Galectin 3 and its binding protein in rheumatoid arthritis.
(33/201)
OBJECTIVE: To characterize the expression pattern and role of galectin 3 and galectin 3 binding protein (G3BP) in rheumatoid arthritis (RA), in comparison with galectin 1, and to explore whether soluble galectin 3 and G3BP, investigated in serum, synovial fluid, or cell culture supernatant, are associated with disease. METHODS: Synovial tissues from patients with RA or osteoarthritis (OA), as well as from healthy controls, were analyzed for galectins 1 and 3 and G3BP by in situ hybridization and immunohistochemistry. Levels of galectin 3 and G3BP in serum and synovial fluid from patients with RA and OA and controls, as well as in cell culture supernatants, were determined by enzyme-linked immunosorbent assay (ELISA). In vitro, the intracellular expression of galectin 3 in RA and OA synovial fibroblasts after modulation with tumor necrosis factor alpha (TNFalpha), interleukin-1beta (IL-1beta), and anti-CD40 monoclonal antibodies was measured by flow cytometry. RESULTS: In RA, galectin 3 messenger RNA and protein stained throughout the synovial membrane, whereas G3BP was particularly expressed at sites of bone destruction. In contrast, the expression of galectin 1 was not uniform in different RA specimens, and was never found at sites of invasion. In OA and normal synovial tissues, only a small number of cells were positive for galectins and/or G3BP. Galectin 3 was elevated in RA sera and synovial fluids, whereas G3BP was increased in RA synovial fluids only. In RA, serum galectin 3 correlated with C-reactive protein levels, whereas G3BP was associated with joint destruction and/or synovial cell activation as measured by the levels of cartilage oligomeric matrix protein. In vitro, RA synovial fibroblasts showed an increased release of galectin 3 into culture medium, as measured by ELISA, but decreased secretion of G3BP. In RA synovial fibroblasts with low basal expression of galectin 3, TNFalpha increased its intracellular level in a dose-dependent manner. In contrast, IL-1beta or anti-CD40 monoclonal antibodies showed no effect. CONCLUSION: Our data indicate that galectin 3 and G3BP are not only involved in inflammation, but also contribute to the activation of synovial fibroblasts. The intracellular accumulation of galectin 3 can be enhanced by TNFalpha. Thus, galectin 3 and G3BP represent novel markers of disease activity in RA. (+info)
Characterization of a pseudoachondroplasia-associated mutation (His587-->Arg) in the C-terminal, collagen-binding domain of cartilage oligomeric matrix protein (COMP).
(34/201)
We have introduced a pseudoachondroplasia-associated mutation (His(587)-->Arg) into the C-terminal collagen-binding domain of COMP (cartilage oligomeric matrix protein) and recombinantly expressed the full-length protein as well as truncated fragments in HEK-293 cells. CD spectroscopy revealed only subtle differences in the overall secondary structure of full-length proteins. Interestingly, the mutant COMP did not aggregate in the presence of calcium, as does the wild-type protein. The binding site for collagens was recently mapped to amino acids 579-595 and it was assumed that the His(587)-->Arg mutation influences collagen binding. However full-length mutant COMP bound to collagens I, II and IX, and the binding was not significantly different from that of wild-type COMP. Also a COMP His(587)-->Arg fragment encompassing the calcium-binding repeats and the C-terminal collagen-binding domain bound collagens equally well as the corresponding wild-type protein. The recombinant fragments encompassing the C-terminal domain alone showed multiple bands following SDS/PAGE, although their theoretical molecular masses could be verified by MS. A temperature-induced conformational change was observed in CD spectroscopy, and negative-staining electron microscopy demonstrated that both wild-type and mutant proteins formed defined elongated aggregates after heating to 60 degrees C. Our results suggest that the His(587)-->Arg mutation is not itself deleterious to the structure and collagen-binding of COMP. (+info)
Hyaluronic acid levels may have predictive value for the progression of knee osteoarthritis.
