Examination of corneal proteoglycans and glycosaminoglycans by rotary shadowing and electron microscopy

Proteoglycans (PGs) from cornea and their relevant glycosaminoglycan (GAG) chains, dermatan sulphate (DS) and keratin sulphate (KS), were examined by electron microscopy following rotary shadowing, and compared with hyaluronan (HA), chondroitin sulphate (CS), alginate, heparin, heparan sulphate (HS) and methyl cellulose. Corneal DS PG had the tadpole shape previously seen in scleral DS FG, and the images from corneal KS PG could be interpreted similarly, although the GAG (KS) chains were very much fainter than those of DS PG GAG. Isolated GAG (KS, DS, CS, HA, etc.) examined in the same way showed images that decreased very significantly in clarity and contrast, in the sequence HA greater than DS greater than CS greater than KS. The presence of secondary and tertiary structures in the GAGs may be at least partly responsible for these variations. HA appeared to be double stranded, and DS frequently self-aggregated, KS and HS showed tendencies to coil into globular shapes. It is concluded that it is unsafe to assume the absence of GAGs, based on these techniques, and quantitative measurements of length may be subject to error. The results on corneal DS PG confirm and extend the hypothesis that PGs specifically associated with collagen fibrils are tadpole shaped.     

Identification, quantification and fine structural characterization of glycosaminoglycans from uterine leiomyoma and normal myometrium

The type, amount and fine chemical composition of glycosaminoglycans (GAGs) present both in human normal myometrium and uterine leiomyoma have been studied. GAGs were fractionated by ion-exchange chromatography on DEAE-Sephacel, isolated by gel-permeation chromatography on Sepharose CL-6B and characterized using electrophoresis in cellulose acetate membranes, specific enzymic treatments and analysis by high-performance capillary electrophoresis (HPCE). No statistical intrabatch differences in total GAG content in both tissues were identified, whereas significant interbatch differences between normal myometrium and uterine leiomyoma were recorded. Hyaluronan (HA), chondroitin sulphate (CS), dermatan sulphate (DS), heparan sulphate (HS) and keratan sulphate (KS) were identified in both tissues. Statistically significant (P 相似文献   

Distinctive fibroblastic subpopulations in skin and oral mucosa demonstrated by differences in glycosaminoglycan content

Summary The glycosaminoglycan (GAG) content of rabbit skin, oral mucosa, and cultured [³H]-glucosamine-labeled dermal and submucosal fibroblasts was compared. Skin contained predominantly dermatan sulfate (DS) and a small amount of hyaluronic acid (HA), whereas mucosa contained primarily keratan sulfate (KS) and smaller quantities of HA and DS. Culture medium from dermal and submucosal fibroblasts contained GAGs co-electrophoresing with DS, HA, and chondroitin sulfate (CS), although the relative proportions of these GAG differed. CS isolated from dermal and mucosal fibroblast culture medium co-electrophoresed with chondroitin 4-sulfate (C4-S) on cellulose acetate, whereas dermal medium CS was resistant to digestion by chondroitinase ABC, and mucosal medium CS was chondroitinase ABC-susceptible. The pericellular matrix of dermal fibroblasts contained primarily DS and C4-S/C6-S, as confirmed by chondroitinase ABC digestion; the corresponding fraction of mucosal fibroblasts contained HS and a GAG co-electrophoresing with a C6-S standard, yet resistant to digestion by chondroitinase ABC. Thus the GAG content of dermal and mucosal fibroblasts differed both qualitatively in terms of the type of GAG secreted into the culture medium and pericellular matrix, and quantitatively, in terms of the relative proportions of these GAGs in both fractions. These differences support the concept of distinctive fibroblastic subpopulations in skin and mucosal tissue, inasmuch as the cells were subjected to identical culturing conditions. This work was supported by research grant 15878 (C.N.B.) from the Shriners Hospitals for Crippled Children and DE 07803 (C.N.B.) from the National Institute of Dental Research, National Institutes of Health, Bethesda, MD.     

