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GAG
Terms modified by GAG Selected AbstractsIncreased Glycosaminoglycans Production in Sclerosing Basal Cell Carcinoma-Derived Fibroblasts and Stimulation of Normal Skin Fibroblast Glycosaminoglycans Production by a Cytokine-Derived from Sclerosing Basal Cell CarcinomaDERMATOLOGIC SURGERY, Issue 11 2000Ronald L. Moy MD Sclerosing basal cell carcinoma (S-BCC) is characterized by an abundant stroma. There is evidence that some tumor cells secrete cytokines that are mitogenic for stromal fibroblasts (FBs). From this study we report increased glycosaminoglycan (GAG) production by cultures of S-BCC FBs in comparison to cultures of nodular BCC (N-BCC) FBs and normal skin FBs. GAG production was measured by cetylpyridinium chloride precipitation of incorporated [3H]-glucosamine. The sclerosing BCC FBs demonstrated a significant increase in production of GAG over control FBs (P < .001) and over N-BCC FBs (P < .001). Values reported as a mean percentage ± SEM for GAG production by S-BCC over control normal skin FBs are 359 ± 28 and over N-BCC FBs are 266 ± 27. In additional experiments, cell extract dilutions from S-BCC tumor, normal dermis, and normal epidermis were incubated with cultures of normal skin FBs. S-BCC-conditioned media was also incubated with normal FBs and GAG production was measured. For both S-BCC extracts and conditioned media, a dose response curve was established showing increased GAG production by normal FBs in relation to increasing the concentration of S-BCC extract or conditioned media. When S-BCC extract was added to normal FBs there was increased GAG production in comparison to normal FBs incubated with dermal or epidermal extracts (P < .001) for both. Two growth factors, transforming growth factor-, (TGF-,) and platelet-derived growth factor (PDGF), already known to be mitogenic for FBs, were incubated with N-BCC and normal FBs in an effort to elucidate the potential cytokine(s) released by S-BCC, causing increased GAG production by surrounding FBs. Neither of these cytokines proved to be effective in promoting a significant increase in GAG production. Our findings support the hypothesis that BCCs release factors that alter stromal FB production of GAG. [source] Improved workup for glycosaminoglycan disaccharide analysis using CE with LIF detectionELECTROPHORESIS, Issue 22 2008Alicia M. Hitchcock Abstract This work describes improved workup and instrumental conditions to enable robust, sensitive glycosaminoglycan (GAG) disaccharide analysis from complex biological samples. In the process of applying CE with LIF to GAG disaccharide analysis in biological samples, we have made improvements to existing methods. These include (i) optimization of reductive amination conditions, (ii) improvement in sensitivity through the use of a cellulose cleanup procedure for the derivatization, and (iii) optimization of separation conditions for robustness and reproducibility. The improved method enables analysis of disaccharide quantities as low as 1,pmol prior to derivatization. Biological GAG samples were exhaustively digested using lyase enzymes, the disaccharide products and standards were derivatized with the fluorophore 2-aminoacridone and subjected to reversed polarity CE-LIF detection. These conditions resolved all known chondroitin sulfate (CS) disaccharides or 11 of 12 standard heparin/heparan sulfate disaccharides, using 50,mM phosphate buffer, pH 3.5, and reversed polarity at 30,kV with 0.3,psi pressure. Relative standard deviation in migration times of CS ranged from 0.1 to 2.0% over 60 days, and the relative standard deviations of peak areas were less than 3.2%, suggesting that the method is reproducible and precise. The CS disaccharide compositions are similar to those obtained by our group using tandem MS. The reversed polarity CE-LIF disaccharide analysis protocol yields baseline resolution and quantification of heparin/heparan sulfate and CS/dermatan sulfate disaccharides from both standard preparations and biologically relevant proteoglycan samples. The improved CE-LIF method enables disaccharide quantification of biologically relevant proteoglycans from small samples of intact tissue. [source] A novel ultra-sensitive method for the quantification of glycosaminoglycan disaccharides using an automated DNA sequencerELECTROPHORESIS, Issue 7 2006Kay Vogel Abstract Analysis of glycosaminoglycans (GAGs) is of increasing importance concerning alterations in extracellular matrix composition and selectivity of glomerular basement membrane. In this report we describe the analysis of chondroitin sulfate disaccharides as an example of GAG ,disaccharide analysis using standard DNA sequencing equipment (DNA sequencer-assisted GAG disaccharide separation, DSA-GAGS). The presented methodology allows nanomolar quantification of 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-derived GAG disaccharides. In comparison to RP-HPLC the established method is much more sensitive, showing detection limits of 38,fmol/,L. Variation coefficients were approximately 10%, enabling exact quantifications after run times of 17,min at 30°C and an electrophoresis voltage of 15,kV; using a capillary DNA sequencer, available in many molecular laboratories, presented advantages like automated sample injection, opportunity of high-throughput analyses, separation of even sulfated disaccharide epimers, and the possibility of using APTS-derived fucose as an internal standard. Furthermore, highly reproducible retention times rendered easy identification of specific signals (SD,0.02). With regard to these results, the described method is a useful tool for the quantification of GAG disaccharides in low amounts, indicating advantages of obverse RP-HPLC and slab gel polyacrylamide electrophoresis in sensitivity, error-proneness, automation, and handling. [source] Construction and characterization of Bacillus subtilis deletion mutants lacking the prophage 2 - trnS regionFEMS MICROBIOLOGY LETTERS, Issue 2 2006Genki Akanuma Abstract During development of a novel method for constructing a series of deletions in Bacillus subtilis using an isogenic set of gene-disrupted mutants created by integration of pMutin, deletion of the trnS operon, consisting of seven tRNA genes, was found to affect cell growth, development of competence and spore formation. A suppressor (sts1) of the ,trnS mutant was isolated, sequenced and found to have undergone a single base change, CAG to GAG, in the first anticodon of tRNALeu, in the trnB operon. [source] Haemoglobin H disease due to (,,,SEA) ,-globin gene deletion and ,2-codon 30 (,GAG) mutation: a family studyINTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY, Issue 5 2001S.K. Ma A Chinese family in which two siblings suffer