least some of the aggrecan complexes.

Objective: To investigate potential synergy of low dosages glucocorticosteroids (GC's) and interleukin-10 (IL-10), using established murine collagen-induced arthritis (CIA) as a model.¶Methods: DBA-1J/BOM mice were immunized with bovine type II collagen and boosted at day 21. Mice with established CIA were selected and treated for at least 7 days with either prednisolone (0.05–5 mg/kg), IL-10 (0.1–5 μg/day) or the combination of prednisolone/IL-10 (0.05/1 and 0.05/5). Arthritis score was monitored visually, and joint pathology was examined by histology, and serum cartilage oligomeric matrix protein (COMP) measured.¶Results: Amelioration of CIA was found with dosages of 1 and 5 mg/kg prednisolone, while a dose of 0.05 mg/kg prednisolone was ineffective. Treatment of CIA with 5 μg/day IL-10 resulted in a mild, but significant suppression. Synergistic effects were seen with the combination of low dose prednisolone and IL-10 (0.05 mg/kg, 1 μg/day). Both arthritis score and joint pathology were significantly reduced. Moreover, COMP levels were significantly decreased after IL-10/prednisolone treatment, confirming decreased cartilage involvement. Of great interest, treatment of CIA with prednisolone/IL-10 markedly reduced IL-1β and enhanced IL-10 production by synovial tissue. In addition, synovial mRNA levels for IL-1β were decreased, while mRNA levels for IL-10 and IL-1Ra were upregulated by combined treatment.¶Conclusion: This study demonstrates synergistic effects of combined treatment with prednisolone and IL-10 on suppressing disease activity of CIA as well as reducing cartilage.

SUMMARY In rheumatoid arthritis peripheral cartilaginous joints are inflamed and eroded. One driving factor may be an immune response towards proteins in the cartilage. Here it is shown that cartilage oligomeric matrix protein (COMP), expressed specifically in cartilage, is arthritogenic in the rat. Both native and denatured rat COMP induced severe arthritis in selected rat strains. The arthritis occurred only in peripheral joints which were attacked by an erosive inflammatory process similar to that seen in the human disease. The disease was self-limited and no permanent destruction of joints was seen macroscopically. Disease development appeared to be dependent on an immune response to autologous (rat) COMP and not on cross-reactivity to other cartilage rat collagens (types II, IX and XI). The disease and the immune response to COMP were genetically controlled by the MHC; the RT1u and RT1l haplotypes were more susceptible than the a, c, d, f and n haplotypes. Both LEW and E3 gene backgrounds were highly permissive for disease induction. These findings suggest that the induction of arthritis with rat COMP represents a unique pathogenesis which is controlled by different genes compared with collagen-induced arthritis or adjuvant-induced arthritis.

Hyaluronan is a major component of the vitreous gel. Hyaluronan-binding macromolecules, including the aggregating proteoglycans, have been shown to perform an important role in maintaining the structural integrity of a number of tissues. However, there have not previously been any biochemical data to establish the presence of these types of macromolecules in vitreous. Bovine vitreous gel was solubilized (apart from a residual collagenous pellet) in 4 M guanidine hydrochloride and after dialysis into phosphate buffered saline analyzed by gel filtration chromatography with in-line measurement of refractive index and multi-angle laser light scattering. The concentration of hyaluronan in whole vitreous was found to be 0.57 mg/ml. The average molecular weight of the hyaluronan was found to be 170,000 (after isolation of the vitreous hyaluronan by isopycnic centrifugation in 0.5 M guanidine hydrochloride and papain digestion). Following Superose 12 gel filtration chromatography of the Streptomyces hyaluronan lyase digested vitreous extract, a pool of material was identified at or near the void volume of the column, and this material was shown to contain sulphated proteoglycans. Analysis of fractions following Superose 12 gel filtration chromatography by Western blotting showed that this pool of material contained the chondroitin sulphate proteoglycans versican and type IX collagen. Link protein was also identified in vitreous extracts by Western blotting. In whole vitreous, the concentration of versican was found to be 21.4+/-2.8 microg/ml and of link protein 0.62+/-0.07 microg/ml. Versican and link protein were thus present in approximately 1:1 molar ratios, but hyaluronan was present in a molar excess of 150 times. Therefore, aggregating proteoglycans are present in vitreous but, assuming that they bind to hyaluronan in-vivo, their overall density along the hyaluronan is much lower than that found in other tissues.