Carriers for enzymatic attachment of glycosaminoglycan chains to peptide

In the previous study, we have found that the endo-beta-xylosidase from Patinopecten had the attachment activities of glycosaminoglycan (GAG) chains to peptide. As artificial carrier substrates for this reaction, synthesis of various GAG chains having the linkage region tetrasaccharide, GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl, between GAG chain and core protein of proteoglycan was investigated. Hyaluronic acid (HA), chondroitin (Ch), chondroitin 4-sulfate (Ch4S), chondroitin 6-sulfate (Ch6S), and desulfated dermatan sulfate (desulfated DS) as donors and the 4-metylumbelliferone (MU)-labeled hexasaccharide having the linkage region tetrasaccharide at its reducing terminals (MU-hexasaccharide) as an acceptor were subjected to a transglycosylation reaction of testicular hyaluronidase. The products were analyzed by high-performance liquid chromatography and enzyme digestion, and the results indicated that HA, Ch, Ch4S, Ch6S, and desulfated DS chains elongated by the addition of disaccharide units to the nonreducing terminal of MU-hexasaccharide. It was possible to custom-synthesize various GAG chains having the linkage region tetrasaccharide as carrier substrates for enzymatic attachment of GAG chains to peptide.     

Changes in CD of hyaluronates and chondroitins upon periodate oxidation

Changes in conformation of hyaluronate and chondroitin sulfates following periodate oxidation were studied by CD. We monitored the progressive oxidation of these polymers by periodic acid at 4°C in pH 5.6 buffer. The negative CD band of hyaluronate at 208 nm decreased in intensity upon oxidation and changed its sign after 16 h of oxidation. In contrast, the 208-nm CD band of chondroitin sulfates decreased, but showed no change in sign even after 48 h of oxidation. A specific difference in solution conformation between hyaluronate and chondroitins may be responsible for the difference in oxidation-induced dichroic behavior. The results are discussed in terms of available x-ray diffraction analyses of these polymers.     

Effect of sulfated GAGs on the expression and activation of MMP-2 and MMP-9 in corneal and dermal explant cultures

It has been shown previously that hyaluronan (HA) added to fibroblast and keratocyte cell cultures or corneal explant cultures produces an up-regulation of MMP-2 and MMP-9 expression and activation. Here, we examine the effect of sulfated GAG-s, chondroitin 4 and 6 sulfate (CS4, CS6), dermatan sulfate (DS), keratan sulfate (KS) and heparan sulfate (HS) on MMP-2 and 9 expression and activation under the same culture conditions. It appears that CS4 has only minor effects, KS inhibits MMP-2 activation and CS6, DS and HS increase MMP-2 activation in corneal explant cultures. For skin explant cultures, DS, KS and HS strongly increase MMP-9 activation, whereas KS inhibits and DS increases MMP-2 activation. All these effects can be strongly inhibited by the addition of an antibody to CD44, except CS6 and DS. Activation by these two GAGs was only slightly affected, supporting the contention that the effects of HA, CS4, KS and HS are mediated by one of the isoforms of this CD44 receptor. The physio-pathological significance of these results is discussed for cornea and skin ageing, because of the divergent evolution with in vitro ageing of the relative proportions of GAGs synthesised by these two cell types.     