from haemogloblin (Hb) H disease due to (,,,SEA) ,-globin gene deletion and ,2-codon 30 (,GAG) mutation is described. Both siblings are transfusion-independent and have survived to adulthood. In contrast to previous report of hydrops fetalis associated with ,-,-thal-1 and ,2-codon 30 (,GAG) mutation, the ,-globin genes are intact in the two siblings, which most probably alleviates the ,-chain excess and protects the fetus from severe anaemia. Correlation of genotype with phenotype in Hb H disease is important for genetic counselling, especially in the antenatal setting. [source] Urinary macromolecules and renal tubular cell protection from oxalate injury: Comparison of normal subjects and recurrent stone formersINTERNATIONAL JOURNAL OF UROLOGY, Issue 3 2006MASAO TSUJIHATA Aim:, To determine whether urinary macromolecules (UMM), which are the high molecular weight substances in urine, can provide protection against the oxalate-associated injury to the renal tubular cells. Methods:, UMM were extracted from 24-h urine of 12 healthy adult male volunteers and 13 recurrent-stone-former male patients. Urine parameters in relation to urolithiasis were measured, including the level of glycosaminoglycans (GAG) in the UMM. Madin-Darby canine kidney (MDCK) cells were used to evaluate the protective activity of UMM from oxalate-induced cytotoxicity by LDH release measurement and methyl-thiazolyl tertrazolium (MTT) assay. Results:, Considering urinary parameters, citrate was significantly higher in urine from normal subjects than stone-former subjects; the other parameters show no differences between the groups. Total UMM and the level of GAG in the UMM were also significantly higher in the normal subject group. Compared with normal subject and stone-former subject UMM, after cells were treated with the UMM and then exposed to oxalate solution, LDH release was significantly higher in stone-former group. In the MTT assay, we found that more viable cells were observed after treatment with UMM compared to control in both groups. Moreover, UMM from the normal subjects showed higher protective activity against oxalate-related cytotoxicity than UMM from the stone-former subjects. Conclusion:, UMM protected renal epithelial cells from oxalate-related injury. This protective activity was found to be higher in normal subject UMM than stone-former UMM. Among other factors, a higher concentration of GAG and citrate in normal subject UMM might affect some parts in this finding. [source] A histochemical study of the reproductive structures in the flatworm Dugesia leporii (Platyhelminthes, Tricladida)INVERTEBRATE BIOLOGY, Issue 2 2006Gavina Corso Abstract. The functional morphology and the topographic distribution of tissues in the reproductive system of specimens of Dugesia leporii, an endemic Sardinian free-living planarian, are investigated. Data are provided on the nature of epithelial and glandular secretions, spermatophores, and cocoons by histochemistry, light microscopy, and scanning electron microscopy. All secreting epithelial cells produce strongly acidic sulfated glycoproteins. Glandular cells secrete strongly acidic sulfated glycoproteins or keratohyalin-like material in the penis bulb, and prekeratin-like material in atrial glands. Secretions of the bursa copulatrix may be involved in the activation of sperm while material produced by the bursa canal and oviducts probably serves to propel spermatophores or sperm and eggs. Mucous secretion of the seminal vesicle may serve to dilute and activate sperm before copulation. The viscous secrete of the ejaculatory duct and vasa deferentia may play a protective role to maintain sperm viability. Materials produced by the penis papilla and atrium probably lubricate the epithelial surface. The bilayered wall of spermatophore made of keratohyalin-like material and strongly acidic sulfated glycoproteins is produced by two gland types of the penis bulb. The bilayered shell of cocoon made of prekeratin-like and keratohyalin-like materials is secreted by both atrial glands and vitelline cells. The cocoon stalk is made of keratohyalin-like material produced by cement glands. Shell glands, producing GAG, are not involved in cocoon formation, but they may be implicated in the dilution and activation of seminal material to favor sperm movement toward the oviducts. [source] Characterization of cartilagenous tissue formed on calcium polyphosphate substrates in vitroJOURNAL OF BIOMEDICAL MATERIALS RESEARCH, Issue 3 2002Stephen D. Waldman Abstract Successful joint resurfacing by tissue-engineered cartilage has been limited, in part, by an inability to secure the implant to bone. To overcome this, we have developed the methodology to form a cartilage implant in vitro consisting of a layer of cartilagenous tissue overlying a porous, biodegradable calcium polyphosphate (CPP) substrate. As bone will grow into the CPP after implantation, it will result in anchorage of the cartilage. In this study, the cartilagenous tissue formed in vitro after 8 weeks in culture was characterized and compared to native articular cartilage. Light microscopic examination of histological sections showed that there was a continuous layer of cartilagenous tissue on, and integrated with the subsurface of, the CPP substrate. The in vitro -formed tissue achieved a similar thickness to native articular cartilage (mean ± SEM: in vitro = 0.94 ± 0.03 mm; ex vivo = 1.03 ± 0.01 mm). The cells in the in vitro -formed tissue synthesized large proteoglycans (Kav ± SEM: in vitro = 0.27 ± 0.01; ex vivo = 0.27 ± 0.01) and type II collagen similar to the chondrocytes in the ex-vivo cartilage. The in vitro -formed tissue had a similar amount of proteoglycan (GAG ,g/mg dry wt.: in vitro = 198 ± 10; ex vivo = 201 ± 13) but less collagen than the native cartilage (hydroxyproline ,g/mg dry wt.: in vitro = 21 ± 1; ex vivo = 70 ± 8). The in vitro -formed tissue had only about 3% of the load-bearing capacity and stiffness of the native articular cartilage, determined from unconfined mechanical compression testing. Although low, this was within the range of properties reported by others for tissue-engineered cartilage. It is possible that the limited load-bearing capacity is the result of the low collagen content and further studies are required to identify the conditions that will increase collagen synthesis. © 2002 Wiley Periodicals, Inc. J Biomed Mater Res 62:323,330, 2002 [source] Regulated expression of syndecan-4 in rat calvaria osteoblasts induced by fibroblast growth factor-2JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2007Shu Jun Song Abstract Fibroblast growth factor-2 (FGF2) is a member of a prominent growth factor family that drives proliferation in a wide variety of cell types, including osteoblasts. The