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.     

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.     

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.     

Glycosaminoglycans of Rat Cerebellum: I. Quantitative Analysis of the Main Constituents at Postnatal Day 6

Abstract: Isolated glycosaminoglycans (GAGs) were quantified biochemically in the cerebella of 6-day-old rats. ¹⁴C-Labeled hyaluronic acid (HA) and chondroitin-4-sulfate (C-4-S), added prior to isolation of GAGs from tissue, served as internal standards to allow correction for unknown losses during the purification procedure and exact quantification of GAGs in the intact tissue. Three main constituents—HA, chondroitin sulfate (CS), and heparan sulfate (HS)—were found at concentrations of 1.82, 1.52, and 0.76 μg/mg protein amounting to 44%, 37%, and 19% of the total GAG fraction, respectively. Incorporation of [³H]glucosamine precursor into GAGs was higher for HS (56% of incorporated precursor) and lower for HA (29%) and CS (15%). The specific activities of individual GAGs were 64.7 nCi/μg for HS, 14.2 for HA, and 8.3 for CS.     

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.     

Relation between hyaluronan and sulphated glycosaminoglycan synthesis and degradation in cultured embryonic fibroblasts. Effect of concanavalin A and ammonium chloride administration.

In order to evaluate the relationship between glycosaminoglycan (GAG) synthesis and degradation, the effect of NH4Cl, which inhibits lysosomal degradation, on GAG production was analysed in vitro in concanavalin A (Con A) stimulated fibroblasts from 7 and 14-day-old chick embryos. 35SO4 incorporation into total proteoglycan (PG), 3H incorporation into individual GAG classes, beta-N-acetyl-D-glucosaminidase and beta-D-glucuronidase activity were determined. The results indicate a correlation between Con A and NH4Cl effects: NH4Cl induced a reduction principally in the GAG classes most stimulated by Con A. Thus HA and DS are much more stimulated by Con A and inhibited by NH4Cl than are CS and HS.     

伴刀豆蛋白A及其衍生物对培养软骨细胞蛋白多糖代谢的影响

观察了ＣｏｎＡ对培养软骨细胞ＰＧ合成代谢的影响。证实ＣｏｎＡ能够使培养的软骨细胞高分子硫酸化ＰＧ的合成增加３～４倍,其分子量、硫酸化部位和硫酸化程度与对照组相比无明显差异,是具有正常结构的软骨型ＰＧ。ＣｏｎＡ对低分子型ＰＧ的合成未见明显的影响。ＣｏｎＡ促进ＰＧ合成的作用可由ＭｅＭａｎ完全解除,比具有同样效应的激素、生长因子都强,并有明显的凝集素特异性。推测ＣｏｎＡ的作用可能与软骨细胞膜或细胞内的分化诱导因子的受体或软骨中存在的ＣｏｎＡ软骨细胞分化因子有关。     

Characterization of proteoglycans synthesized by rat thyroid cells in culture and their response to thyroid-stimulating hormone

A Fisher rat thyroid cell line was maintained in culture and the cells were labeled with [3H]glucosamine, [35S]sulfate, and [35S]cysteine to examine the synthesis of proteoglycans. 3H and 35S radioactivity from these precursors were incorporated into both chondroitin sulfate (CS) and heparan sulfate (HS) proteoglycans. CS proteoglycans were almost exclusively secreted into the medium while HS proteoglycans remained mainly associated with the cell layer. Single chain glycosaminoglycans released by papain digestion or alkaline borohydride treatment of either the CS or HS proteoglycans had average molecular weights of approximately 30,000 on Sepharose CL-6B chromatography. Both CS and HS proteoglycans were relatively small and contained only one or two glycosaminoglycans chains. 3H and 35S incorporation into both CS and HS proteoglycans were increased by thyroid-stimulating hormone (TSH) in a dose-dependent manner, which is in part explained by an adenylate cyclase-dependent mechanism as indicated by a similar effect in response to dibutyryl cAMP. TSH enhanced the incorporation of 35S into CS from [35S]cysteine about 1.5-fold and that from [35S]sulfate about 2-fold. This result demonstrated that the increased 35S incorporation from the [35S]sulfate precursor reflects an actual increase in sulfate incorporation and is not simply a result from an apparent increase in specific activity of the phosphoadenosine phosphosulfate donor. Analysis of disaccharides from chondroitinase digests revealed that the proportion of non-sulfated, 4-sulfated, and 6-sulfated disaccharides was not altered appreciably by TSH. These results, together with the disproportionate increase in 3H incorporation into CS from [3H]glucosamine, indicated that TSH increased the specific activity of the 3H label as well. Chase experiments revealed that CS proteoglycans were rapidly (t1/2 = 15 min) secreted into the medium and that the degradation of cell-associated proteoglycans was enhanced by TSH.     