A novel, somewhat basic noncollagenous protein was purified from guanidine hydrochloride extracts of human articular cartilage using cesium chloride density gradient centrifugation, followed by ion-exchange chromatography at pH 5, and gel filtration on two serially coupled columns of Superose 6 and Superdex 200. The protein of 91.5 kDa contains a single polypeptide chain substituted with N-linked oligosaccharides. It appeared unique to cartilage as studied by enzyme-linked immunosorbent assay and immunoblots of various tissue extracts. Its concentration in articular cartilages showed some variability with age being lower in young individuals. It represents a chondrocyte product, since it is synthesized by articular chondrocytes in explant cultures. Interestingly, the distribution of the protein in the articular cartilage provides important information on the nature of chondrocytes at different compartments in the tissue. Thus, chondrocytes in the middle/deeper layers of the tissue in particular, appeared to have produced the protein and deposited it in the interterritorial matrix. The protein was neither seen in the superficial nor in the deepest regions of the articular cartilage. Based on its immunolocalization we have named this protein CILP (cartilage intermediate layer protein).

Cartilage and tendon extracellular matrices are composed of collagens, proteoglycans, and a number of noncollagenous proteins. Cartilage oligomeric matrix protein (COMP) is a prominent such protein, structurally related to the thrombospondins. We found that native COMP binds to collagen I/II and procollagen I/II and that the interaction is dependent on the divalent cations Zn2+ or Ni2+, whereas Ca2+, Mg2+, and Mn2+ did not promote binding. Using a solid phase assay, Scatchard analysis identified one class of binding site with a dissociation constant (Kd) close to 1.5 nM in the presence of Zn2+. The results were confirmed by studies using surface plasmon resonance. Furthermore, metal chelate chromatography demonstrated that COMP bound Zn2+ and Ni2+. Electron microscopy showed that the interaction occurred at four defined sites on the 300-nm collagen and procollagen molecules. Two were located close to each end, and two at 126 and 206 nm, respectively, from the C-terminal. COMP interacted via its C-terminal globular domain and significantly only in the presence of Zn2+.

Osteoadherin is a recently described bone proteoglycan containing keratan sulfate. It promotes integrin (alphav beta3)-mediated cell binding (Wendel, M., Sommarin, Y., and Heinegârd, D. (1998) J. Cell Biol. 141, 839-847). The primary structure of bovine osteoadherin has now been determined by nucleotide sequencing of a cDNA clone from a primary bovine osteoblast expression library. The entire translated primary sequence corresponds to a 49,116-Da protein with a calculated isoelectric point for the mature protein of 5.2. The dominating feature is a central region consisting of 11 B-type, leucine-rich repeats ranging in length from 20 to 30 residues. The full, primary sequence contains four putative sites for tyrosine sulfation, three of which are at the N-terminal end of the molecule. There are six potential sites for N-linked glycosylation present. Osteoadherin shows highest sequence identity, 42%, to bovine keratocan and 37-38% identity to bovine fibromodulin, lumican, and human PRELP. Unique to osteoadherin is the presence of a large and very acidic C-terminal domain. The distribution of cysteine residues resembles that of other leucine-rich repeat proteins except for two centrally located cysteines. Northern blot analysis of RNA samples from various bovine tissues showed a 4.5-kilobase pair message for osteoadherin to be expressed in bone only. Osteoadherin mRNA was detected by in situ hybridization in mature osteoblasts located superficially on trabecular bone.

Chondroadherin is a cartilage protein with cell binding properties. The expression of chondroadherin was studied in rat tissues and during postnatal femoral head development. For design of oligonucleotide probes and primers a 1664 bp, full length, rat chondroadherin cDNA was isolated from a rat chondrosarcoma library and sequenced. Northern blot analysis showed chondroadherin mRNA to be present in femoral head and rib cartilage, as well as in tendon. More sensitive reverse-transcriptase PCR additionally identified the mRNA in calvaria, long bone and bone marrow. Localization of chondroadherin by immunocytochemistry in the developing femoral head from postnatal day 14 to day 60 showed presence of the protein in cartilaginous regions. With increasing age a very distinct localization of chondroadherin was seen in the territorial matrix around late proliferative cells in the growth plate as well as in the developing articular cartilage in the maturing femoral head. Localization of chondroadherin mRNA by in situ hybridization was in agreement with immunocytochemistry with strong hybridization signals in late proliferative cells in the growth plate. In the articular cartilage the expression was restricted to cells in the lower regions. A three-fold increase of cartilage chondroadherin content in the growing femoral head was demonstrated by Western blot analysis. The high expression of this cell binding protein in a dynamic region of cartilage suggests an important role for chondroadherin in the regulation of chondrocyte growth and proliferation.