binding and signal transduction triggered by these mitogens is dependent on glycosaminoglycan (GAG) sugars, particularly of the heparan sulfate (HS) class. These are secreted in proteoglycan (PG) complexes, some of which become FGF co-receptors. The syndecans, the transmembrane forms of HSPG of which there are four members, act as multifunctional receptors for a variety of ligands involved in cell-extracellular matrix (ECM) adhesion as well as growth factor binding. To understand the role of syndecans in developing osteoblasts, the effects of exogenous FGF2 on syndecan expression were examined using primary rat calvarial osteoblasts. All four syndecan mRNAs were expressed in the osteoblasts, although only syndecan-4 was upregulated by FGF2 treatment in a dose-dependent manner. This upregulation could be abrogated by pretreatment with the protein synthesis inhibitor cycloheximide, suggesting that the upregulation of syndecan-4 by FGF2 is not a primary response. Osteoblast proliferation and mineralization were enhanced by exogenous FGF2 treatment, but could be specifically diminished by anti-syndecan-4 antibody pretreatment. This treatment also blocked FGF2-induced extracellular signal-regulated kinase activation, but not the expression of the bone-specific transcription factor Runx2. These results demonstrate that mitogen-triggered syndecan-4 expression is an intrinsic part of the pathways subtending osteoblast proliferation and mineralization. J. Cell. Biochem. 100: 402,411, 2007. © 2006 Wiley-Liss, Inc. [source] Enhanced cartilage tissue engineering by sequential exposure of chondrocytes to FGF-2 during 2D expansion and BMP-2 during 3D cultivationJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2001Ivan Martin Abstract Bovine calf articular chondrocytes, either primary or expanded in monolayers (2D) with or without 5 ng/ml fibroblast growth factor-2 (FGF-2), were cultured on three-dimensional (3D) biodegradable polyglycolic acid (PGA) scaffolds with or without 10 ng/ml bone morphogenetic protein-2 (BMP-2). Chondrocytes expanded without FGF-2 exhibited high intensity immunostaining for smooth muscle ,-actin (SMA) and collagen type I and induced shrinkage of the PGA scaffold, thus resembling contractile fibroblasts. Chondrocytes expanded in the presence of FGF-2 and cultured 6 weeks on PGA scaffolds yielded engineered cartilage with 3.7-fold higher cell number, 4.2-fold higher wet weight, and 2.8-fold higher wet weight glycosaminoglycan (GAG) fraction than chondrocytes expanded without FGF-2. Chondrocytes expanded with FGF-2 and cultured on PGA scaffolds in the presence of BMP-2 for 6 weeks yielded engineered cartilage with similar cellularity and size, 1.5-fold higher wet weight GAG fraction, and more homogenous GAG distribution than the corresponding engineered cartilage cultured without BMP-2. The presence of BMP-2 during 3D culture had no apparent effect on primary chondrocytes or those expanded without FGF-2. In summary, the presence of FGF-2 during 2D expansion reduced chondrocyte expression of fibroblastic molecules and induced responsiveness to BMP-2 during 3D cultivation on PGA scaffolds. © 2001 Wiley-Liss, Inc. [source] Oversulfated chondroitin sulfate-E binds to BMP-4 and enhances osteoblast differentiationJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2008Tatsuya Miyazaki Small leucine-rich proteoglycans, such as biglycan, and their side chain sulfated glycosaminoglycans (GAGs), have been suggested to be involved in bone formation and mineralization processes. The present study was designed to investigate whether chondroitin sulfate (CS), one of the GAG, and its oversulfated structures coupled with bone morphogenetic protein-4 (BMP-4) alter the differentiation and subsequent mineralization of MC3T3-E1 osteoblastic cells. CS-E, one of the oversulfated CS structure, enhanced cell growth, alkaline phosphatase (ALP) activity, collagen deposition, and mineralization whereas heparin enhanced only ALP activity and mineralization. As well as CS-E, CS-H, and CPS also enhanced the mineralization of the cells. CS-E enhanced the mineralization of the cells by interacting with protein in the conditioned medium. CS-E induced mineralization was significantly inhibited by an antibody against BMP-4. The addition of exogenous BMP-4 further increased the capacity of CS-E to enhance mineralization. Fluorescence correlation spectroscopy method using fluoresceinamine-labeled GAG revealed that the oversulfated GAGs have a high affinity for BMP-4. The disaccharide analysis of the cells indicated that MC3T3-E1 cells are capable of producing oversulfated structures of CS by themselves. The lack of CS from the cells after chondroitinase treatment resulted in the inhibition of mineralization. These results in the present study indicate that oversulfated CS, which possesses 4,6-disulfates in N -acetyl-galactosamine, binds to BMP-4 and promotes osteoblast differentiation and subsequent mineralization. J. Cell. Physiol. 217: 769,777, 2008. © 2008 Wiley-Liss, Inc. [source] Biglycan binds to ,- and ,-sarcoglycan and regulates their expression during development,JOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2006Michael S. Rafii The dystrophin-associated protein complex (DAPC), which links the cytoskeleton to the extracellular matrix, is essential for muscle cell survival, and is defective in a wide range of muscular dystrophies. The DAPC contains two transmembrane subcomplexes,the dystroglycans and the sarcoglycans. Although several extracellular binding partners have been identified for the dystroglycans, none have been described for the sarcoglycan subcomplex. Here we show that the small leucine-rich repeat (LRR) proteoglycan biglycan binds to ,- and ,-sarcoglycan as judged by ligand blot overlay and co-immunoprecipitation assays. Our studies with biglycan-decorin chimeras show that ,- and ,-sarcoglycan bind to distinct sites on the polypeptide core of biglycan. Both biglycan proteoglycan as well as biglycan polypeptide lacking glycosaminoglycan (GAG) side chains are components of the dystrophin glycoprotein complex isolated from adult skeletal muscle membranes. Finally, our immunohistochemical and biochemical studies with biglycan null mice show that the expression of ,- and ,-sarcoglycan is selectively reduced in muscle from young (P14-P21) animals, while levels in adult muscle (,P35) are unchanged. We conclude that biglycan is a ligand for two members of the sarcoglycan complex and regulates their expression at discrete developmental ages. J. Cell. Physiol. 