Alterations in human gingival glycosaminoglycan pattern in inflammation and in phenytoin induced overgrowth

Glycosaminoglycans were extracted from normal, inflamed and phenytoin induced overgrowth of human gingival tissue by proteolysis and alcohol precipitation. Extracts were run in a Dowex-1 column and the fractions were treated with mucopolysaccharidases. Cellulose acetate electrophoresis was carried out with or without enzyme digestion for identification of individual glycosaminoglycans. Glycosaminoglycans were found to be decreased in inflammation but were observed to increase in the overgrowth. Hyaluronic acid was found to be increased in both the pathological conditions. Dermatan sulphate, chondroitin sulphate and heparan sulphate were observed to be decreased in inflammation. In overgrowth, dermatan sulphate and chondroitin sulphate were found to increase while the presence of heparan sulphate was not significant. The changes in the pattern of individual glycosaminoglycan in the two varied conditions are discussed.Abbreviations GAG glycosaminoglycan - MPS mucopolysaccharide - DS dermatan sulphate - HS heparan sulphate - CS chondroitin sulphate - HA hyaluronic acid - KS keratan sulphate     

Easy HPLC-based separation and quantitation of chondroitin sulphate and hyaluronan disaccharides after chondroitinase ABC treatment

The sulphation patterns of glycosaminoglycan (GAG) chains are decisive for the biological activity of their proteoglycan (PG) templates for sugar chain polymerization and sulphation. The amounts and positions of sulphate groups are often determined by HPLC analysis of disaccharides resulting from enzymatic degradation of the GAG chains. While heparan sulphate (HS) and heparin are specifically degraded by heparitinases, chondroitinases not only degrade chondroitin sulphate (CS) and dermatan sulphate (DS), but also the protein-free and unsulphated GAG hyaluronan (HA). Thus, disaccharide preparations derived by chondroitinase degradation may be contaminated by HA disaccharides. The latter will often comigrate in HPLC chromatograms with unsulphated disaccharides derived from CS. We have investigated how variation of pH, amount of enzyme, and incubation time affects disaccharide formation from CS and HA GAG chains. This allowed us to establish conditions where chondroitinase degrades CS completely for quantification of all the resulting disaccharides, with negligible degradation of HA, allowing subsequent HA analysis. In addition, we present simple methodology for disaccharide analysis of small amounts of CS attached to a hybrid PG carrying mostly HS after immune isolation. Both methods are applicable to small amounts of GAGs synthesized by polarized epithelial cells cultured on permeable supports.     

Glycosaminoglycans of Rat Cerebellum: II. A Developmental Study

Total and individual glycosaminoglycans (GAGs) were determined in rat cerebellum in tissue explants at various postnatal ages. The major constituents of GAGs were chondroitin sulfate (CS), hyaluronic acid (HA), and heparan sulfate (HS). Dermatan sulfate (DS) and keratan sulfate (KS) could not be detected and therefore each amounts to less than 5% of all GAGs at all ages studied. HA was the prominent GAG during postnatal development and only a minor constituent at adult ages, whereas CS was the predominant GAG in adulthood. HS remained relatively constant throughout development. The incorporation of [3H]glucosamine into individual GAGs was highest for HS at postnatal day 6, whereas HA showed intermediate and CS the lowest levels of incorporation during the first postnatal week. All major GAGs showed the lowest incorporation values at adult ages.     

Characterization of glycosaminoglycans from human normal and scoliotic nasal cartilage with particular reference to dermatan sulfate.

The composition and the distribution of glycosaminoglycans (GAGs) present in normal human nasal cartilage (HNNC), were examined and compared with those in human scoliotic nasal cartilage (HSNC). In both tissues, hyaluronan (HA), keratan sulfate (KS) and the galactosaminoglycans (GalAGs)--chondroitin sulfate (CS) and dermatan sulfate (DS)--were identified. The overall GAG content in HSNC was approx. 30% higher than the HNNC. Particularly, a 114% increase in HA, and 46% and 86% in KS and DS, respectively, was recorded. CS was the main type of GAG in both tissues with no significant compositional difference. GalAG chains in HSNC exhibited an altered disaccharide composition which was associated with significant increases of non-sulfated and 6-sulfated disaccharides. DS, which was identified and quantitated for the first time in HNNC and HSNC, contained low amounts of iduronic acid (IdoA), 18% and 28% respectively. In contrast to other tissues, where IdoA residues are organized in long IdoA rich repeats, the IdoA residues of DS in human nasal cartilage seemed to be randomly distributed along the chain. DS chains in HSNC were of larger average molecular size than those from HNNC. These results clearly indicate the GAG content and pattern in both HNNC and HSNC and demonstrate that scoliosis of nasal septum cartilage is related to quantitative and structural modifications at the GAG level.     

