Distribution Of Glycosaminoglycans Research Articles

Effect of postoperative immobilisation on the regrowth of the knee joint semilunar cartilage: an experimental study.

Bilateral total medial meniscectomy was undertaken in the knee joints of 12 adult greyhounds. One-half of the animals had one meniscectomised joint immobilised by an external fixation system for 5 weeks following meniscectomy. All animals were sacrificed 6 months after surgery. The menisci (regrown and normal) were examined for collagen content and analysed histochemically to assess collagen assembly and glycosaminoglycan (GAG) distribution. Although the collagen content of regrown tissues were not statistically different from normal medial or lateral menisci, the fibre development and GAGs were abnormal. It is concluded that movement following meniscectomy is likely beneficial to the matrix formation in the regenerating tissue, but this together with weight bearing may have a detrimental effect on articular cartilage of the medial joint compartment.

Matrix synthesis in high density cultures of bovine epiphyseal plate chondrocytes.

Bovine epiphyseal plate chondrocytes were cultured by a method combining both suspension culture and high density monolayers. The matrix synthesized by the cultured cells was analyzed at fifteen days for glycosaminoglycan, proteoglycan, and collagen content. In the cell culture product glycosaminoglycan distribution was: 65% chondroitin-6-sulfate, 18% chondroitin-4-sulfate, 15% keratan sulfate, and less than 2% dermatan sulfate. Essentially all the radioactive sulfate in labelled specimens was present in high molecular weight aggregates. The collagen which was synthesized co-migrated with Type II collagen standard. Parallel analysis showed the matrix of cultured cells to be similar to that of intact epiphyseal plate tissue. This study demonstrates the ability to grow epiphyseal plate chondrocytes in a cell culture system which allows matrix synthesis similar to that seen in vivo.

Isolation of arterial wall proteoglycans

Proteoglycans of the arterial media-intima were extracted with associative and dissociative salt solutions. A two step isolation procedure, involving initial precipitation with cetylpyridinium chloride and subsequent CsCl isopycnic density gradient centrifugation was adopted for their effective isolation and purification. The majority of the proteoglycans were: 1. 1. Obtained between buoyant densities 1.60 and 1.77 g ml; 2. 2. Possessed a uronic acid to protein ratio of 2.9 or 1.8 respectively for associatively and dissociatively extracted proteoglycans and 3. 3. Showed a glycosaminoglycan distribution pattern similar to that for the total tissue.

Glycosaminoglycans in Embryonic Mouse Teeth and the Dissociated Dental Constituents

The nature, amounts, and distribution of glycos-aminoglycans (GAG) before and during odontoblast terminal differentiation were studied. GAG have been isolated from intact mouse tooth germs and from dissociated dental epithelia and dental papillae after labeling with [ 3H] glucos-amine or 35SO 4 2− as precursor. The kinds and relative amounts of 3H-labeled GAG were analyzed by chromatography on a DEAE-cellulose column and cellulose thin-layer sheets. The amounts of individual GAG relative to total GAG were determined from the elution profiles, whereas their nature was identified by the selective removal of chromatographic peaks after enzymatic or chemical degradation. We found hyaluronate and probably a minute quantity of heparan sulfate in the dental epithelium, while hyaluronate, heparan sulfate, and chondroitin sulfate were the main types of GAG in the dental papilla. The chondroitin sulfate recovered was further fractionated by cellulose thin-layer chromatography into two isomers, namely chondroitin-4-sulfate (the major component) and chondroitin-6-sulfate. Changes in the elution profile from DEAE-cellulose chromatography of tooth GAG extracted from different developmental stages suggest that modifications of GAG occur during odontogenesis. Alcian blue staining localized large amounts of hyaluronate and sulfated GAG along the epithelio-mesenchymal junction. Tissue specificity and changing patterns of GAG were demonstrated during odontogenesis.

Effect of anti-inflammatory agents on the glycosaminoglycan metabolism in cultured human synovial cells.

The interaction of various anti-inflammatory agents with the metabolism of proteoglycans (glycosaminoglycans) was studied by determination of the distribution of radiolabelled glycosaminoglycans into the intracellular, pericellular and extracellular compartments of cultured synovial cells. The agents interfere dose dependently with different metabolic processes: Benzarone and flufenamic acid (10−4 mol/l) show an inhibition of the synthesis of glycosaminoglycans probably operative at an early phase of the metabolism (transcription or translation process). Colchicine causes an inhibition of both the biosynthesis and secretion of glycosaminoglycans at concentrations ≧10−7 mol/l. An inhibition of the degradation is assumed at a lower concentration (10−8 mol/l). Exposure of the cells to glycosaminoglycan polysulfate leads to an inhibition of the synthesis and degradation as well, the latter being the dominant effect at lower concentrations (0.3–30 mg/l). The secretion of hyaluronate and chondroitin sulfate and the proteolytic degradation of proteochondroitin sulfate seems to be inhibited by the proteinase inhibitor gabexate mesilate at concentrations ≧10−3 mol/l.

Dimethyl sulfoxide affects colony morphology on agar and alters distribution of glycosaminoglycans and fibronectin.