209: 439,447, 2006. © 2006 Wiley-Liss, Inc. [source] Novel glycosaminoglycan mimetic (RGTA, RGD120) contributes to enhance skeletal muscle satellite cell fusion by increasing intracellular Ca2+ and calpain activityJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2005M. Zimowska Glycosaminoglycans (GAG) are classes of molecules that play an important role in cellular processes. The use of GAG mimetics called regenerating agent (RGTA) represents a tool to investigate the effect of GAG moiety on cellular behavior. A first member of the RGTA family (RG1192), a dextran polymers with defined amounts of sulfate, carboxymethyl, as well as hydrophobic groups (benzylamide), was shown to stimulate skeletal muscle repair after damage and myoblast differentiation. To obtain a comprehensive insight into the mechanism of action of GAG mimetics, we investigated the effect on myoblast differentiation of a novel RGTA, named RGD120, which was devoid of hydrophobic substitution and had ionic charge similar to heparin. Myoblasts isolated from adult rat skeletal muscles and grown in primary cultures were used in this study. We found that chronic treatment with RGD120 increased the growth of adult myoblasts and induced their precocious fusion into myotubes in vitro. It also partially overcame the inhibitory effect of the calpain inhibitor N -acetyl-leu-leu-norleucinal (ALLN) on these events. Western blot and zymography analyses revealed that milli calpain was slightly increased by RGD120 chronic treatment. In addition, using fluorescent probes (Indo-1 and Boc-leu-met-MAC), we demonstrated that RGD120 added to prefusing myoblast cultures accelerates myoblast fusion into myotubes, induced an increase of cytosolic free calcium concentration, and concomitantly an increase of intracellular calpain protease activity. Altogether, these results suggested that the efficiency of RGD120 in stimulating myogenesis might be in part explained through its effect on calcium mobilization as well as on the calpain amount and activity. © 2005 Wiley-Liss, Inc. [source] Control of human articular chondrocyte differentiation by reduced oxygen tensionJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2004Christopher L. Murphy Cell number is often a limiting factor in studies of chondrocyte physiology, particularly for human investigations. Chondrocytes can be readily proliferated in monolayer culture, however, differentiated phenotype is soon lost. We therefore endeavored to restore normal phenotype to human chondrocytes after serial passage in monolayer culture by manipulating cell morphology and oxygen tension towards the in vivo state. Third passage cells were encapsulated in alginate and exposed to either 20% or more physiologic 5% oxygen tensions. To assess cell phenotype, gene expression was measured using TaqMan real-time PCR. Encapsulated, primary chondrocytes cultured in 20% oxygen were used as a positive reference. Passaged human chondrocytes were fibroblastic in appearance and had lost normal phenotype as evidenced by a decrease in expression of collagen II, aggrecan, and sox9 genes of 66, 6, and 14 fold, respectively; with concomitant high expression of type I collagen (22 fold increase). A partial regaining of the differentiated phenotype was observed by encapsulation in 20% oxygen; however, even after 4 weeks, collagen II gene expression was not fully restored. Collagen II and aggrecan expression were increased, on average, 3 fold, in 5% oxygen tension compared to 20% cultures. Furthermore, matrix glycosaminoglycan (GAG) levels were significantly increased in reduced oxygen. In fact, after 4 weeks in 5% oxygen, encapsulated third passage cells had collagen II expression fully regained and aggrecan and sox9 levels actually exceeding primary cell levels in 20% oxygen. Our results show that the phenotype of serially passaged human articular chondrocytes is more fully restored by combining encapsulation with culture in more physiological levels of oxygen. Sox9, an essential transcription factor for chondrocyte differentiation is strongly implicated in this process since its expression was upregulated almost 27 fold. These findings have implications for the optimal conditions for the in vitro culture of chondrocytes. © 2004 Wiley-Liss, Inc. [source] TorsinA in PC12 cells: Localization in the endoplasmic reticulum and response to stressJOURNAL OF NEUROSCIENCE RESEARCH, Issue 2 2003Jeffrey Hewett Abstract Most cases of early-onset torsion dystonia are caused by deletion of GAG in the coding region of the DYT1 gene encoding torsinA. This autosomal dominant neurologic disorder is characterized by abnormal movements, believed to originate from neuronal dysfunction in the basal ganglia of the human brain. The torsins (torsinA and torsinB) are members of the "ATPases associated with a variety of cellular activities" (AAA+) superfamily of proteins that mediate chaperone and other functions involved in conformational modeling of proteins, protection from stress, and targeting of proteins to cellular organelles. In this study, the intracellular localization and levels of endogenous torsin were evaluated in rat pheochromocytoma PC12 cells following differentiation and stress. TorsinA, apparent MW 37 kDa, cofractionates with markers for the microsomal/endoplasmic reticulum (ER) compartment and appears to reside primarily within the ER lumen based on protease resistance. TorsinA immunoreactivity colocalizes with the lumenal ER protein protein disulfide isomerase (PDI) and extends throughout neurites. Levels of torsinA did not increase notably in response to nerve growth factor-induced differentiation. None of the stress conditions tested, including heat shock and the unfolded protein response, affected torsinA, except for oxidative stress, which resulted in an increase in the apparent MW of torsinA and redistribution to protrusions from the cell surface. These findings are consistent with a relatively rapid covalent modification of torsinA in response to oxidative stress causing a change in state. Mutant torsinA may interfere with and/or compromise ER functions, especially in dopaminergic neurons, which have high levels of torsinA and are intrinsically vulnerable to oxidative stress. © 2003 Wiley-Liss, Inc. [source] MMP-mediated collagen breakdown induced by activated protein C in equine cartilage is reduced by corticosteroidsJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 3 2010Elaine R. Garvican Abstract The plasma serine protease activated protein C (APC) is synthesized by human chondrocytes at sites of pathological cartilage fibrillation. APC levels are increased in osteoarthritis (OA) synovial fluid, and in vitro APC has been shown to synergize with interleukin-1, (IL-1) to promote degradation from ovine cartilage. A model of equine cartilage degradation was established and used to explore corticosteroid activities. Intraarticular corticosteroids are a commonly prescribed treatment for joint disease, however their role in disease modification remains unclear. APC synergized with IL-1 or tumor necrosis factor-, (TNF,), promoting significant collagen degradation from equine cartilage explants within 4 days, but did not augment glycoaminoglycan (GAG) release. APC activated pro-matrix metalloproteinases (MMP)-2 but not pro-MMP-9, as assessed by gelatin zymography. APC did not directly activate pro-MMP-13. Dexamethasone, triamcinolone, and methylprednisolone acetate (MPA) were evaluated at concentrations between 10, 5M and 10,10M. High concentrations significantly increased GAG release from IL-1+APC,treated explants. With the exception of MPA at 10,10M, all concentrations of corticosteroids caused significant decreases in IL-1+APC-driven hydroxyproline loss. Treatment with corticosteroids suppressed expression of MMP-1, -3, and -13 mRNA. The collagenolysis associated with IL-1+APC synergy, and the inhibition of this effect by corticosteroids may involve gelatinase activation and downregulation of MMP expression, respectively. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:370,378, 2010 [source] Magnetic resonance imaging of cartilage glycosaminoglycan: Basic principles, imaging technique, and clinical applications,JOURNAL OF ORTHOPAEDIC RESEARCH, Issue 3 2008Martha L. Gray Abstract Many new therapeutic strategies have been and are being developed to prevent, correct, or slow the progression of osteoarthritis. Our ability to evaluate the efficacy of these techniques, or to determine the situations for which they might provide the most benefit, critically depends on diagnostic measures that can serve as proxies for the present or predicted state of the cartilage. We focus here on a body of work surrounding the development of magnetic resonance imaging (MRI) techniques to noninvasively image the glycosaminoglycan (GAG) concentration of articular cartilage. These techniques are based on the concept of fixed charge in cartilage resulting from the glycosaminoglycans. Starting with sodium MRI, and the subsequent development of delayed gadolinium-enhanced MRI of cartilage (dGEMRIC) based on proton MRI, these techniques permit "visualization" of the charged GAG distribution in cartilage in vitro or in vivo. The dGEMRIC technique has been used in preliminary clinical studies to understand treatment strategies and to monitor disease, and as such is allowing studies that a decade ago would have been impossible. This new technical capability offers the promises of speeding development of effective therapies and focusing their use in areas where they can be most successful. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:281,291, 2008 [source] Microenvironment regulation of PRG4 phenotype of chondrocytesJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 5 2007Megan E. Blewis Abstract Articular cartilage is a heterogeneous tissue with superficial (S), middle (M), and deep (D) zones. Chondrocytes in the S zone secrete the lubricating PRG4 protein, while chondrocytes from the M and D zones are more specialized in producing large amounts of the glycosaminoglycan (GAG) component of the extracellular matrix. Soluble and insoluble chemicals and mechanical stimuli regulate cartilage development, growth, and homeostasis; however, the mechanisms of regulation responsible for the distinct PRG4-positive and negative phenotypes of chondrocytes are unknown. The objective of this study was to determine if interaction between S and M chondrocytes regulates chondrocyte phenotype, as determined by coculture in monolayer at different ratios of S:M (100:0, 75:25, 50:50, 25:75, 0:100) and at different densities (240,000, 120,000, 60,000, and 30,000 cells/cm2), and by measurement of PRG4 secretion and expression, and GAG accumulation. Coculture of S and M cells resulted in significant up-regulation in PRG4 secretion and the percentage of cells expressing PRG4, with simultaneous down-regulation of GAG accumulation. Tracking M cells with PKH67 dye in coculture revealed that they maintained a PRG4-negative phenotype, and proliferated less than S cells. Taken together, these results indicate that the up-regulated PRG4 expression in coculture is a result of preferential proliferation of PRG4-expressing S cells. This finding may have practical implications for generating a large number of phenotypically normal S cells, which can be limited in source, for tissue engineering applications. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 25:685,695, 2007 [source] Anti-inflammatory effects of continuous passive motion on meniscal fibrocartilageJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 5 2005Mario Ferretti Abstract Motion-based therapies have been applied to promote healing of arthritic joints. The goal of the current study was to determine the early molecular events that are responsible for the beneficial actions of motion-based therapies on meniscal fibrocartilage. Rabbit knees with Antigen-Induced-Arthritis (AIA) were exposed to continuous passive motion (CPM) for 24 or 48 h and compared to immobilized knees. The menisci were harvested and glycosaminoglycans (GAG), interleukin-1, (IL-1,), matrix metalloproteinase-1 (MMP-1), cyclooxygenase-2 (COX-2), and interleukin-10 (IL-10) were determined by histochemical analysis. Within 24 h, immobilized knees exhibited marked GAG degradation. The expression of proinflammatory mediators MMP-1, COX-2, and IL-1, was notably increased within 24 h and continued to increase during the next 24 h in immobilized knees. Knees subjected to CPM revealed a rapid and sustained decrease in GAG degradation and the expression of all proinflammatory mediators during the entire period of CPM treatment. More importantly, CPM induced synthesis of the anti-inflammatory cytokine IL-10. The results demonstrate that mechanical signals generated by CPM exert potent anti-inflammatory signals on meniscal fibrochondrocytes. Furthermore, these studies explain the molecular basis of the beneficial effects of CPM observed on articular cartilage and suggest that CPM suppresses the inflammatory process of arthritis more efficiently than immobilization. © 2005 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved. [source] Spatially-localized correlation of dGEMRIC-measured GAG distribution and mechanical stiffness in the human tibial plateauJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 1 2005Joseph T. Samosky Abstract The concentration of glycosaminoglycan (GAG) in articular cartilage is known to be an important determinant of tissue mechanical properties based on numerous studies relating bulk GAG and mechanical properties. To date limited information exists regarding the relationship between GAG and mechanical properties on a spatially-localized basis in intact samples of native tissue. This relation can now be explored by using