CD44 in inflammation and metastasis

CD44 is a major cell surface receptor for the glycosaminoglycan, hyaluronan (HA). CD44 binds HA specifically, although certain chondroitin-sulfate containing proteoglycans may also be recognized. CD44 binding of HA is regulated by the cells in which it is expressed. Thus, CD44 expression alone does not correlate with HA binding activity. CD44 is subject to a wide array of post-translational carbohydrate modifications, including N-linked, O-linked and glycosaminoglycan side chain additions. These modifications, which differ in different cell types and cell activation states, can have profound effects on HA binding function and are the main mechanism of regulating CD44 function that has been described to date. Some glycosaminoglycan modifications also affect ligand binding specificity, allowing CD44 to interact with proteins of the extracellular matrix, such as fibronectin and collagen, and to sequester heparin binding growth factors. It is not yet established whether the HA binding function of CD44 is responsible for its proposed involvement in inflammation. It has been shown, however, that CD44/HA interactions can mediate leukocyte rolling on endothelial and tissue substrates and that CD44-mediated recognition of HA can contribute to leukocyte activation. Changes in CD44 expression (mainly up-regulation, occasionally down-regulation, and frequently alteration in the pattern of isoforms expressed) are associated with a wide variety of cancers and the degree to which they spread; however, in other cancers, the CD44 pattern remains unchanged. Increased expression of CD44 is associated with increased binding to HA and increased metastatic potential in some experimental tumor systems; however, in other systems increased HA binding and metastatic potential are not correlated. CD44 may contribute to malignancy through changes in the regulation of HA recognition, the recognition of new ligands and/or other new biological functions of CD44 that remain to be discovered. Abbreviations: aa, amino acid(s); CS, chondroitin sulfate; CSPG, chondroitin sulfate containing proteoglycan; CD44H, ‘hematopoietic’, also called ‘standard’, isoform of CD44 which contains none of the alternatively spliced variant exons; CD44-Rg, CD44 receptor globulin, a secreted chimaeric protein composed of the external domain of the adhesion receptor CD44 and the hinge, CH2 and CH3 regions of human immunoglobulin-G heavy chain; ECM, extracellular matrix; GAG, glycosaminoglycan; HA, hyaluronan; HS, heparan sulfate; KS, keratan sulfate; PB, peripheral blood; PBL, peripheral blood lymphocytes This revised version was published online in November 2006 with corrections to the Cover Date.     

Cell surface glycosaminoglycans (GAGs) of cultured chick fibroblasts : Modifications in relation to the stage of embryo development

We have investigated the changes in glycosaminoglycan (GAG) composition between cultured fibroblasts derived from 8- and 16-day chick embryos. GAG composition has been studied after [3H]glucosamine and [35S]sulfate labeling. Both the 8- and 16-day embryo fibroblasts were found to contain hyaluronic acid (HA), dermatan sulfate (DS), heparan sulfate (HS) and chondroitin sulfates (CS), the latter being the major component in 8- and 16-day cells. These four GAGs were quantified after their separation using cellulose acetate electrophoresis. The amounts of HA and CS were respectively shown to increase 2-fold and 4-fold between the 8th and 16th day of development, whereas the amounts of HS and DS resp. diminished 2.5-fold and 1.2-fold. These results show that the relative proportions of the different GAGs alter during embryo development. The fibroblasts from 8-day-old embryos detached more rapidly from the culture dishes than the cells from 16-day-old embryos when treated with trypsin. However, this difference was not directly related to the different GAG content.     

Studies on cartilage formation. XVIII. Changes of the composition of glycosaminoglycans in the regenerating articular surface.