A protein prominent in guanidine hydrochloride extracts of adult bovine and equine digital flexor tendons was confirmed to be Cartilage Oligomeric Matrix Protein (COMP) by non-reducing and reducing SDS-PAGE, reaction with rabbit anti-COMP polyclonal antiserum on Western blots, trypsin digestion followed by HPLC on a C2/C18 column, and identification of COMP mRNA from tendon on Northern blots. Immunohistochemistry and Western blots of extracts showed COMP to be present in all regions of digital flexor tendons. Equine tendon COMP was purified by ion exchange chromatography and gel filtration and used in a heterologous inhibition ELISA to quantify COMP in equine digital flexor tendons at different ages, and in other tendons and ligaments. Mean COMP levels in digital flexor tendon were approximately 2-5mg/g wet weight, but they showed a large variation. Levels were low in neonatal tendon but rose rapidly during growth, with the metacarpal (tensional) superficial digital flexor tendon having the highest levels (approximately 10mg/g wet weight). Levels subsequently declined in this region, while in areas which experience a variable amount of compression, levels increased less but then remained constant. Extensor tendons and collateral ligaments, which experience less loading in vivo, had levels similar to those in neonatal tendon. COMP was identified in scarred skin and granulation tissue but not in normal skin, chronic fibrosis, or a fibrosarcomatous skin growth. A unilateral non-weight-bearing growing animal contained three to six times more COMP in the weight-bearing digital flexor tendons compared to the paralyzed limb, while the extensor tendons had similar amounts in both limbs. With the recent discovery of a COMP gene mutation causing pseudoachondroplasia (Hecht et al., 1995), in which lax tendons and ligaments are a feature, the present data suggest that COMP is synthesized in response to, and is necessary for tendon to resist, load.

The mouse chondroadherin gene was isolated from a cosmid genomic library by the use of a rat chondroadherin cDNA probe. Southern blot analysis of mouse genomic DNA revealed a simple pattern of hybridization indicating a single copy gene for chondroadherin. The mouse chondroadherin gene encompasses 4.1 kb and consists of four exons separated by one large intron of 1929 bp followed by two smaller introns of 247 and 225 bp, respectively. Most of the translated region, including the start codon and the main part of a leucine-rich region, is contained within the first exon. Two small exons of 164 and 146 bp encode the rest of the protein. Interestingly, 4 bases from the stop codon, in the 3′-UTR, a third intron is located. A putative promoter region of 669 bp was sequenced and shown to contain a potential TATAA-box signal 29 bp upstream of the transcription start site and several recognition sites for transcription factors. The exon/intron organization of the chondroadherin gene differs from those of the other known genes of the leucine-rich repeat (LRR) family in the extracellular matrix. Taken together with comparison of protein sequences of other members of the LRR family in the extracellular matrix, the data suggest that chondroadherin has evolved along a different pathway. The chondroadherin gene was mapped to mouse chromosome 11, near D11Mit14, by single-strand conformation polymorphism linkage analysis.

Chondroadherin (the 36-kD protein) is a leucine-rich, cartilage matrix protein known to mediate adhesion of isolated chondrocytes. In the present study we investigated cell surface proteins involved in the interaction of cells with chondroadherin in cell adhesion and by affinity purification. Adhesion of bovine articular chondrocytes to chondroadherin-coated dishes was dependent on Mg2+ or Mn2+ but not Ca2+. Adhesion was partially inhibited by an antibody recognizing beta1 integrin subunit. Chondroadherin-binding proteins from chondrocyte lysates were affinity purified on chondroadherin-Sepharose. The beta1 integrin antibody immunoprecipitated two proteins with molecular mass approximately 110 and 140 kD (nonreduced) from the EDTA-eluted material. These results indicate that a beta1 integrin on chondrocytes interacts with chondroadherin. To identify the alpha integrin subunit(s) involved in interaction of cells with the protein, we affinity purified chondroadherin-binding membrane proteins from human fibroblasts. Immunoprecipitation of the EDTA-eluted material from the affinity column identified alpha2beta1 as a chondroadherin-binding integrin. These results are in agreement with cell adhesion experiments where antibodies against the integrin subunit alpha2 partially inhibited adhesion of human fibroblast and human chondrocytes to chondroadherin. Since alpha2beta1 also is a receptor for collagen type II, we tested the ability of different antibodies against the alpha2 subunit to inhibit adhesion of T47D cells to collagen type II and chondroadherin. The results suggested that adhesion to collagen type II and chondroadherin involves similar or nearby sites on the alpha2beta1 integrin. Although alpha2beta1 is a receptor for both collagen type II and chondroadherin, only adhesion of cells to collagen type II was found to mediate spreading.