The influence of high ambient glucose level on the production of pericellular glycosaminoglycans by cultured endothelial cells

The 14C-acetate metabolic labeling of glycosaminoglycans (GAGs) was used to investigate the effect of high glucose level on the production of hyaluronic acid (HA), heparan sulphate (HS), chondroitin sulphate (CS) and dermatan sulphate (DS) by human immortalized umbilical vein endothelial cells. It is demonstrated that 30 mM glucose decreased the accumulation of HS and increased the accumulation of CS and DS in the cell layer, pericellular matrix and conditioned medium in 48 h of incubation. The modulation of the overall metabolism of sulphated GAGs by high glucose is in contrast to the observed redistribution of HA from the conditioned medium to the pericellular matrix of endothelial cells. The preincubation at 30 mM glucose increased also the attachment of hyaluronidase-treated endothelial cells to HA-coated surface and had no effect on the cell attachment to poly-D-lysine, indicating the alterations of CD44 binding to immobilized HA. The treatment of endothelial cells with p-nitrophenyl-beta-D-xylopyranoside, which inhibits the coupling of CS to the core protein, attenuated high glucose-induced pericellular HA accumulation and decreased cell attachment to HA-coated surface. It is supposed the implication of CD44-related CS in the accumulation of pericellular HA by endothelial cells exposed to high glucose level.     

Effects of 4-deoxy-L-threo-pentose, a novel carbohydrate, on neural cell proteoglycan synthesis and function.

A novel carbohydrate, 4-deoxy-L-threo-pentose (4-deoxyxylose), was synthesized by way of reductive dechlorination of a chlorodeoxy sugar. This carbohydrate, an analogue of xylose which is required for the initiation of glycosaminoglycan (GAG) synthesis, was used to explore the function of GAG side chains in neurite outgrowth on a laminin substrate. 4-Deoxyxylose inhibited the incorporation of 35SO4 into the GAGs of neuronal and astrocytic proteoglycans, with no effect being seen on the incorporation of [3H]glucosamine into proteoglycan. Direct analysis of the heparan sulphate fraction from such cells using nitrous acid digestion confirmed that the GAGs were undersulphated. No inhibition of either 35SO4 or [3H]glucosamine incorporation was observed in primary mouse hepatocytes exposed to 4-deoxyxylose. 4-Deoxyxylose produced a direct dose-dependent inhibition of neurite outgrowth by sensory neurons, and medium conditioned by neurons or astrocytes in the presence of 4-deoxyxylose displayed less laminin-complexed neurite-promoting activity than medium conditioned in its absence. These data suggest that 4-deoxyxylose inhibits neurite outgrowth by altering the sulphation of the GAGs of heparan sulphate proteoglycans.     

