Stromelysin 1, neutrophil collagenase, and collagenase 3 do not play major roles in a model of chondrocyte mediated cartilage breakdown.
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AIMS: To determine the collective roles of stromelysin 1, neutrophil collagenase, and collagenase 3 in chondrocyte mediated cartilage proteoglycan and type II collagen degradation in tissue culture model systems. METHODS: Bovine nasal cartilage explants were cultured with and without recombinant human interleukin 1 alpha (IL-1 alpha), recombinant human tumour necrosis factor alpha, or retinoic acid. Proteoglycan and type II collagen release were determined by colorimetric assay and immunoassay, respectively, in the absence and presence of matrixin inhibitors. Potential toxic effects of the inhibitors were assessed by measuring rates of glycolysis. RESULTS: Loss of proteoglycan and type II collagen from nasal cartilage was inhibited by batimastat, a broad spectrum matrixin inhibitor. BB-3437, a selective inhibitor of stromelysin, neutrophil collagenase, and collagenase 3, at the concentrations used in this study, showed a weak but dose dependent inhibitory effect on the IL-1 stimulated degradation of type II collagen, but had virtually no effect on proteoglycan breakdown. Neither inhibitor affected rates of glycolysis. CONCLUSIONS: Stromelysin 1, neutrophil collagenase, and collagenase 3 are unlikely to contribute to chondrocyte mediated proteoglycan degradation in our model system. The modest effect of a selective inhibitor of these enzymes on IL-1 stimulated collagen breakdown suggests a minor role for one or more of these proteinases; potent inhibition by an inhibitor of interstitial collagenase and the gelatinases suggests that these enzymes play a major role in IL-1 stimulated, chondrocyte mediated type II collagen breakdown from nasal cartilage. (+info)
In vitro endothelial differentiation of long-term cultured murine embryonic yolk sac cells induced by matrigel.
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The yolk sac of an early mammalian embryo contains progenitors of hematopoietic cells and vascular endothelial cells. We established a cell line, YS4, from murine embryonic yolk sac 10 years ago. The line has been successfully cultured since then. To determine whether these long-term cultured yolk sac cells still have the potential to differentiate into endothelial cells, an in vitro model of yolk sac cell differentiation into tubeforming endothelial cells was established in the present study by culturing the yolk sac cells on basement membrane proteins (Matrigel). The results indicate that upon plating onto Matrigel, YS4 cells attach quickly, align in tandem, and form a complete network of capillary structures within 12 h. By using antibodies against the known components of Matrigel in a tube formation inhibition assay, we found that extracellular matrix proteins such as laminin, collagen IV, vitronectin, and fibronectin are the most important components in the Matrigel which induce the yolk sac cells to undergo endothelial differentiation. New basement membrane proteins are also required for the endothelial differentiation process, as indicated by the fact that base membrane protein synthesis inhibitor, D609, can block the differentiation process. Furthermore, our experiments revealed the involvement of several signal transduction pathways, such as protein kinase A, C and protein tyrosine kinase in this differentiation process. (+info)
Glypican-1 is a VEGF165 binding proteoglycan that acts as an extracellular chaperone for VEGF165.
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Glypican-1 is a member of a family of glycosylphosphatidylinositol anchored cell surface heparan sulfate proteoglycans implicated in the control of cellular growth and differentiation. The 165-amino acid form of vascular endothelial growth factor (VEGF165) is a mitogen for endothelial cells and a potent angiogenic factor in vivo. Heparin binds to VEGF165 and enhances its binding to VEGF receptors. However, native HSPGs that bind VEGF165 and modulate its receptor binding have not been identified. Among the glypicans, glypican-1 is the only member that is expressed in the vascular system. We have therefore examined whether glypican-1 can interact with VEGF165. Glypican-1 from rat myoblasts binds specifically to VEGF165 but not to VEGF121. The binding has an apparent dissociation constant of 3 x 10(-10) M. The binding of glypican-1 to VEGF165 is mediated by the heparan sulfate chains of glypican-1, because heparinase treatment abolishes this interaction. Only an excess of heparin or heparan sulfates but not other types of glycosaminoglycans inhibited this interaction. VEGF165 interacts specifically not only with rat myoblast glypican-1 but also with human endothelial cell-derived glypican-1. The binding of 125I-VEGF165 to heparinase-treated human vascular endothelial cells is reduced following heparinase treatment, and addition of glypican-1 restores the binding. Glypican-1 also potentiates the binding of 125I-VEGF165 to a soluble extracellular domain of the VEGF receptor KDR/flk-1. Furthermore, we show that glypican-1 acts as an extracellular chaperone that can restore the receptor binding ability of VEGF165, which has been damaged by oxidation. Taken together, these results suggest that glypican-1 may play an important role in the control of angiogenesis by regulating the activity of VEGF165, a regulation that may be critical under conditions such as wound repair, in which oxidizing agents that can impair the activity of VEGF are produced, and in situations were the concentrations of active VEGF are limiting. (+info)
Distinct secondary structures of the leucine-rich repeat proteoglycans decorin and biglycan. Glycosylation-dependent conformational stability.