delayed gadolinium-enhanced MRI of cartilage (dGEMRIC,a recently available non-destructive magnetic resonance imaging method for measuring glycosaminoglycan concentration) combined with non-destructive mechanical indentation testing. In this study, three tibial plateaus from patients undergoing total knee arthroplasty were imaged by dGEMRIC. At 33,44 test locations for each tibial plateau, the load response to focal indentation was measured as an index of cartilage stiffness. Overall, a high correlation was found between the dGEMRIC index (T) and local stiffness (Pearson correlation coefficients r = 0.90, 0.64, 0.81; p < 0.0001) when the GAG at each test location was averaged over a depth of tissue comparable to that affected by the indentation. When GAG was averaged over larger depths, the correlations were generally lower. In addition, the correlations improved when the central and peripheral (submeniscal) areas of the tibial plateau were analyzed separately, suggesting that a factor other than GAG concentration is also contributing to indentation stiffness. The results demonstrate the importance of MRI in yielding spatial localization of GAG concentration in the evaluation of cartilage mechanical properties when heterogeneous samples are involved and suggest the possibility that the evaluation of mechanical properties may be improved further by adding other MRI parameters sensitive to the collagen component of cartilage. © 2004 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved. [source] A comparison of the , -D-xyloside, odiparcil, to warfarin in a rat model of venous thrombosisJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 9 2006J. R. TOOMEY Summary,Background:,A significant need exists for new chronic oral anticoagulation therapies to replace warfarin. Previous studies have shown that , -D-xylosides, which prime glycosaminoglycan (GAG) synthesis, have antithrombin and antithrombotic activity. In the following report, a new orally active , -D-xyloside (odiparcil) has been characterized in a rat model of venous thrombosis and its efficacy and bleeding liability compared to warfarin. Additionally, studies were conducted to investigate odiparcil's ex vivo antithrombin and antiplatelet activity, and also to explore the potential utility of protamine sulfate as a neutralizing agent. Methods and results:,In vivo thrombosis studies were conducted in a rat inferior vena cava model, and bleeding studies in a rat tail transection model. Following oral dosing, warfarin and odiparcil produced dose-related suppression of thrombus formation. A therapeutically relevant dose of warfarin in this model (international normalized ratio; INR 3.0) achieved ,65% inhibition of thrombus formation. Warfarin caused dose-related significant increases in bleeding indices. Odiparcil antithrombotic activity was limited by its mechanism to a maximum suppression of thrombus formation of 65,70%, and did not prolong bleeding indices. Additionally, odiparcil-induced heparin cofactor II (HCII)-dependent antithrombin activity was shown to be a function of dermatan sulfate-like GAG production. Other than thrombin-related effects, no odiparcil effects on platelet function were observed. In antidote studies, it was demonstrated that odiparcil-induced antithrombotic activity could be partially neutralized by protamine sulfate. Conclusions:,These experiments suggest that an antithrombotic approach based upon xyloside induction of circulating GAGs may have the potential to approximate the efficacy of warfarin and yet with a reduced risk to hemostasis. [source] Performance of new gellan gum hydrogels combined with human articular chondrocytes for cartilage regeneration when subcutaneously implanted in nude miceJOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, Issue 7 2009J. T. Oliveira Abstract Gellan gum is a polysaccharide that has been recently proposed by our group for cartilage tissue-engineering applications. It is commonly used in the food and pharmaceutical industry and has the ability to form stable gels without the use of harsh reagents. Gellan gum can function as a minimally invasive injectable system, gelling inside the body in situ under physiological conditions and efficiently adapting to the defect site. In this work, gellan gum hydrogels were combined with human articular chondrocytes (hACs) and were subcutaneously implanted in nude mice for 4 weeks. The implants were collected for histological (haematoxylin and eosin and Alcian blue staining), biochemical [dimethylmethylene blue (GAG) assay], molecular (real-time PCR analyses for collagen types I, II and X, aggrecan) and immunological analyses (immunolocalization of collagen types I and II). The results showed a homogeneous cell distribution and the typical round-shaped morphology of the chondrocytes within the matrix upon implantation. Proteoglycans synthesis was detected by Alcian blue staining and a statistically significant increase of proteoglycans content was measured with the GAG assay quantified from 1 to 4 weeks of implantation. Real-time PCR analyses showed a statistically significant upregulation of collagen type II and aggrecan levels in the same periods. The immunological assays suggest deposition of collagen type II along with some collagen type I. The overall data shows that gellan gum hydrogels adequately support the growth and ECM deposition of human articular chondrocytes when implanted subcutaneously in nude mice. Copyright © 2009 John Wiley & Sons, Ltd. [source] Surface Plasmon Resonance Spectroscopy as a Tool to Study Polyplex-Glycoaminoglycan InteractionsMACROMOLECULAR RAPID COMMUNICATIONS, Issue 12 2005Peter Dubruel Abstract Summary: This article reports the application of surface plasmon resonance (SPR) to monitor the interaction between polymer-DNA complexes and glycoaminoglycans (GAG). The GAG selected was hyaluronic acid (HA). First a HA derivative containing a disulfide linkage was synthesized, enabling chemisorption onto a gold surface. Next, the interaction between different complexes (prepared using PEI or PDMAEMA) and HA was studied using SPR. This study clearly indicates that GAG-polyplex interactions depend on the type of polymer selected and on the charge ratio of the polyplexes prepared. The derivative developed opens up new perspectives in the field of nonviral gene delivery. [source] Variable small protein (Vsp)-dependent and Vsp-independent pathways for glycosaminoglycan recognition by relapsing fever spirochaetesMOLECULAR MICROBIOLOGY, Issue 4 2000Loranne Magoun Tick-borne relapsing fever, caused by pathogenic Borrelia such as B. hermsii and B. turicatae, features recurrent episodes of bacteraemia, each of which is caused by a population of spirochaetes that expresses a different variable major