Introduction Complexes between cartilage oligomeric matrix protein (COMP) and the complement activation product C3b have been found in the circulation of patients with rheumatoid arthritis and systemic lupus erythematosus. In systemic sclerosis (SSc) COMP expression in the skin is upregulated both in lesional and non-lesional skin, which is also reflected in an increased amount of circulating COMP. We investigated the presence of COMP-C3b complexes in serum and skin biopsies of patients with SSc. Methods The presence of COMP and COMP-C3b complexes in the serum of 80 patients with limited cutaneous SSc (lcSSc, n = 40) and diffuse cutaneous SSc (dcSSc, n = 40) and 97 healthy controls was measured by ELISA and correlated to different clinical parameters. Samples were collected both at baseline and after three to five years to assess longitudinal changes in COMP-C3b complex levels. Furthermore, skin biopsies from seven patients with dcSSc and three healthy controls were analyzed for expression of COMP and deposition of C3b and IgG. Results Serum levels of COMP-C3b were found to be elevated in both dcSSc and lcSSc compared to healthy controls and decreased at the second measurement in patients on immunosuppressive therapy. No co-localization of COMP and C3b was found in the skin biopsies, indicating that the COMP-C3b complexes are formed upon release of COMP into the circulation. Conclusion COMP-C3b complexes are found in the serum of patients with SSc. The lack of co-localization between COMP and C3b in the skin suggests that COMP does not drive complement activation in the skin in SSc.

Distribution and sites of synthesis of a cartilage extracellular matrix protein, cartilage oligomeric matrix protein (COMP), and of a bone extracellular matrix protein, bone sialoprotein (BSP), were studied in the femoral head of growing Wistar rats from day 14 to day 60 by immunocytochemistry and in situ hybridization. This period includes formation of the secondary ossification center and differentiation of articular cartilage. At early stages, immunoreactivity for COMP was pronounced throughout the cartilage. The localization of COMP was predominantly territorial in the center of the immature femoral head and in the growth plate at all ages studied. In the superficial parts, a shift from a uniform extracellular matrix staining at day 14 to an interterritorial localization at day 33 to day 60 was seen, apparently concurrent with formation of articular cartilage. COMP staining, representing cartilage remnants, also extended into the center of the trabecular bone in the primary spongiosa. In the secondary ossification center, the staining for COMP decreased at the onset of calcification. The protein was only synthesized by chondrocytes, as shown by in situ hybridization. The highest level of COMP mRNA was detected in chondrocytes in the central region of the growth plate. In the layer corresponding to the articular cartilage of the femoral head, mRNA levels for COMP were low from day 14 to day 33 but were increased on day 60. This shows substantial synthesis in the developing articular cartilage. Immunoreactivity for BSP was detected in bone trabeculae of primary spongiosa. In situ hybridization showed the highest levels of BSP mRNA in regions of newly formed bone. BSP mRNA was detected in hypertrophic chondrocytes in the secondary ossification center as early as day 18, well before the appearance of immunochemically detectable BSP. Interestingly, simultaneous expression of COMP and BSP mRNA was seen after day 18 in hypertrophic chondrocytes of the growth plate and later also in hypertrophic chondrocytes close to the mineralization zone of the articular cartilage.