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Biglycan and decorin have been overexpressed in eukaryotic cells and two major glycoforms isolated under native conditions: a proteoglycan substituted with glycosaminoglycan chains; and a core protein form secreted devoid of glycosaminoglycans (Hocking, A. M., Strugnell, R. A., Ramamurthy, P., and McQuillan, D. J. (1996) J. Biol. Chem. 271, 19571-19577; Ramamurthy, P., Hocking, A. M., and McQuillan, D. J. (1996) J. Biol. Chem. 271, 19578-19584). Far-UV CD spectroscopy of decorin and biglycan proteoglycans indicates that, although they are predominantly beta-sheet, biglycan has a significantly higher content of alpha-helical structure. Decorin proteoglycan and core protein are very similar, whereas the biglycan core protein exhibits closer similarity to the decorin glycoforms than to the biglycan proteoglycan form. However, enzymatic removal of the chondroitin sulfate chains from biglycan proteoglycan does not induce a shift to the core protein structure, suggesting that the final form is influenced by polysaccharide addition only during biosynthesis. Fluorescence emission spectroscopy demonstrated that the single tryptophan residue, which is at a conserved position at the C-terminal domain of both biglycan and decorin, is found in similar microenvironments. This indicates that in this specific domain the different glycoforms do exhibit apparent conservation of structure. Exposure of decorin and biglycan to 10 M urea resulted in an increase in fluorescent intensity, which indicates that the emission from tryptophan in the native state is quenched. Comparison of urea-induced protein unfolding curves provide further evidence that decorin and biglycan assume different structures in solution. Decorin proteoglycan and core protein unfold in a manner similar to a classic two-state model, in which there is a steep transition to an unfolded state between 1 and 2 M urea. The biglycan core protein also shows a similar steep transition. However, biglycan proteoglycan shows a broad unfolding transition between 1 and 6 M urea, probably indicating the presence of stable unfolding intermediates. (+info)
Expression of NG2/human melanoma proteoglycan in human adult articular chondrocytes.
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OBJECTIVE: NG2 is a transmembrane chondroitin sulfate (CS) rich proteoglycan originally identified in rats. It has recently been shown to be identical to human melanoma proteoglycan (HMPG). In rats NG2 has a limited distribution in adult tissues, being expressed predominantly by neuronal and glial cells whereas during development it is also expressed in developing mesenchyme including cartilage. NG2/HMPG has putative roles in interactions between glial and melanoma cells with extracellular matrix (ECM) molecules. This study was undertaken to assess whether NG2/HMPG was expressed by normal and osteoarthritic human articular chondrocytes. DESIGN: Cryostat sections of human fetal knee joints and normal and osteoarthritic articular cartilage were immunostained with antibodies against rat NG2 (N143.8) and HMPG (M28B5, 9.2.27). Immunoprecipitation and Western blotting was carried out on protein extracts of chondrocytes from normal and osteoarthritic cartilage. Immunofluorescence of NG2 and potential ligands was carried out in vitro on cells from normal and osteoarthritic cartilage. RESULTS: Fetal and both normal and osteoarthritic adult cartilage showed strong immunoreactivity for NG2/HMPG. Western blotting showed a smeared component of molecular weight greater than 400 kDa and a faint band at 250 kDa which became predominant upon digestion with chondroitinase ABC. Immunofluorescence of chondrocytes in vitro showed NG2 to be distributed in a punctate pattern without co-localization of actin or several ECM proteins including fibronectin and type VI collagen. CONCLUSION: NG2/HMPG is expressed by human fetal and adult chondrocytes and in adult articular chondrocytes the core protein is chondroitin sulfated. The function of this molecular in human articular cartilage remains to be defined. (+info)
Differential effects of local application of BMP-2 or TGF-beta 1 on both articular cartilage composition and osteophyte formation.