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   

Fibrin D-dimer impairs the accumulation and anticoagulant properties of heparan sulphate and stimulates secretion of plasminogen activator inhibitor-1 by rabbit coronary endothelial cells

Fibrin split product D-dimer (DD) is most probably involved in the development of vascular disorders. At 1.5 microM concentration DD inhibited the incorporation of D-[1-(3)H]glucosamine hydrochloride and [2-(14)C]acetate x Na into pericellular heparan sulphate (HS) of rabbit coronary endothelial cells without affecting other groups of glycosaminoglycans (GAGs). At the same time, DD reduced HS ability to bind antithrombin (AT) and suppressed NO production. The effect of DD on pericellular GAGs was similar to that of N(omega)-methyl-L-arginine, the competitive inhibitor of endothelial NO synthase (eNOS). L-Ascorbic acid, eNOS activator, increased the level of endogenous NO in the DD-treated cells, and restored HS accumulation and antithrombin binding. It is suggested that DD influence on endothelial HS may be mediated by NO production. Another effect of DD, namely, stimulation of plasminogen activator inhibitor-1 (PAI-1) secretion did not depend on the NO level. The decreased HS content, reduced anticoagulant properties of HS, and increased PAI-1 secretion disorganized the endothelial matrix, and promoted fibrin formation and vascular damage. This points to DD as an important factor in the development of vascular disorders.     

Proteoglycan biosynthesis by chick embryo retina glial-like cells

In this report we present biochemical evidence that purified cultures of chick embryo retina glial-like cells actively synthesize heparan sulfate (HS) and chondroitin sulfate/dermatan sulfate (CS/DS) proteoglycans as well as hyaluronic acid. Glial-like cell cultures were metabolically labeled with [3H]glucosamine and 35SO4, and the medium, cell layer, and substratum-bound fractions were analyzed separately. Proteoglycans were characterized according to charge, apparent molecular size, and glycosaminoglycan (GAG) composition and were found to be differentially distributed among the cellular compartments. HS was the predominant GAG overall and was the major species found in the cell layer and substratum-bound fractions. CS/DS was also present in each fraction and comprised the largest proportion of GAGs in the medium. The major GAG-containing material resolved into three different size classes. The first, found in the cell layer and substratum-bound fractions, contained both CS/DS and HS and was of large size. A second, intermediately sized class with a higher CS/DS:HS ratio was found in the medium. The smallest class was found in the cell layer fraction and comprised HS, most likely present as free GAG chains. In addition, each fraction contained hyaluronic acid. Characteristics of these macromolecules differ from those produced by purified cultures of chick embryo retina neurons and photoreceptors in terms of size, compartmental distribution, and presence of hyaluronic acid.     

Hyaluronic acid synthesis is absent in normal human endothelial cells irrespective of hyaluronic acid synthetase inhibitor activity, but is significantly high in transformed cells

The characteristics of glycosaminoglycan (GAG) synthesis in normal and transformed human endothelial cells were analyzed by the incorporation of [3H]glucosamine and by the activities of GAG synthetases. The GAG synthesized by normal endothelial cells consisted of mainly heparan sulfate (HS) and chondroitin sulfate/dermatan sulfate but little hyaluronic acid (HA) (less than 1%). The characteristics of GAG synthesis by normal cells reflected the synthetic enzyme activities for each individual GAG: the activity of HA synthetase was very low. In spite of this, the activity of HA synthetase inhibitor, induced in growth-retarded fibroblasts with low HA synthetase activity (Matuoka et al. (1987 J. Cell Biol., 104, 1105-1115), was very low in endothelial cells. In contrast to normal cells, transformed endothelial (ECV304) cells synthesized mainly HA (62% of total GAGs). These findings suggest that the regulatory system of GAG metabolism is cell type specific, and that transformation is accompanied by high levels of HA synthesis in endothelial cells.     

Enhanced channelling of sulphate through a rapidly exchangeable sulphate pool in response to stimulated glycosaminoglycan synthesis in pancreatic epithelial cells.