We have determined the primary structure of a connective tissue matrix protein from the nucleotide sequence of a clone isolated from a human articular chondrocyte cDNA library. The major part of the amino acid sequence has also been determined by direct protein sequencing. The translated primary sequence corresponds to 382 amino acid residues, including a 20-residue signal peptide. The molecular mass of the mature protein is 41,646 Da. The main part of the protein consists of 10 leucine-rich repeats ranging in length from 20 to 26 residues, with asparagine at position 10 (B-type). The N-terminal part is unusual in that it is basic and rich in arginine and proline. There are four potential N-linked glycosylation sites present. In three of these sites, post-translational modifications are likely to be present since Asn was not found by direct protein sequencing. The amino- and carboxyl-terminal parts contain four and two cysteine residues, respectively, probably forming disulfide bonds by analogy with the other members of this family. The protein shows highest identity (36%) to fibromodulin and 33% to bovine lumican, two other leucine-rich repeat connective tissue proteins. Northern blot analysis showed the presence of an approximately 3.8-kilobase mRNA in different types of bovine cartilage and cultured osteoblasts, whereas RNAs isolated from bovine kidney, skin, spleen, thymus, and trabecular bone and rat calvaria were negative. Human articular chondrocyte and rat chondrosarcoma cell RNAs contained an additional mRNA of approximately 1.6 and 1.8 kilobases, respectively.

Decorin and biglycan are structurally related interstitial proteoglycans synthesized in connective tissues like skin, tendon, and cartilage. Despite the conspicuous sequence similarities, where about 55% of the amino acid residues in decorin and biglycan are located in identical positions, the two proteoglycans show differences in their interaction with collagen. Decorin binds to collagen type I, whereas biglycan in several assay systems shows no affinity for this collagen type. Here we have made use of these structural similarities and affinity differences in studies of the collagen binding properties of decorin. Recombinant biglycan/decorin chimeras were produced in mammalian cells and analyzed for their capacity to bind collagen. In the chimeras, biglycan contributes sequences crucial for synthesis and export from the mammalian cells, and decorin provides potential collagen-binding properties. By using this approach we show that decorin binds to the collagen primarily via leucine-rich repeats 4-5 composed of some 40 amino acid residues. Proteoglycan chimeras containing decorin sequences from the N terminus to leucine-rich repeat 3 or sequences from leucine-rich repeat 6 to the C terminus do not show any detectable binding to collagen. A proteoglycan chimera containing decorin leucine-rich repeats 4-5 flanked by biglycan sequences binds to collagen. However, this chimera binds to collagen with somewhat lower affinity than wild type decorin, suggesting that additional low affinity binding sites may be located in other parts of decorin. Alternatively, the conformation of the collagen binding leucine-rich repeats 4-5 are different in decorin and in the biglycan/decorin chimera, leading to a lower collagen affinity for the latter.

Aggregates of proteoglycans from the Swarm rat chondrosarcoma reassembled in vitro have been studied by rotary-shadowing electron microscopy, and shown to be similar to native structures that have never been dissociated [Mörgelin, Engel, Heinegård and Paulsson (1992) J. Biol. Chem. 267, 14275-14284]. A hyaluronate with defined chain length (HAshort) has now been prepared by autoclaving high-Mr hyaluronate and fractionation to a narrow size distribution by gel filtration. Proteoglycan monomers, core protein, hyaluronate-binding region and link protein were combined with HAshort. Free chains of HAshort and reconstituted complexes with proteoglycan, link protein and aggrecan fragments were examined by electron microscopy after rotary shadowing. Length measurements showed that the hyaluronate was condensed to about half of its original length on binding intact aggrecan monomers, any aggrecan fragment or link protein alone. This strongly implies that hyaluronate adopts a defined spatial arrangement within the central filament of the aggregate, probably different from its secondary structure in solution. No differences in length were observed between link-free and link-stabilized aggregates.

Introduction: Cartilage oligomeric matrix protein (COMP) is found at elevated concentrations in sera of patients with joint diseases such as rheumatoid arthritis (RA) and osteoarthritis (OA). We recently showed that COMP activates complement via the alternative pathway and that COMP-C3b complexes are present in sera of RA patients, but not in healthy controls. We now set out to elaborate on the information provided by this marker in a variety of diseases and larger patient cohorts. Methods: COMP-C3b levels in sera were measured by using an enzyme-linked immunosorbent assay (ELISA) capturing COMP and detecting C3b. Serum COMP was measured by using ELISA. Results: COMP-C3b levels were significantly elevated in patients with RA as well as in systemic lupus erythematosus (SLE), compared with healthy controls. SLE patients with arthritis had significantly higher COMP-C3b levels than did those without. COMP-C3b was furthermore elevated in patients with ankylosing spondylitis (AS), psoriatic arthritis (PsA), reactive arthritis, systemic sclerosis, and OA. COMP-C3b did not correlate with COMP in any of the patient groups. COMP-C3b correlated with disease activity in RA, but not in other diseases. COMP-C3b levels in RA patients decreased on treatment with tumor necrosis factor (TNF)-alpha inhibitors, whereas the levels increased in patients with AS or PsA. The changes of COMP-C3b did not parallel the changes of C-reactive protein (CRP). Conclusions: COMP-C3b levels are elevated in several rheumatologic diseases and correlate with inflammatory measures in RA. COMP-C3b levels in RA decrease during TNF-alpha inhibition differently from those of CRP, suggesting that formation of COMP-C3b relates to disease features not reflected by general inflammation measures.

