"GAG-ing with the neuron": The role of glycosaminoglycan patterning in the central nervous system.

Proteoglycans (PGs) are a diverse family of proteins that consist of one or more glycosaminoglycan (GAG) chains, covalently linked to a core protein. PGs are major components of the extracellular matrix (ECM) and play critical roles in development, normal function and damage-response of the central nervous system (CNS). GAGs are classified based on their disaccharide subunits, into the following major groups: chondroitin sulfate (CS), heparan sulfate (HS), heparin (HEP), dermatan sulfate (DS), keratan sulfate (KS) and hyaluronic acid (HA). All except HA are modified by sulfation, giving GAG chains specific charged structures and binding properties. While significant neuroscience research has focused on the role of one PG family member, chondroitin sulfate proteoglycan (CSPG), there is ample evidence in support of a role for the other PGs in regulating CNS function in normal and pathological conditions. This review discusses the role of all the identified PG family members (CS, HS, HEP, DS, KS and HA) in normal CNS function and in the context of pathology. Understanding the pleiotropic roles of these molecules in the CNS may open the door to novel therapeutic strategies for a number of neurological conditions.

Aortic valve leaflet glycosaminoglycans composition and modification in severe chronic valve regurgitation.

BACKGROUND AND AIM OF THE STUDY: The surgical segments of aortic valve leaflets from patients with severe chronic aortic regurgitation were analyzed (by percentage and structure) for their content of complex polysaccharides and glycosaminoglycans (GAGs), and compared with control segments. METHODS: The GAG, hyaluronic acid (HA), chondroitin sulfate (CS) and dermatan sulfate (DS) and disaccharide contents were determined in segments (leaflet, root attachment region and belly) of aortic valve leaflets (non-coronary, left coronary and right coronary) using a multi-analytical approach. RESULTS: The aortic valve leaflets showed the presence of HA and CS/DS, with an overall charge density of -0.51-0.55. The CS/DS polymers showed a 4-sulfated/6-sulfated ratio of -0.70-0.77 in the belly, and -1.60-1.72 in commissure parts (-/+124%). The total amount of GAGs was -1.60-2.40 microg/mg of tissue. A significant increase in sulfated GAGs was observed in all valve parts in patients suffering from severe aortic insufficiency, as well as an increase in the 4-sulfated/6-sulfated ratio in the leaflet belly (-/+102%). CONCLUSION: It is speculated that differences in 4-sulfated/6-sulfated ratio determined in the belly and leaflet attachment region-commissure parts of the leaflets may correlate with the tensile or compressive loading of normal aortic valve regions. At the same time, it may be assumed that the increase in sulfated GAGs and 4-sulfated/6-sulfated ratio in the leaflet belly of valves taken from patients suffering severe aortic insufficiency was consistent with an altered matrix microstructure capable of influencing the hydration of these pathological tissues, and of conditioning their mechanical weakness.

Myxoid tumours of soft tissue: the so-called myxoid extracellular matrix is heterogeneous in composition.

AIM: Myxoid tumours of soft tissue are characterized by their so-called 'myxoid' extracellular matrix. The aim was to investigate the composition and possible function of this matrix which is poorly understood. METHODS AND RESULTS: Using Alcian Blue staining with and without pretreatment with hyaluronidase and application of the critical electrolyte concentration method followed by densitometry, the glycosaminoglycan composition of three different myxoid tumours was studied. The composition of glycosaminoglycans varied with tumour type and grade, despite their general characterization as myxoid tumours. Intramuscular myxoma contained similar amounts of the various glycosaminoglycans as grade I myxofibrosarcoma; grade III myxofibrosarcoma contained less hyaluronic acid and more heparan sulphate, whereas extraskeletal myxoid chondrosarcoma contained predominantly chondroitin-4 and -6 sulphates. Western blot identified albumin as a major protein in tumour lysates, and its presence in the extracellular matrix and cytoplasm of the majority of tumours was demonstrated by immunohistochemistry; production of albumin by the tumour cells was confirmed by quantitative polymerase chain reaction. CONCLUSIONS: The extracellular matrix of myxoid tumours of soft tissue has a heterogeneous composition consisting of, amongst others, glycosaminoglycans and albumin, which appear to play an active role in their morphogenesis.

[Natural and synthetic glycosaminoglycans. Molecular characteristics as the basis of distinct drug profiles]. / Natürliche und synthetische Glykosaminoglykane. Molekulare Charakteristika als Grundlage unterschiedlicher Arzneistoffprofile.