protein. Relapsing fever is also associated with the infection of a variety of tissues, such as the central nervous system. In this study, we show that glycosaminoglycans (GAGs) mediate the attachment of relapsing fever spirochaetes to mammalian cells. B. hermsii strain DAH bound to immobilized heparin, and heparin and dermatan sulphate blocked bacterial binding to host cells. Bacterial binding was diminished by inhibition of host cell GAG synthesis or sulphation, or by the enzymatic removal of GAGs. GAGs mediated the attachment of relapsing fever spirochaetes to potentially relevant target cells, such as endothelial and glial cells. B. hermsii was able to attach to GAGs independently of variable major proteins, because strains expressing the variable major proteins Vsp33, Vlp7 or no variable major protein at all each recognized GAGs. Nevertheless, we found that a variable major protein of B. turicatae directly promoted GAG binding by this relapsing fever spirochaete. B. turicatae strain Oz1 serotype B, which expresses the variable major protein VspB, bound to GAGs more efficiently than did B. turicatae Oz1 serotype A, which expresses VspA. Recombinant VspB, but not VspA, bound to heparin and dermatan sulphate. Previous studies have shown that strain Oz1 serotype B grows to higher concentrations in the blood than does Oz1 serotype A. Thus, relapsing fever spirochaetes have the potential to express Vsp-dependent and Vsp-independent GAG-binding activities and, for one pair of highly related B. turicatae strains, differences in GAG binding correlate with differences in tissue tropism. [source] Biomimetic modification of chitosan with covalently grafted lactose and blended heparin for improvement of in vitro cellular interactionPOLYMERS FOR ADVANCED TECHNOLOGIES, Issue 1 2008Huaping Tan Abstract Lactose- and heparin-modified chitosan films were prepared and their physical and biological properties were compared with chitosan, chitosan-g-heparin, and chitosan-g-lactose films. Atomic force microscopy (AFM) measurement showed that all these films in the dry state were rather flat with a roughness smaller than 20,nm. While the chitosan-g-lactose/heparin and chitosan-g-lactose films have the highest swelling and weight loss ratios, the chitosan and chitosan-g-heparin films have the lowest. The chitosan-g-lactose/heparin film showed stronger ability to induce chondrocyte attachment, proliferation, viability, and glycosaminoglycan (GAG) secretion than that of the chitosan, chitosan-g-heparin, and chitosan-g-lactose films. Chondrocyte aggregates and nodules were observed on the chitosan-g-lactose/heparin and chitosan-g-lactose films, which still preserved viable metabolic ability. These results show that the lactose-modified and heparin-incorporated chitosan film can enhance the cell,biomaterial interaction synchronously. The resulting chitosan-g-lactose/heparin material is more bioactive that might be applicable as promising scaffold for chondrogenesis. Copyright © 2007 John Wiley & Sons, Ltd. [source] Reduction of GAG storage in MPS II mouse model following implantation of encapsulated recombinant myoblastsTHE JOURNAL OF GENE MEDICINE, Issue 11 2005Adelaide Friso Abstract Background Hunter syndrome, mucopolysaccharidosis type II (MPS II), is a X-linked inherited disorder caused by the deficiency of the enzyme iduronate-2-sulfatase (IDS), involved in the lysosomal catabolism of the glycosaminoglycans (GAG) dermatan and heparan sulfate. Such a deficiency leads to the intracellular accumulation of undegraded GAG and eventually to a progressive severe clinical pattern. Many attempts have been made in the last two to three decades to identify possible therapeutic strategies for the disorder, including gene therapy and somatic cell therapy. Methods In this study we evaluated the intraperitoneal implantation of allogeneic myoblasts over-expressing IDS, enclosed in alginate microcapsules, in the MPS II mouse model. Animals were monitored for 8 weeks post-implantation, during which plasma and tissue IDS levels, as well as tissue and urinary GAG contents, were measured. Results and conclusions Induced enzyme activity occurred both in the plasma and in the different tissues analyzed. A significant decrease in urinary undegraded GAG between the fourth and the sixth week of treatment was observed. Moreover, a biochemical reduction of GAG deposits was measured 8 weeks after treatment in the liver and kidney, on average 30 and 38%, respectively, while in the spleen GAG levels were almost normalized. Finally, the therapeutic effect was confirmed by histolochemical examination of the same tissues. Such effects were obtained following implantation of about 1.5 × 106 recombinant cells/animal. Taken together, these results represent a clear evidence of the therapeutic efficacy of this strategy in the MPS II mouse model, and encourage further evaluation of this approach for potential treatment of human beings. Copyright © 2005 John Wiley & Sons, Ltd. [source] Transcriptional profiling and biochemical analysis of mechanically induced cartilaginous tissues in a rat modelARTHRITIS & RHEUMATISM, Issue 4 2010Kristy T. Salisbury Palomares Objective To characterize patterns of molecular expression that lead to cartilage formation in vivo in a postnatal setting, by profiling messenger RNA expression across the time course of mechanically induced chondrogenesis. Methods Retired breeder Sprague-Dawley rats underwent a noncritical-sized transverse femoral osteotomy. Experimental animals (n = 45) were subjected to bending stimulation (60° cyclic motion in the sagittal plane for 15 minutes/day) of the osteotomy gap beginning on day 10 after the operation. Control animals (n = 32) experienced continuous rigid fixation. Messenger RNA isolated on days 10, 17, 24, and 38 after surgery was analyzed using a microarray containing 608 genes involved in skeletal development, tissue differentiation, fracture healing, and mechanotransduction. The glycosaminoglycan (GAG) content in the stimulated tissues was compared with that in native articular cartilage as a means of assessing the progression of chondrogenic development of the tissues. Results The majority of the 100 genes that were differentially expressed were up-regulated in response to mechanical stimulation. Many of these genes are associated with articular cartilage development and maintenance, diarthrodial joint development, cell adhesion, extracellular matrix synthesis, signal transduction, and skeletal development. Quantitative real-time polymerase chain reaction results were consistent with the microarray findings. The GAG content of the stimulated tissues increased over time and was no different from that of articular cartilage on day 38 after surgery. Conclusion Our findings indicate