The cartilaginous articular surface of the distal part of the femur of adult dogs was removed and the composition of GAGs was determined in the granulation tissue adhering to the bone wound and in that adhering to the articular capsule 7, 33, and 70 days after operation. The articular cartilage and the synovial layer of the articular capsule of intact adult dogs were also studies. The materials were digested with papain and the released GAGs were fractionated according to Svejcar and Robertson's method. The articular cartilage of non-operated dogs contained, on the average, 65.3% ChS, 13% KS, 5.8% HA and 15.8% GAG of lower molecular weight. The synovial layer of the capsule contained 41.1% HA, 15.3% Ch4-S and Ch6-S, 13.7% DS, 21.7% KS, 2% H and 6% GAG of lower molecular weight. The granulation tissue of the articular surface and that adhering to the capsule show a different developmental course. The former differentiates into cartilage, whereas the latter is simply added to the tissue of the capsule. The two tissues are different in GAG composition as early as on the 7th postoperative day. With time an increase of Ch4-S, Ch6-S and KS can be observed in the tissue of the articular surface. The tissue adhering to the capsule is characterized by a high HA and an increasing DS content. From the study of the composition of GAG's (proportion of GAG building stones) a deeper insight can be obtained into the details of GAG biosynthesis characteristic of cartilage than from the analysis of quantitative data of ChS. In the development of GAG composition characteristic of the tissue, the epimerase reactions participating in GAG biosynthesis, and the mechanisms regulating their activities seem to play a primary role.     

Variation of glycosaminoglycan in the growth of transplantable tumors derived from a spontaneous ddY mouse mammary tumor

The components and variations of glycosaminoglycan (GAG) in the growth of transplantable tumors derived from a spontaneous mouse mammary tumor were investigated. A 45-week-old ddY female mouse, obtained from Shizuoka Laboratory Animal Center, was found to have a bean-sized mass at the third mammary gland of the left side. The tumor mass was surgically excised and used for transplantation in the present study. This mammary tumor was histologically found to be Type B-adenocarcinoma. Transplantable mammary tumors consisted of the fibrous or edematous interstitium contained a large amount of GAG components, which was mainly hyaluronic acid (HA), dermatan sulfate (DS) and chondroitin sulfate A/C (ChS). In the analysis of GAG components, HA content was present in a large amount in logarithmic growth phase of transplanted mammary tumors, but it was markedly decreased in stationary phase. On the other hand, the contents of DS and ChS increased in stationary phase of the tumor growth, and these increases corresponded, histologically, with the propagation of the fibrous interstitial tissues.     

Binding of calcium to glycosaminoglycans: an equilibrium dialysis study

Binding of calcium to the glycosaminoglycans (GAGs) heparin, chondroitin sulfate (CS), keratan sulfate (KS), and hyaluronic acid (HA) has been studied by equilibrium dialysis using exclusion of sulfate to correct for Gibbs-Donnan effects. Calcium binding occurs to all of these GAG species, suggesting that both sulfate and carboxylate groups are involved in cation binding. For all GAGs, the binding stoichiometry is consistent with a calcium-binding "site" consisting of two anionic groups. The order of calcium binding affinities is heparin greater than CS greater than KS greater than HA, and is critically dependent upon charge density; heparin binds calcium with 10-fold higher affinity than CS. The mode of calcium binding to GAGs is consistent with a recently proposed mechanism of growth plate calcification which states that cartilage proteoglycan functions as a reservoir of calcium for calcification of epiphyseal cartilage.     