For several decades, anticoagulants based on glycosaminoglycans (GAG) are drugs of choice in the therapy and prophylaxis of thromboembolic diseases. In principle, it has to be differentiated between the natural GAG-anticoagulants, which are molecular mixtures with complex composition, and the synthetic GAG-anticoagulants, which are chemically defined oligosaccharides. The former include unfractionated heparin, the various low molecular weight heparins and danaparoid. Representatives of the second group are fondaparinux, idraparinux and the hexadecasaccharide SR123781A. They share a common mechanism of action together with the endogenous antithrombotic heparan sulfate, i.e. the catalysis of the antithrombin-mediated inhibition of factor Xa. Besides, considerable structural differences between the various GAG-anticoagulants result in rather distinct product characteristics. This concerns their pharmacodynamics, pharmacokinetics as well as practice-related aspects such as dosage, monitoring, accumulation tendency, antagonisation and HIT-Typ II.

Effects of sterilization on an extracellular matrix scaffold: part I. Composition and matrix architecture.

The impact of peracetic acid (PAA), lyophilization, and ethylene oxide (EO) sterilization on the composition and three dimensional matrix structure of small intestinal submucosa (SIS), a biologic scaffold used to stimulate the repair of damaged tissues and organs, was examined. Fibronectin and glycosaminoglycans are retained in SIS following oxidation by peracetic acid and alkylation using ethylene oxide gas. Significant amounts of FGF-2 are also retained, but VEGF is susceptible to the effects of PAA and is dramatically reduced following processing. Further, matrix oxidation, lyophilization, and sterilization with EO can be performed without irreversibly collapsing the three dimensional structure of the native SIS. These structural features and growth promoting extracellular matrix constituents are likely to be important variables underlying cellular attachment, infiltration and eventual incorporation of SIS into healing host tissues.

Glycomics approach to structure-function relationships of glycosaminoglycans.

Extracellular modulation of phenotype is an emerging paradigm in this current postgenomics age of molecular and cell biology. Glycosaminoglycans (GAGs) are primary components of the cell surface and the cell-extracellular matrix (ECM) interface. Advances in the technology to analyze GAGs and in whole-organism genetics have led to a dramatic increase in the known important biological role of these complex polysaccharides. Owing to their ubiquitous distribution at the cell-ECM interface, GAGs interact with numerous proteins and modulate their activity, thus impinging on fundamental biological processes such as cell growth and development. Many recent reviews have captured important aspects of GAG structure and biosynthesis, GAG-protein interactions, and GAG biology. GAG research is currently at a stage where there is a need for an integrated systems or glycomics approach, which involves an integration of all of the above concepts to define their structure-function relationships. Focusing on heparin/heparan (HSGAGs) and chondroitin/dermatan sulfate (CSGAGs), this review highlights the important aspects of GAGs and summarizes these aspects in the context of taking a glycomics approach that integrates the different technologies to define structure-function relationships of GAGs.

Interaction of chemokines and glycosaminoglycans: a new twist in the regulation of chemokine function with opportunities for therapeutic intervention.

Despite their key role in inflammation, the apparent redundancy in the chemokine system is often cited as an argument against probing chemokines as therapeutic targets for inflammation. However, this in vitro redundancy frequently does not translate to the in vivo situation, as exemplified by the use of specific receptor antagonists, ligand neutralizing or receptor blocking antibodies and gene-deleted mice in models of human disease. Specificity may be conferred onto the chemokine system by fine-tuning of responses both temporally and spatially through their highly specific interactions with glycosaminoglycans (GAGs). In this survey, we present evidence for specificity in the interaction and introduce emerging technologies that enable detailed assessment of protein-GAG interactions. Finally, we address the issue of exploitation of this interaction for therapeutic advantage.

Classification of chondroitin sulfate A, chondroitin sulfate C, glucosamine hydrochloride and glucosamine 6 sulfate using chemometric techniques.