OBJECTIVE--The release of fragments of cartilage oligomeric matrix protein (COMP) and aggrecan into knee joint fluid and serum after joint injury and in post-traumatic and primary osteoarthritis was monitored in a cross-sectional study. METHODS--Samples of joint fluid and serum were obtained from healthy volunteers and from patients with arthroscopically verified injury to anterior cruciate ligament and or meniscus of the knee at different times after injury and with different degrees of post-traumatic osteoarthritis. Samples were also obtained from patients with primary osteoarthritis. Fragments of COMP were quantified in joint fluid and in serum by enzyme-linked immunoassay. Fragments of aggrecan were quantified in joint fluid by enzyme-linked immunoassay. RESULTS--Concentrations of COMP and aggrecan fragments in joint fluid in the study groups were increased over the reference levels of 47 (range 10-109) and 34 (range 6-59) micrograms/mL, respectively, in the volunteers with healthy knees. The ratios (w/w) of aggrecan to COMP fragments in joint fluid were also increased in all study groups over that in the reference group. Average concentrations of COMP in joint fluid were high shortly after injury and decreased with time but remained increased over reference levels for many years. Joint fluid COMP concentrations were also increased in early stage post-traumatic and primary osteoarthritis, but not in advanced osteoarthritis. CONCLUSION--Increased amounts of aggrecan and COMP fragments are released into joint fluid after joint injury and in early stage osteoarthritis. The ratios of aggrecan to COMP in joint fluid vary with time after injury and with osteoarthritis disease stage. Although both molecules are sensitive to the agents active in matrix breakdown in joint disease, differences may thus exist in the release mechanisms and attempted repair. The concentrations of the individual markers in body fluids and their ratios may serve to monitor treatment efficacy, disease progression and repair in osteoarthritis and other joint diseases.

Background and objectives Systemic sclerosis is a heterogeneous disease characterised both by inflammation and fibrosis. TLR4-dependent signalling has been proposed to play a significant role in the connection between inflammation and fibrosis in SSc. We have previously found increased levels of the TLR4-agonist S100A8/A9 in both serum and faeces of patients with SSc. In this study, we examined the role of S100A8/A9 in the pathogenesis of inflammation induced skin fibrosis in the bleomycin mouse model. Materials and methods S100A9 -/- mice were kept on a C57 Bl/6 background. These mice lack biologically active S100A8/A9 protein complex. Wild type (WT) littermates were used as controls. In total, 40 age matched female mice were used. Skin fibrosis was induced by 12 subcutaneous injections of bleomycin over a 4 week period. Dermal skin thickness was evaluated by light microscopy and skin collagen content by measurement of hydroxyproline. Skin concentration of water and fat was evaluated after freeze-drying and fat extraction of skin biopsies with acetone respectively. Punch biopsies were taken for later mRNA quantification by rtPCR and for electron microscopy. Results Skin thickness increased to a similar extent in WT and S100A9 -/- mice compared to saline injected animals after bleomycin treatment (262 ± 11 μm vs 263 ± 12 μm). The amount of collagen also increased to the same level in WT and S100A9 -/- mice (53 ± 1.6 vs 53 ± 0.9 μg/mm 2 ). Compared to saline injected control mice, bleomycin treatment resulted in an increase in skin water content in WT (70 ± 0.6 vs 65 ± 1.3 %; p -/- mice (68 ± 1.3 vs 68 ± 0.5 %). Bleomycin treatment caused a more pronounced reduction of dry skin fat content in WT (41 ± 2.6 in saline vs 17 ± 1.6 % in bleomycin treated mice; p -/- mice (36 ± 1.4 vs 23 ± 2.2 %; p = 0.002). Conclusion S100A9 deficiency does not protect mice from bleomycin induced skin fibrosis. S100A9 deficiency seems to attenuate inflammatory changes of skin composition in the bleomycin mouse model of skin fibrosis.