Identification,structural analysis and function of hyaluronan in developing fish larvae (leptocephali)

Hyaluronan (HA) has been identified as the principal glycosaminoglycan (CAG) in the highly hydrated, extracellular body matrix of the larval stage (leptocephalus) of seven species of true eels (Teleostei: Elopomorpha: Anguilliformes) and the ladyfish Elops saurus (Elopiformes), and was found as a minor GAG component in the bonefish Albula sp. (Albuliformes). Identification was based on: (1) HPLC separation of unsaturated disaccharides derived from chondroitinase ABC digests of whole-body GAG extracts; (2) 1H NMR analyses of native GAG polymers; and (3) degradation of GAG extracts by Streptomyces hyaluronan lyase. The unsaturated disaccharide 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-D-glucose (DeltaDi-HA) accounted for 92.4-99.8% of the total disaccharides in chondroitinase digests. Trace amounts of unsaturated disaccharides of chondroitin sulfate were also present. Two-dimensional gCOSY spectra of the native HA polymer were similar for all species. Proton assignments for the HA disaccharide repeat (GlcAbeta1-3GlcNAcbeta1-4) in D(2)O, based on gCOSY, DQF-COSY and TOCSY analyses for the eel Ahlia egmontis, were concordant with published chemical shifts for HA oligosaccharides. In addition to its presumed role in maintaining the structural integrity and hydration of the gelatinous body of the leptocephalus, HA is postulated to function as a storage polysaccharide in those species in which it is the predominant GAG.     

Different contents of glycosaminoglycans in a human neoplastic salivary duct cell line and its subclone with a myoepithelial phenotype

The glycosaminoglycans (GAG) biosynthesized by a neoplastic human salivary duct cell line, HSGc, and by its nontumorigenic subclone, HSGc-E1, having a myoepithelial-like phenotype, were examined by incorporation of [3H]-acetate into GAG. The rate of GAG radiolabeling in HSGc-E1 was significantly greater than that in HSGc. The radiolabeled GAG recovered from HSGc-E1 showed a distribution of 22-32% in the cells and 68-78% secreted into the medium, while the amounts of GAG in the cells and medium of HSGc were equal. Two-dimensional electrophoresis of GAG extracted from the cells demonstrated that HSGc-E1 contained a much greater amount of heparan sulfate (HS, 53.5% of total), while HSGc synthesized hyaluronic acid (HA, 17.5%), HS 38.8%, chondroitin sulfate (Ch-S, 27.6%) and dermatan sulfate (DS, 16.1%). Moreover, treatment of HSGc with sodium butyrate or dibutyryl cyclic AMP (each is a potent inducer of differentiation to myoepithelial-like cells) strongly enhanced GAG synthesis, while dexamethasone (an inducer of differentiation to a more functional duct epithelium) did not stimulate GAG synthesis. These findings suggest that biosynthetic changes in the GAG content of neoplastic salivary cells are associated with their myoepithelial differentiation.     

Effect of the gonadal status and the gender on glycosaminoglycans profile in the lower urinary tract of dogs

Glycosaminoglycans (GAGs) form a functional component of connective tissues that affect the structural and functional integrity of the lower urinary tract (LUT). The specific GAGs of physiological relevance are both nonsulfated (hyaluronan) and sulfated GAGs (chondroitin sulphate [CS], dermatan sulphate [DS], keratan sulphate [KS], and heparan sulphate [HS]). As GAG composition in the LUT is hormonally regulated, we postulated that gonadectomy-induced endocrine imbalance alters the profile of GAGs in the canine LUT. Four regions of the LUT (body and neck of the bladder as well as the proximal and distal urethra) from 20 clinically healthy dogs (5 intact males, 5 intact anoestrus females, 4 castrated males, and 6 spayed females) were collected, wax-embedded and sectioned. Alcian blue staining at critical electrolyte concentrations was performed on the sections to determine total GAGs, hyaluronan, total sulfated GAGs, combined components of CS and DS, as well as KS and HS. The amount of staining was evaluated in 3 tissue layers, i.e., epithelium, subepithelial stroma and muscle within a region. Overall, hyaluronan (67.1%) was the predominant GAG in the LUT. Among sulfated GAGs, a combined component of KS and HS was found to be 61.8% and 38.2% for CS and DS. Gonadal status significantly affected GAG profiles in the LUT (P < 0.01). All GAG components were lower (P < 0.05) in body of the bladder of gonadectomized dogs. Total sulfated GAGs and a combined component of KS and HS were lower (P < 0.05) in all 4 regions of gonadectomized dogs. Except for a combined component of CS and DS, decreases in all GAGs were found more consistently in the muscle compared to other tissue layers. Differences between genders became obvious only when considered along with the effect of gonadal status. In gonadectomized dogs, changes in GAG components in the LUT were more consistent in females compared to males; this may partly explain different levels of risk in the development of urinary incontinence between genders. Quantitative differences in GAG profiles found between intact and gonadectomized dogs indicate a potential role of gonadectomy-induced endocrine imbalance in modifying GAG composition in the canine LUT. Profound alteration in the pattern of GAGs in gonadectomized dogs may compromise structural and functional integrity of the LUT and is possibly involved in the underlying mechanism of urinary incontinence post neutering.     