(35/201)
STUDY OBJECTIVES: To study prognostic value of different biochemical markers for morphological progression of early knee osteoarthritis. DESIGN: A total of 89 patients with knee osteoarthritis (OA) were enroled into the study. The follow-up period was 2 years. Radiological OA progression was evaluated by measuring joint space width. Pentosidine was detected using the HPLC method described earlier, cartilage oligomeric matrix protein (COMP) using the method published by our team. MMP-9, tissue inhibitors of metalloproteinases (TIMP), YKL-40 and hyaluronic acid were detected using commercially available kits. RESULTS: In the group of patients suffering from knee OA, higher serum levels of pentosidine (P=0.04), MMP-9 (P=0.02), TIMP (P=0.04) and COMP (P=0.05) were detected compared with healthy control subjects. Using a correlation analysis method, it has been found that the patients with higher basic serum levels of hyaluronic acid had a faster radiological progression (r=0.56, P<0.005), as well as the patients with higher basic serum pentosidine levels (r=0.30, P<0.005). Other biochemical markers had no statistically significant prognostic value. CONCLUSIONS: In our study, serum levels of hyaluronic acid and pentosidine had a predictive value for further development of knee OA in that further joint space narrowing was detected in the patients with knee OA in the next 2 years. (+info)
COMP-Ang1: a designed angiopoietin-1 variant with nonleaky angiogenic activity.
(36/201)
Angiopoietin-1 (Ang1) has potential therapeutic applications in inducing angiogenesis, enhancing endothelial cell survival, and preventing vascular leakage. However, production of Ang1 is hindered by aggregation and insolubility resulting from disulfide-linked higher-order structures. Here, by replacing the N-terminal portion of Ang1 with the short coiled-coil domain of cartilage oligomeric matrix protein (COMP), we have generated a soluble, stable, and potent Ang1 variant, COMP-Ang1. This variant is more potent than native Ang1 in phosphorylating the tyrosine kinase with Ig and epidermal growth factor homology domain 2 (Tie2) receptor and Akt in primary cultured endothelial cells, enhancing angiogenesis in vitro and increasing adult angiogenesis in vivo. Thus, COMP-Ang1 is an effective alternative to native Ang1 for therapeutic angiogenesis in vivo. (+info)
Interactions between the cartilage oligomeric matrix protein and matrilins. Implications for matrix assembly and the pathogenesis of chondrodysplasias.
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The cartilage oligomeric matrix protein (COMP) and matrilins are abundant non-collagenous proteins in the cartilage extracellular matrix. In the presence of calcium, COMP and matrilin-1 elute together in the gel filtration of cartilage extracts and can be co-immunoprecipitated. In a screen for ligands of matrilin-1, -3, and -4 using an ELISA-style binding assay, COMP was identified as a prominent binding partner for all three, indicating a conservation of the COMP interaction among matrilins. The interaction of COMP and matrilin-4 is saturable, and an apparent K(D) of 1 nm was determined. However, only the full-length COMP and the full-length matrilin-4 proteins showed a strong interaction, indicating that the oligomeric structures markedly increase the affinity. Mutations in COMP or matrilin-3 cause related forms of human chondrodysplasia, and the COMP mutation D469Delta, which is found in patients with pseudoachondroplasia, has been shown to cause a reduced calcium binding. Despite this, the mutation causes only a slight decrease in matrilin-4 binding. This indicates that impaired binding of COMP to matrilins does not cause the pseudoachondroplasia phenotype but rather that matrilins may be coretained in the rough endoplasmatic reticulum where COMP accumulates in the chondrocytes of patients. (+info)
Suggestion of nonlinear or phasic progression of knee osteoarthritis based on measurements of serum cartilage oligomeric matrix protein levels over five years.
(38/201)
OBJECTIVE: In many patients with knee osteoarthritis (OA), the disease progresses, and there is loss of cartilage; in others, the disease stabilizes with time. Previous studies have demonstrated that concentrations of serum proteins that reflect joint tissue metabolism can identify knees that will deteriorate, leading to the suggestion that OA disease activity is phasic or cyclical. The aim of the current study was to determine whether longitudinal measurements of one such protein, serum cartilage oligomeric matrix protein (COMP), are related to disease outcome over a 5-year period. METHODS: Serum COMP levels were measured by enzyme-linked immunosorbent assay at study entry and every 6 months thereafter in 115 patients with knee pain and OA of mainly the tibiofemoral joint. Cartilage loss was determined from knee radiographs taken at entry and at 24, 36, and 60 months. Disease progression was defined as either a reduction in the tibiofemoral joint space width by at least 2 mm or total knee replacement (TKR) in either knee at followup. COMP concentrations at baseline and the area under the curve (AUC) of measurements obtained over 5 years were compared between progressors and nonprogressors by Student's 2-tailed t-test. The patterns and probability of progression according to TKR or > or =2 mm of narrowing of the tibiofemoral joint space were analyzed by logistic regression models. RESULTS: The mean +/- SD ages of the progressors and nonprogressors were 64.2 +/- 7.8 years and 63.3 +/- 10.6 years, respectively, and the proportion of females was 51% and 56%, respectively. Of the 37 patients whose OA progressed (22 by TKR and 15 by > or =2-mm reduction in tibiofemoral joint space), 13 lost cartilage during the first 2 years, and 18 lost cartilage during the last 2 years. The mean +/- SD serum COMP concentration at baseline was significantly higher in the progressors compared with the nonprogressors (14.12 +/- 3.39 units/liter versus 12.62 +/- 3.25 units/liter; P < 0.036). Serum COMP levels rose significantly after TKR; however, after allowing for the effect of TKR, the AUC/month was significantly higher in the progressors compared with the nonprogressors (12.52 +/- 2.71 versus 10.82 +/- 2.71; P < 0.003). Serum COMP concentrations were higher during periods of radiographic progression and identified periods of progression that were nonlinear. Logistic regression analysis showed that on average, a 1-unit increase in serum COMP levels increased the probability of radiographic progression by 15%. CONCLUSION: The data suggest that serum COMP is related to progressive joint damage in knee OA. The patterns of progression for the early and late progressors are consistent with the hypothesis that knee OA progression is episodic or phasic. Large between-subject variation precludes the use of individual values to predict progression with confidence. However, sequential measurements of serum COMP levels may identify patients whose OA is likely to progress over the next year or two. (+info)
Galectin-3 is induced in rheumatoid arthritis synovial fibroblasts after adhesion to cartilage oligomeric matrix protein.