Chondroitin sulfate A, chondroitin sulfate C, glucosamine hydrochloride and glucosamine sulfate are natural products that are becoming increasingly popular in the treatment of arthritis. They belong to a class of compounds known as glycosaminoglycans (GAGs). They are available over the counter as nutritional supplements. However, increasing use has led to increasing scrutiny of the quality of products on the market. There is also interest in the pharmacological properties of these compounds. To facilitate this, there is a need for better qualitative and quantitative methods of analysis. This paper describes methods for achieving the qualitative identification of chondroitin sulfate A, chondroitin sulfate C, glucosamine hydrochloride or glucosamine sulfate. Fourier transform infrared spectroscopy coupled with a variety of chemometric methods successfully classified these compounds. Using soft independent modeling of class analogies (SIMCA), hierarchical cluster analysis (HCA) and principal components analysis (PCA) samples were classified as either chondroitin sulfate A, chondroitin sulfate C, glucosamine hydrochloride or glucosamine sulfate. This work also examined the discriminating ability of different sections of the spectrum. It was found that for the classification of these compounds that using the finger print region of the spectrum (below 2000 cm(-1)) gave the best discrimination.

Platelet factor 4 neutralizes heparan sulfate-enhanced antithrombin inactivation of factor Xa by preventing interaction(s) of enzyme with polysaccharide.

Platelet factor 4 (PF4) is a heparin-binding protein which exhibits anti-heparin activities through the inhibition of antithrombin (AT)-dependent reactions with the serine proteases thrombin and factor Xa. PF4 also neutralizes heparan sulfate (HS), a glycosaminoglycan (GAG) present on the surface of endothelial cells, thereby possibly modulating an anticoagulant response. Previous models of PF4 mechanism did not distinguish whether PF4 causes steric hindrance of AT binding to fXa or of AT binding to the surface of the GAG chain. To shed light on the mechanism of PF4, studies of HS/heparin-catalyzed fXa inactivation by AT were undertaken. The results were consistent with PF4 directly interfering with AT binding to fXa rather than AT binding to the GAG chain, since PF4 did not prevent the heparin-dependent increase in AT intrinsic fluorescence. In fact, PF4 mechanism was competitive with respect to AT and non-competitive with respect to fXa, suggesting inhibition of important regulatory/catalytic interactions of fXa with the polysaccharide. Altogether, the results suggested a model by which PF4 bound to proximal (but distinct) sites to AT, resulting in steric interference of fXa binding to both polysaccharide and AT. It is proposed that PF4 inhibited the sequence of events recapitulated in the template mechanism describing heparin-dependent inhibition of fXa.

Glycosaminoglycan profiles of myxomatous mitral leaflets and chordae parallel the severity of mechanical alterations.

OBJECTIVES: This biochemical study compared the extracellular matrix of normal mitral valves and myxomatous mitral valves with either unileaflet prolapse (ULP) or bileaflet prolapse (BLP). BACKGROUND: Myxomatous mitral valves are weaker and more extensible than normal valves, and myxomatous chordae are more mechanically compromised than leaflets. Despite histological evidence that glycosaminoglycans (GAGs) accumulate in myxomatous valves, previous biochemical analyses have not adequately examined the different GAG classes. METHODS: Leaflets and chordae from myxomatous valves (n = 41 ULP, 31 BLP) and normal valves (n = 27) were dried, dissolved, and assayed for deoxyribonucleic acid, collagen, and total GAGs. Specific GAG classes were analyzed with selective enzyme digestions and fluorophore-assisted carbohydrate electrophoresis. RESULTS: Biochemical changes were more pronounced in chordae than in leaflets. Myxomatous leaflets and chordae had 3% to 9% more water content and 30% to 150% higher GAG concentrations than normal. Collagen concentration was slightly elevated in the myxomatous valves. Chordae from ULP had 62% more GAGs than those from BLP, primarily from elevated levels of hyaluronan and chondroitin-6-sulfate. CONCLUSIONS: The GAG classes elevated in the myxomatous chordae are associated with matrix microstructure and elastic fiber deficiencies and may influence the hydration-related "floppy" nature of these tissues. These abnormalities may be related to the reported mechanical weakness of myxomatous chordae. The biochemical differences between ULP and BLP confirm previous mechanical and echocardiographic distinctions.

Differential expression of glycosaminoglycans and proteoglycans in the migratory pathway of the primordial germ cells of the mouse.

