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.

Distinctive populations of basement membrane and cell membrane heparan sulfate proteoglycans are produced by cultured cell lines.

We have investigated the nature and distribution of different populations of heparan sulfate proteoglycans (HSPGs) in several cell lines in culture. Clone 9 hepatocytes and NRK and CHO cells were biosynthetically labeled with 35SO4, and proteoglycans were isolated by DEAE-Sephacel chromatography. Heterogeneous populations of HSPGs and chondroitin/dermatan proteoglycans (CSPGs) were found in the media and cell layer extracts of all cultures. HSPGs were further purified from the media and cell layers and separated from CSPGs by ion exchange chromatography after chondroitinase ABC digestion. In all cell types, HSPGs were found both in the cell layers (20-70% of the total) as well as the medium. When the purified HSPG fractions were further separated by octyl-Sepharose chromatography, very little HSPG in the incubation media bound to the octyl-Sepharose, whereas 40-55% of that in the cell layers bound and could be eluted with 1% Triton X-100. This hydrophobic population most likely consists of membrane-intercalated HSPGs. Basement membrane-type HSPGs were identified by immunoprecipitation as a component (30-80%) of the unbound (nonhydrophobic) HSPG fraction. By immunofluorescence, basement membrane-type HSPGs were distributed in a reticular network in Clone 9 and NRK cell monolayers; by immunoelectron microscopy, these HSPGs were localized to irregular clumps of extracellular matrix located beneath and between cells. The cells did not produce a morphologically recognizable basement membrane layer under these culture conditions. When membrane-associated HSPGs were localized by immunoelectron microscopy, they were found in a continuous layer along the cell membrane of all cell types. The results demonstrate that two antigenically distinct populations of HSPG--an extracellular matrix and a membrane-intercalated population--are found at the surface of several different cultured cells lines; these populations can be distinguished from one another by differences in their distribution in the monolayers by immunocytochemistry and can be separated by hydrophobic chromatography; and basement membrane-type HSPGs are secreted and deposited in the extracellular matrix by cultured cells even though they do not produce a bona fide basement membrane-like layer.

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.

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.

[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.

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.

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.

"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.

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.

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.

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.

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.

Epitopes of cartilage core proteins and GAG pattern in human non-Hodgkin lymphoma xenografts.

Glycosaminoglycan and core protein components of proteoglycans (PGs) have been studied in three human non-Hodgkin lymphoma xenografts of B cell origin. Lymphomas showed similar GAG content, but different composition of GAG subtypes. This variability was accompanied by an individual capacity to adhere to extracellular matrix elements. The core proteins identified by monoclonal antibodies raised against human cartilage chondroitin sulfate PG were also distinctly expressed and released. These proteins shared by different cell types may have biological significance.

[Proteoglycans (their structure, function and role in liver diseases)]. / Proteoglikánok (struktúra, funkció, szerepük májbetegségekben).

Proteoglycans are macromolecules containing a core protein to which glycosaminoglycan chains are covalently attached. The family contains several members with different structures and various functions. Some of them are elements of the extracellular matrix, while others are located to the cell surface playing important role in cell-cell and cell-extracellular matrix interactions. Present paper discusses the possible consequences of the alterations of proteoglycans observed in liver cirrhosis and liver tumors. It has to be emphasized however, that they are also involved in the pathomechanism of arteriosclerosis, Alzheimer-disease, immune diseases, arthritis, tumor progression and metastasis formation.

[Study on mucopolysaccharides in human lung carcinoma tissue--characteristics in histological types (author's transl)].

The mucopolysaccharides were prepared from human lung carcinomas of three histologically different types and the control tissue by exhaustive proteolytic digestion, quaternary ammonium chloride fractionation and column chromatography on Dowex 1 (Cl-). They were identified by chemical, enzymic and electrophoretic methods, as hyaluronic acid (HA), chondroitin sulfate (ChS), dermatan sulfate (DS), heparan sulfate (HS) and over-sulfated ChS and/or DS. Qualitatively they were not differed in tumor and normal tissues. However, the amounts of whole mucopolysaccharide were much increased in carcinomas than those of normal control in order of squamous cell carcinoma greater than small cell undifferentiated carcinoma greater than or equal to adenocarcinoma. The increment of mucopolysaccharide contents in carcinoma are largely due to increased amounts of HA and ChS. Carcinoma-type characteristic pattern was also demonstrated in terms of relative amounts of non-sulfated (HA) and sulfated (ChS, DS, HS) mucopolysaccharides: In squamous cell carcinoma and adenocarcinoma sulfated mucopolysaccharides were predominant (73 to 78% of total mucopolysaccharides), whereas in small cell undifferentiated carcinoma sulfated ones were diminished (25% of total mucopolysaccharides). In normal lung tissue sulfated mucopolysaccharide comprised 64% of total mucopolysaccharides. The presence of over-sulfated ChS and/or DS, which have not until now been found in lung tissue, was higher in carcinoma tissue as compared to the normal control. Total glycopeptides which were derived from tissue glycoproteins and not in detail characterized in this study were decreased in carcinomas of any histological types as compared to those of normal lung tissue, when expressed by hexosamine content. Biological and clinical significance of mucopolysaccharides in carcinoma state was discussed.