(39/201)
BACKGROUND: Galectin-3 is expressed in the synovial tissue of patients with rheumatoid arthritis (RA), particularly at sites of joint destruction. OBJECTIVE: To explore the possibilities that galectin-3 is induced either by proinflammatory cytokines or by adhesion to cartilage components. METHODS: Cell culture plates were coated with fibronectin, collagens I-VI, or cartilage oligomeric matrix protein (COMP), and the suspended cells were then added. The medium was changed after 1 hour at 37 degrees C. Adherent cells were further incubated for 18 hours in the presence or absence of tumour necrosis factor alpha (TNF alpha) or interleukin 1 beta. Cells were pretreated with murine IgG1, anti-CD29, -CD51, -CD61 (integrins), or -CD3 monoclonal antibodies and transferred to culture plates coated with COMP. Adherent cells were counted by light microscopy. The expression of intracellular galectin-3, or cell surface CD29, CD51, and CD61 was determined by flow cytometry before and after adhesion. RESULTS: Four times more RA synovial fibroblasts (SF) than osteoarthritis SF adhered to COMP. RA SF presented more cell surface integrins, and monoclonal antibodies against CD51 inhibited the adhesion to COMP by 80%. TNF alpha reduced the expression of CD61 and the adhesion to COMP, but did not reverse the adhesion once it had taken place. The adhesion of RA SF to COMP was found to increase the intracellular level of galectin-3. In contrast, intracellular galectin-3 decreased after exposure to TNF alpha. CONCLUSION: The increase of galectin-3 occurs after adhesion to COMP, and the alpha V beta 3 receptor (CD51/CD61) has a pivotal role in this process. (+info)
Identification of subpopulations with characteristics of mesenchymal progenitor cells from human osteoarthritic cartilage using triple staining for cell surface markers.
(40/201)
We first identified and isolated cellular subpopulations with characteristics of mesenchymal progenitor cells (MPCs) in osteoarthritic cartilage using fluorescence-activated cell sorting (FACS). Cells from osteoarthritic cartilage were enzymatically isolated and analyzed directly or after culture expansion over several passages by FACS using various combinations of surface markers that have been identified on human MPCs (CD9, CD44, CD54, CD90, CD166). Culture expanded cells combined and the subpopulation derived from initially sorted CD9+, CD90+, CD166+ cells were tested for their osteogenic, adipogenic and chondrogenic potential using established differentiation protocols. The differentiation was analyzed by immunohistochemistry and by RT-PCR for the expression of lineage related marker genes. Using FACS analysis we found that various triple combinations of CD9, CD44, CD54, CD90 and CD166 positive cells within osteoarthritic cartilage account for 2-12% of the total population. After adhesion and cultivation their relative amount was markedly higher, with levels between 24% and 48%. Culture expanded cells combined and the initially sorted CD9/CD90/CD166 triple positive subpopulation had multipotency for chondrogenic, osteogenic and adipogenic differentiation. In conclusion, human osteoarthritic cartilage contains cells with characteristics of MPCs. Their relative enrichment during in vitro cultivation and the ability of cell sorting to obtain more homogeneous populations offer interesting perspectives for future studies on the activation of regenerative processes within osteoarthritic joints. (+info)