Primordial germ cells are an embryonic cell line that give rise to gametes in vertebrates. They originate outside the embryo proper and migrate by a well-defined route to the genital ridges. Proteoglycans and glycosaminoglycans have distinctive properties that affect many of the characteristics of the extracellular microenvironment of migratory pathways in a variety of developmental systems. The purpose of this work was to identify the proteoglycans and glycosaminoglycans that are spatially and temporally expressed in the migratory pathway of primordial germ cells. We showed that the expression of proteoglycans and glycosaminoglycans in the primordial germ cells migratory pathway changes according to the different phases of the migratory process. Some molecules such as chondroitin-0-sulfate, decorin, and biglycan are present only in certain phases of the migratory process of primordial germ cells. Heparan sulfate, chondroitin-6-sulfate, versican, perlecan, and syndecan-4, although exhibiting some variation in expression were detected during all phases of the migratory process. Our results indicate that the successive steps of primordial germ cell migration require a coordinated expression of proteoglycans and glycosaminoglycans, that should be present in appropriate levels and in specific areas of the embryo, and that the sequential expression of these extracellular matrix molecules is under a genetic program that appears to be common to a variety of cell types during embryonic development.

Tissue structure-specific distribution of glycosaminoglycans in the human penis.

The aim of this work was to isolate and characterise the glycosaminoglycans present in the different tissue structures of the human penis in view of their potentially significant role in the physiology of erection. Penile tissue samples were obtained from patients who underwent penectomy and were subsequently dissected into individual tissue structures. Total glycosaminoglycans were isolated and purified from tunica albuginea, corpora cavernosa and corpus spongiosum, following tissue mincing, ultrasonication, lipid extraction, extensive digestion with pronase and DNase, treatment with alkali-borohydride and ethanol precipitation. Isolated glycosaminoglycans were separated by cellulose acetate electrophoresis and fractionated by anion exchange chromatography on DEAE Sephacel columns. Different glycosaminoglycan fractions were identified using glycosaminoglycan-degrading enzymes of known specificity. Gradient polyacrylamide gel electrophoresis was used to determine the average molecular mass of the glycosaminoglycans. The corpus cavernosum and the corpus spongiosum extracts contained almost twice the amount of glycosaminoglycan-associated uronic acids as compared to the tunical extracts (1.47+/-0.09, and 1.49+/-0.15 as opposed to 0.75+/-0.15 microg/mg dry defatted tissue, respectively; S.E.M., n=5). With the exception of hyaluronic acid, the relative amount of individual glycosaminoglycan types varied significantly among extracts of different origin. Heparan sulphate was more abundant in cavernosal, dermatan sulphate in tunical, and chondroitin-6-sulphate in corpus spongiosum extracts. No structure-specific differences were detected with respect to the molecular mass distribution of each glycosaminoglycan type. Our study shows that the different structures of the human penis produce distinct profiles of glycosaminoglycans, which are well suited to the individual functional characteristics of these structures.

Urinary glycosaminoglycan excretion in healthy subjects and in patients with mucopolysaccharidoses.

BACKGROUND: The mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders caused by deficiency of enzymes catalyzing the stepwise degradation of glycosaminoglycans (GAGs), and are transmitted in an autosomal recessive manner, except for Hunter syndrome. METHODS: The levels of GAGs in 150 healthy subjects and 33 patients with MPS were determined, and results were expressed as milligrams of GAGs per grams of creatinine. RESULTS: We found that this ratio decreased with age during the first 15 years of life, but had a constant low rate between the ages of 17-40 years in healthy individuals. A different tendency was present in patients with MPS, because levels of GAG excretion in this group were higher (by four standard deviations up) compared with healthy individuals. The electrophoretic patterns of urinary GAGs in healthy subjects showed that the higher levels detected in urine were chondroitin sulfate (4 and 6) and a smaller quantity of dermatan sulfate, but in each MPS type its characteristic pattern was identified. CONCLUSIONS: This is a simple, reproducible method suitable for routine laboratory separation, identification, and quantity of urinary GAGs and for diagnosing MPS syndromes.

Ion chromatography on anion exchangers modified with mucopolysaccharides.

Anion exchangers modified with mucopolysaccharides, such as chondroitin sulfates and heparin, were used for the stationary phase in ion chromatography. Unusual retention behavior of anions was observed for the modified stationary phases. A 50-microM concentration of tartaric acid could separate inorganic anions in a reasonable time. The retention of analytes could be changed by changing the eluent composition.

Exogenous heparin induces an increase in glycosaminoglycans of the chick embryo chorioallantoic membrane: its possible role in the regulation of angiogenic processes.