The ability of cells to decorate glycosaminoglycans (GAGs) with sulphate in highly specific patterns is important to extracellular matrix biogenesis and placing appropriate glycosulphated ligands on the cell surface. We have examined sulphate metabolism in two pancreatic duct epithelial cell lines - PANC-1 and CFPAC-1 (derived from a cystic fibrosis patient) with a view to understanding how pancreatic cells utilise intracellular sulphate. [35S]Sulphate uptake was rapid and reached near steady state levels within 10 min. However, the intracellular specific activity of [35S]sulphate for PANC-1 and CFPAC-1 reached only 35 and 10%, respectively, of the medium specific activity at 10 min. Therefore, sulphate appears to reside within two compartments; a rapidly exchangeable sulphate pool (RESP) and a slowly exchangeable sulphate pool (SESP). Reducing chloride in the medium, increased the specific activity of [35S]sulphate within cells and increased the size of the inorganic sulphate pool, suggesting that the RESP was enlarged. Sulphate pools were not different in size between the two cell lines in physiological NaCl. Increasing the size of the sulphate pool had no effect on [35S]sulphate:[3H]glucosamine ratios incorporated into glycosaminoglycans (GAGs); however, stimulating the synthesis of GAGs with 4-methylumbelliferyl-beta-d-xyloside, stably elevated [35S]:[3H] ratios. This was due to higher [35S]sulphate incorporation. [35S]Cysteine contributed less than 0.1% of the cells' sulphate requirements. We conclude that in the face of elevated demand for sulphate, pancreatic cells appear to channel a greater proportion through the RESP.     

Studies on growth inhibition by lectins of penicillia and aspergilli

It has previously been shown in our laboratory that wheat germ agglutinin (WGA) binds to Trichoderma viride and inhibits growth of this fungus. Here we report on the effect of WGA, soybean agglutinin (SBA) and peanut agglutinin (PNA) on Penicillia and Aspergilli. Binding of the lectins to the fungi was examined with the aid of their fluorescein isothiocyanate (FITC) conjugated derivatives. FITC-WGA bound to young hyphal walls of all species, in particular to the hyphal tips and septa, in agreement with the chitinous composition of the cell walls of the two genera. Hyphae of all species examined were labelled, though in different patterns, by FITC-SBA and FITC-PNA, suggesting the presence of galactose residues on their surfaces. Young conidiophores, metulae (of the Penicillia), vesicles (of the Aspergilli), sterigmata and young spores, were also labelled. The three lectins inhibited incorporation of [³H]acetate, N-acetyl-D-[³H]glucosamine and D-[¹⁴C]galactose into young hyphae of Aspergillus ochraceus, indicating interference with fungal growth. Inhibition of spore germination by the three lectins was also observed. Preincubation of the lectins with their specific saccharide inhibitors prevented binding and the inhibitory effects. We conclude that lectins are useful tools for the study of fungal cell surfaces, and may also serve as an important aid in fungal classification. The present findings also support the suggestion that one role of lectins in plants is protection against fungal pathogens.Abbreviations Con A concanavalin A - PNA peanut agglutinin - SBA soybean agglutinin - WGA wheat germ agglutinin - FITC fluorescein isothiocyanate - GlcNAc N-acetyl-D-glucosamine - GalNAc N-acetyl-D-galactosamine     

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.     

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.     

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.     

Hypoxia modifies the effect of PDGF on glycosaminoglycan synthesis by primary human lung cells

Hypoxia, a consequence of interstitial lung diseases, may lead to secondary pulmonary hypertension and pulmonary vascular remodeling. Hypoxia induces activation and proliferation of lung cells and enhances the deposition of extracellular matrix including glycosaminoglycans (GAGs). To elucidate the cell biological mechanisms underlying the development of secondary pulmonary hypertension, we studied the effect of hypoxia on GAG synthesis by human lung cells. GAG synthesis was measured by incorporation of [(3)H]glucosamine; GAGs were isolated, purified, and characterized with GAG-degrading enzymes. Fibroblasts and vascular smooth muscle cells (VSMCs) synthesized hyaluronic acid, heparan sulfate, and chondroitin sulfates, whereas dermatan sulfate was found only in fibroblasts. Hypoxia did not influence the size or charge of the individual GAGs. However, hypoxia inhibited platelet-derived growth factor-induced [(3)H]glucosamine incorporation in secreted GAGs, especially hyaluronic acid, in VSMCs. In contrast, it stimulated GAG secretion, specifically heparan sulfate, by fibroblasts. Our results indicate that hypoxia induces modifications in GAG synthesis by human lung VSMCs and fibroblasts that may be correlated to pathophysiological manifestations in lung diseases causing hypoxia.