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OBJECTIVE: The related molecules bone morphogenetic protein-2 (BMP-2) and transforming growth factor beta-1 (TGF-beta 1) have both been shown to stimulate chondrocyte proteoglycan (PG) synthesis in vitro. We investigated the in-vivo effects of these factors on articular cartilage PG metabolism. DESIGN: Several doses of BMP-2 or TGF-beta 1 were injected into the murine knee joint, once or repeatedly. Patellar cartilage PG synthesis was measured by [35S]-sulfate incorporation and reverse transcriptase polymerase chain reaction (RT-PCR). PG content was analyzed by measuring safranin O staining intensity on histologic sections. RESULTS: A single injection of 200 ng BMP-2 induced a much earlier and more impressive stimulation of articular cartilage PG synthesis, than 200 ng TGF-beta 1. RT-PCR revealed that both factors upregulated mRNA of aggrecan more than that of biglycan and decorin. However, 21 days after a single injection of 200 ng TGF-beta 1 PG synthesis still was significantly increased, while stimulation by BMP-2 only lasted for 3 to 4 days. Stimulation by BMP-2 could be prolonged to at least 2 weeks by triple injections of 200 ng each, at alternate days. Remarkably, even after this intense exposure to BMP-2, stimulation of PG synthesis was not reflected in long-lasting enhancement of PG content of articular cartilage. In contrast, even a single injection with 200 ng of TGF-beta 1 induced prolonged enhancement of PG content. After repeated injections, both BMP-2 and TGF-beta 1 induced chondrogenesis at specific sites. 'Chondrophytes' induced by BMP-2 were found predominantly in the region where the growth plates meet the joint space, while those triggered by TGF-beta 1 originated from the periosteum also at sites remote from the growth plates. CONCLUSIONS: BMP-2 and TGF-beta stimulate PG synthesis and PG content with different kinetics, and these factors have different chondro-inductive properties. (+info)
Matrix degradation by chondrocytes cultured in alginate: IL-1 beta induces proteoglycan degradation and proMMP synthesis but does not result in collagen degradation.
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OBJECTIVE: To determine the role of interleukin-1 beta (IL-1 beta) in the degradation of proteoglycans and collagen by articular chondrocytes. DESIGN: Chondrocytes were cultured in alginate beads for 2 weeks to produce extracellular matrix, followed by the addition of IL-1 beta for 1 or 2 days. Breakdown of extracellular matrix (with and without activation of pro-matrix metalloproteinases (MMPs) by APMA) was monitored by release of glycosaminoglycans (GAG, proteoglycans) and hydroxyproline (collagen) from the beads into the medium, and by the amount of damaged collagen in the bead. Levels of (pro)MMPs in the beads were assayed by zymography and their activity was quantified fluorometrically. RESULTS: IL-1 beta induced a profound GAG release (approximately 80% after 2 days at 20 ng/ml IL-1 beta) that was both time and IL-1 beta concentration dependent. Under these conditions no increase in collagen release or damaged collagen in the bead was detected. Zymography demonstrated that the synthesis of a variety of proMMPs was induced by IL-1 beta, without a detectable increase of MMP-activity as measured in the activity assay. After activation of the proMMPs by APMA, a time and IL-1 beta concentration-dependent increase in MMP-activity was found, which resulted in almost complete deterioration of collagen already after 18 h of incubation. In the presence of APMA, GAG release from IL-1 beta treated beads was significantly increased from 24 to 31%. CONCLUSIONS: Our data suggest that proteoglycan and collagen degradation are regulated through different mechanisms: IL-1 beta induces the synthesis of active enzymes that degrade proteoglycans, such as 'aggrecanase', and inactive proMMPs. Thus, IL-1 beta alone is not sufficient to result in collagen-degrading MMPs. Once activated, MMPs may account for up to a quarter of the aggrecan degradation in this model. (+info)
Longitudinal and cross-sectional variability in markers of joint metabolism in patients with knee pain and articular cartilage abnormalities.
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OBJECTIVE: To determine the within- and between-patient variability in the concentrations of synovial fluid, serum and urine markers of joint tissue metabolism in a cohort of patients with knee pain and cartilage changes consistent with early-stage knee osteoarthritis. DESIGN: Samples of synovial fluid, serum, and urine were obtained from 52 patients on eight different occasions during 1 year, as part of a clinical trial in patients with cartilage abnormalities and knee pain. In joint fluid, aggrecan fragments were quantified by dye precipitation and enzyme-linked immunosorbent assay (ELISA), and matrix metalloproteinases-1 and -3, and tissue inhibitor of metalloproteinases-1 by sandwich ELISAs. In serum, keratan sulfate was quantified by ELISA. Type I collagen N-telopeptide cross-links in urine were determined by ELISA. RESULTS: The degree of cross-sectional variability in marker concentrations did not vary between the different sampling occasions, and did not differ between the periods of weeks 0 (baseline), 1-4 (treatment) and 13-26 (follow-up). Both between-patient and within-patient coefficients of variation varied for markers in different body fluid compartments, with the lowest variability for serum keratan sulfate, followed by urine type I collagen N-telopeptide crosslinks, and the highest for synovial fluid markers. For synovial fluid, aggrecan fragments showed the least variability, and matrix metalloproteinases the highest. One patient with septic arthritis showed a fivefold peak increase in joint fluid aggrecan fragment concentrations, while the concentration of matrix metalloproteinase-3 increased 100-fold. CONCLUSIONS: Molecular markers of joint tissue metabolism have been suggested as, for example, outcome measures for clinical trials of disease-modifying drugs in osteoarthritis. This report is the first to present data on between- and within-patient variability for such molecular markers in three different body fluid compartments in stable cohort of patients. The availability of such data enables calculations to determine the number of patients needed in prospective studies using these markers as outcome measures. (+info)