In this study heparin (HE) was injected into the allantoic sac of chick embryo eggs at 5, 9, 14 days of incubation. 48 h after injection glycosaminoglycan (GAG) concentration was measured in the chorioallantoic membrane (CAM) in order to verify if HE-related CAM angiogenic activity previously demonstrated [Ribatti et al: Acta Anat 1987; 130:257-263] might be correlated with changes in GAG concentration. The results showed that HE inoculation induced an increase of 3H-glucosamine incorporation into total GAGs in comparison to control CAMs. Furthermore, HE altered the balance between the GAG classes, and in particular it produced a significant increase in the accumulation of hyaluronic acid and heparan sulfate between 7 and 11 days of incubation in comparison to control CAMs.

Maturation-related biochemical changes in swine anterior cruciate ligament and tibialis posterior tendon.

In order to determine the differences between ligaments and tendons in terms of change in biochemical composition during maturation, the biochemical characteristics of the anterior cruciate ligament and tibialis posterior tendon of swine were studied. The collagen content of the tibialis posterior tendon was found to increase rapidly with growth of the body, reaching a plateau prior to maturation. In contrast, the rate of increase in the anterior cruciate ligament was slow, indicating that maturation of this tissue is delayed. The quantity of glycosaminoglycan in both the anterior cruciate ligament and the tibialis posterior tendon decreased with growth. In mature animals, glycosaminoglycans in the anterior cruciate ligament included chondroitin sulfate, hyaluronic acid, and dermatan sulfate, but only trace amounts of chondroitin sulfate were found in the tibialis posterior tendon. Although the ratio of dermatan sulfate to hyaluronic acid generally increased with growth, this increase was more conspicuous in the tibialis posterior tendon than in the anterior cruciate ligament. The anterior cruciate ligament and tibialis posterior tendon both contained collagen of types I, III, and V. In mature swine, type III was increased in the anterior cruciate ligament but not in the tibialis posterior tendon. These findings demonstrate slower maturation for ligament than for tendon with regard to the changes in biochemical constituents, especially those in collagen type and glycosaminoglycan, during the growth process, and also suggest that the composition of these tissues changes in accordance with their changing functional requirements.

Differential growth factor-induced modulation of proteoglycans synthesized by normal human renal versus cyst-derived cells.

In polycystic kidney disease (PKD), there is an insiduous enlargement of the kidneys and dilation of the renal tubules associated with extracellular matrix (ECM) alterations. The latter include thickening of tubular basement membrane and decreased synthesis of sulfated proteoglycan (PG). Because PKD is believed to be a disorder of cell growth and deranged ECM metabolism, it is conceivable that the formation of cystic tubules may be modulated by certain growth factors (GF) that influence the synthesis of ECM glycoproteins. In this study, the effect of various GF, i.e., epidermal, hepatocyte (HGF) and transforming (TGF), and triiodothyronine on the PG synthesized by normal human kidney (NK) epithelial cells and cells derived from cysts of patients with autosomal dominant PKD (ADPKD) was assessed. (35S) sulfate incorporation studies revealed that, among various GF, HGF and TGF-beta 1 had the maximal stimulatory effect on the synthesis of PG extracted from ADPKD cells. A minimal increase in the PG synthesis was observed in NK cells; however, PG synthesized under the influence of HGF or TGF-beta 1 were of relatively higher molecular weight, with a shift of K(av) from 0.28 to 0.12, as ascertained by Sepharose-6B chromatography. PG synthesized by ADPKD cells had a K(av) = 0.18, and it did not change with the GF treatment. The charge-density characteristics of PG of ADPKD cells were relatively lower than those of NK cells, and they were unaffected by HGF or TGF-beta 1 treatment. Interestingly, both the HGF and TGF-beta 1 significantly affected posttranslational modifications of PG.(ABSTRACT TRUNCATED AT 250 WORDS)

The nomenclature of glycosaminoglycans and proteoglycans.

1. The proposed terms and codes provide concise, quantitative information on the polymer backbone, state of oxidation, patterns of sulphation and epimerization, and proportions of monomeric units. Oversulphated domains and special units are easily recognized. 2. They are intermediate between general statements (e.g. 'chondroitin sulphate') and detailed primary structures. 3. They bring terminology into line with current analytical techniques, which depend largely on enzymes developed by S. Suzuki and co-workers. Current enzyme nomenclature (chondroitinase ABC, AC) can still be used. 4. They are not primarily intended to be spoken, although some codes are easily articulated. 5. They have capacity and flexibility to accommodate future developments.