that mechanical stimulation causes up-regulation of genes that are principally involved in joint cavity morphogenesis and critical to articular cartilage function. Further study of this type of stimulation may identify key signaling events required for postnatal hyaline cartilage formation. [source] Mechanical injury potentiates proteoglycan catabolism induced by interleukin-6 with soluble interleukin-6 receptor and tumor necrosis factor , in immature bovine and adult human articular cartilageARTHRITIS & RHEUMATISM, Issue 10 2009Yihong Sui Objective Traumatic joint injury can damage cartilage and release inflammatory cytokines from adjacent joint tissue. The present study was undertaken to study the combined effects of compression injury, tumor necrosis factor , (TNF,), and interleukin-6 (IL-6) and its soluble receptor (sIL-6R) on immature bovine and adult human knee and ankle cartilage, using an in vitro model, and to test the hypothesis that endogenous IL-6 plays a role in proteoglycan loss caused by a combination of injury and TNF,. Methods Injured or uninjured cartilage disks were incubated with or without TNF, and/or IL-6/sIL-6R. Additional samples were preincubated with an IL-6,blocking antibody Fab fragment and subjected to injury and TNF, treatment. Treatment effects were assessed by histologic analysis, measurement of glycosaminoglycan (GAG) loss, Western blot to determine proteoglycan degradation, zymography, radiolabeling to determine chondrocyte biosynthesis, and Western blot and enzyme-linked immunosorbent assay to determine chondrocyte production of IL-6. Results In bovine cartilage samples, injury combined with TNF, and IL-6/sIL-6R exposure caused the most severe GAG loss. Findings in human knee and ankle cartilage were strikingly similar to those in bovine samples, although in human ankle tissue, the GAG loss was less severe than that observed in human knee tissue. Without exogenous IL-6/sIL-6R, injury plus TNF, exposure up-regulated chondrocyte production of IL-6, but incubation with the IL-6,blocking Fab significantly reduced proteoglycan degradation. Conclusion Our findings indicate that mechanical injury potentiates the catabolic effects of TNF, and IL-6/sIL-6R in causing proteoglycan degradation in human and bovine cartilage. The temporal and spatial evolution of degradation suggests the importance of transport of biomolecules, which may be altered by overload injury. The catabolic effects of injury plus TNF, appeared partly due to endogenous IL-6, since GAG loss was partially abrogated by an IL-6,blocking Fab. [source] Parathyroid hormone 1,34 inhibits terminal differentiation of human articular chondrocytes and osteoarthritis progression in ratsARTHRITIS & RHEUMATISM, Issue 10 2009Je-Ken Chang Objective Parathyroid hormone 1,34 (PTH[1,34]), a parathyroid hormone analog, shares the same receptor, PTH receptor 1, with parathyroid hormone,related peptide (PTHrP). This study was undertaken to address the hypothesis that PTH(1,34) inhibits terminal differentiation of articular chondrocytes and in turn suppresses the progression of osteoarthritis (OA). Methods We studied the effect of PTH(1,34) on human articular chondrocytes with azacytidine (azaC),induced terminal differentiation in vitro and on papain-induced OA in the knee joints of rats. In the in vitro study, we measured the levels of messenger RNA for SOX9, aggrecan, type II collagen, type X collagen, alkaline phosphatase (AP), Indian hedgehog (IHH), Bcl-2, and Bax by real-time polymerase chain reaction, levels of glycosaminoglycan (GAG) by dimethylmethylene blue assay, and rate of apoptosis by TUNEL staining. In the in vivo study, we evaluated the histologic changes in GAG, type II collagen, type X collagen, and chondrocyte apoptosis in the articular cartilage of rat knees. Results AzaC induced terminal differentiation of human chondrocytes, including down-regulation of aggrecan, type II collagen, and GAG and up-regulation of type X collagen, alkaline phosphatase, and IHH. Apoptosis was reversed by 3,10 days of treatment with 10 nM PTH(1,34). SOX9 expression was not changed by either azaC or PTH(1,34) treatment. Bcl-2 and Bax were up-regulated on day 10 and day 14, respectively, after azaC induction of terminal differentiation, but PTH(1,34) treatment did not reverse this effect. Furthermore, PTH(1,34) treatment reversed papain-induced OA changes (decreasing GAG and type II collagen, and increasing type X collagen and chondrocyte apoptosis) in the knee joints of rats. Conclusion Our findings indicate that PTH(1,34) inhibits the terminal differentiation of human articular chondrocytes in vitro and inhibits progression of OA in rats in vivo, and may be used to treat OA. [source] Alteration of articular cartilage frictional properties by transforming growth factor ,, interleukin-1,, and oncostatin MARTHRITIS & RHEUMATISM, Issue 2 2009Jason P. Gleghorn Objective To evaluate the functional effects of transforming growth factor ,1 (TGF,1), interleukin-1, (IL-1,), and oncostatin M (OSM) on the frictional properties of articular cartilage and to determine the role of cytokine-mediated changes in cartilage frictional properties by extracting and redepositing lubricin on the surface of cartilage explants. Methods Neonatal bovine cartilage explants were cultured in the presence or absence of 10 ng/ml of TGF,1, IL-1,, or OSM over 48 hours. Boundary lubrication tests were conducted to determine the effects of endogenously produced surface localized lubricin and of exogenous lubricin at the tissue surface and in the lubricant solution. The initial friction coefficient (,0), equilibrium friction coefficient (,eq), and Young's modulus (EY) were determined from the temporal load data. Results IL-1, and OSM decreased tissue glycosaminoglycan (GAG) content by ,20% over 48 hours and decreased EY to a similar extent (11,17%), but TGF, did not alter GAG content or EY. Alterations in proteoglycan content corresponded to changes in ,0, but endogenous lubricin decreased boundary mode ,eq. The addition of exogenous lubricin, either localized at the tissue surface or in the lubricating solution, did not modulate ,0, but it did lower ,eq in cytokine-treated cartilage. Conclusion This study provides new insight into the functional consequences of cytokine-mediated changes in friction coefficient. In combination with established pathways of cytokine-mediated lubricin metabolism, these data provide evidence of distinct biochemical origins of boundary and biphasic pressure-mediated lubrication mechanisms in cartilage, with boundary lubrication regulated by surface accumulation of lubricants and biphasic lubrication controlled by factors such as GAG content that affect water movement through the tissue. [source] | |||||||||||||||||||||||||||||||