Pancreatic carcinoma is characterized by elevated content of hyaluronan and chondroitin sulfate with altered disaccharide composition

The amount and the types of glycosaminoglycans (GAGs) present in human pancreatic carcinoma were examined and compared with those in normal pancreas. Human pancreatic carcinoma contained increased levels (4-fold) of total GAGs. Particularly, this carcinoma is characterized by a 12-fold increase of hyaluronan (HA) and a 22-fold increase in chondroitin sulfate (CS) content. CS in pancreatic carcinoma exhibited an altered disaccharide composition which is associated with marked increase of non-sulfated and 6-sulfated disaccharides. Dermatan sulfate (DS) was also increased (1.5-fold) in carcinoma, whereas heparan sulfate (HS), the major GAG of normal pancreas, becomes the minor GAG in pancreatic carcinoma without significant changes in the content and in molecular size. In all cases, the galactosaminoglycans (GalGAGs, i.e. CS and DS) derived from pancreatic carcinomas were of lower molecular size compared to those from normal pancreas. The results in this study indicate, for the first time, that human pancreatic carcinoma is characterized by highly increased amounts of HA and of a structurally altered CS.     

Glycosaminoglycans modulate inflammation and apoptosis in LPS‐treated chondrocytes

Previous studies reported that hyaluronic acid (HA), chondroitin sulphate (CS) and heparan sulphate (HS) were able to reduce the inflammatory process in a variety of cell types after lypopolysaccharide (LPS) stimulation. The aim of this study was to investigate the anti‐inflammatory effect of glycosaminoglycans (GAGs) in mouse articular chondrocytes stimulated with LPS. Chondrocyte treatment with LPS (50 µg/ml) generated high levels of TNF‐α, IL‐1β, IL‐6, IFN‐γ, MMP‐1, MMP‐13, iNOS gene expression and their related proteins, increased NO concentrations (evaluated in terms of nitrites formation), NF‐κB activation and IkBα degradation as well as apoptosis evaluated by the increase in caspase‐3 expression and the amount of its related protein. The treatment of chondrocytes using two different doses (0.5 and 1.0 mg/ml) of HA, chondroitin‐4‐sulphate (C4S), chondroitin‐6‐sulphate (C6S), HS, keratan sulphate (KS) and dermatan sulphate (DS) produced a number of effects. HA exerted a very small anti‐inflammatory and anti‐apoptotic effect while it significantly reduced NO levels, although the effect on iNOS expression and activity was extremely slight. C4S and C6S reduced inflammation mediators and the apoptotic process. C6S failed to decrease NO production, although iNOS expression and activity were significantly reduced. HS, like C4S, was able to reduce all the effects stimulated by LPS treatment. KS and DS produced no reduction in any of the parameters considered. These results give further support to the hypothesis that GAGs actively participate in the regulation of inflammatory and apoptotic processes. J. Cell. Biochem. 106: 83–92, 2009. © 2008 Wiley‐Liss, Inc.