We have found striking changes in the morphology of colonies of Chinese hamster ovary cells grown on agar containing low doses of dimethyl sulfoxide. Effects on morphology of cells grown on plastic at the same dimethyl sulfoxide concentrations were not as pronounced. Computer-assisted analysis of darkfield photographs of growing colonies proved very useful in measuring the magnitude of morphological changes at various doses. A large decrease in total cell-bound and released glycosaminoglycans (GAGs) was observed in the presence of dimethyl sulfoxide by measuring incorporation of radiolabeled precursors into cetylpyridinium chloride-precipitable GAGs in Chinese hamster ovary cells. By contrast, dimethyl sulfoxide was found to cause an increase in the network of fibronectin (the large external transformation-sensitive protein) at the cell surface. These observations demonstrate the association of GAGs and fibronectin in processes affecting the three-dimensional growth patterns of aggregates of mammalian cells and also demonstrate the sensitivity of agargrown colonies as model systems for quantitatively measuring the morphological changes induced by exogenous agents such as drugs, hormones, growth factors, mutagens, and carcinogens. These findings might be relevant to the study and treatment of the important class of genetic diseases called mucopolysaccharidoses which result in mental, skeletal, and ocular defects as a consequence of GAG accumulation.

Contents and compositions of glycosaminoglycans in different sites of the human hip joint cartilage.

The distribution of glycosaminoglycans (GAGs) in different functional regions (weight-bearing and nonweight-bearing portions) of the human hip joint cartilage was studied. The results obtained were as follows: (1) Weight-bearing cartilage contains larger amounts of GAGs than nonweight-bearing, cartilage. (2) Weight-bearing cartilage contains keratan sulphate in higher ratio to chondroitin sulphate than nonweight-bearing cartilage. (3) The differences in content and composition of GAGs between the weight-bearing and nonweight-bearing portions are more pronounced in the femoral head than in the acetabulum. The preliminary analyses showed that the chondroitin sulphate from the acetabular cartilage contained exclusively 6-sulphated disaccharide units and there was some heterogeneity in keratan sulphate.

Distribution of heparin and other sulfated glycosaminoglycans in vertebrates

1. 1. A comparative study on the distribution of sulfated glycosaminoglycans in several tissues of six specimens of different vertebrate classes is reported. 2. 2. Each tissue has a characteristic composition differing from each other regarding the relative amount, type and molecular size of chondroitin 4, 6-sulfate, dermatan sulfate and heparan sulfate. 3. 3. The same tissue from different vertebrates has, in general, the same types of sulfated glycosaminoglycans, but with different molecular weights. 4. 4. Exception to this rule was observed for the distribution of heparin which was present only in a few tissues of the 6 vertebrates studied. 5. 5. The possible involvement of the sulfated glycosaminoglycans in cell recognition and/or adhesiveness is discussed in view of this characteristic distribution.

Glycosaminoglycans in emphysematous and fibrotic hamster lungs.

Amounts and distributions of glycosaminoglycans were measured in lungs obtained from hamsters treated intratracheally 21 to 30 days previously with either saline, purified pancreatic elastase, or bleomycin. Normal hamster lungs contained 150 to 200 micrograms glycosaminoglycans per lung. Lungs made fibrotic by bleomycin contained significantly more glycosaminoglycans per lung than saline-treated control lungs; this increase was equally distributed among all glycosaminoglycan subtypes. Emphysematous lungs also contained more glycosaminoglycans than saline control lungs; this increase was less than that of fibrotic lungs and was limited to the dermatan sulfate subtype. The latter was increased in emphysematous lungs when expressed as a percentage of total glycosaminoglycans. In these hamster models of lung diseases that affect the connective tissue framework of the lungs, changes in lung glycosaminoglycans accompany changes in the other connective tissue components.

Glycosaminoglycans in granulation tissue and hypertrophic scars

Abstract The glycosaminoglycans of granulation tissue from regenerating tissue of severely burned patients was investigated and compared with those of (1) hypertrophic scars and keloids, (2) mature scars and (3) normal skin. Results clearly show that granulation tissues have high levels of chondroitin-4-sulphate relative to the other glycosaminoglycans as compared to low levels in mature scar and normal skin. The chondroitin-4-sulphate levels of children's granulation tissue was significantly higher than the levels from hypertrophic scars of children, however the chondroitin-4-sulphate levels of adults' granulation tissue was not significantly different from those of hypertrophic scars. The dermatan sulphate levels of granulation tissue are significantly lower than the dermatan sulphate levels of hypertrophic scars, mature scars or normal skin. This suggests that chondroitin-4-sulphate is related to proliferation of collagen in granulation tissue, and that as the collagen matures and the normal scar forms, chondroitin-4-sulphate levels decrease and dermatan sulphate increases. When the hypertrophic scar develops, the high chondroitin-4-sulphate levels remain high, dermatan sulphate increases, but does not reach the levels of those in the mature scar. Neither hypertrophic scar nor mature scar have levels of hyaluronic acid as high as normal skin. However, our data suggest that adult granulation tissue may have relatively high hyaluronic acid levels. It is suggested that the different distribution of glycosaminoglycans in granulation tissue influences the size and shape of collagen filaments and may also influence the rate of synthesis of collagen.

Interaction of aortic endothelial and smooth muscle cells in culture Effect on glycosaminoglycan levels

Co-cultivation of various intra- and interspecific combinations of pig and rat aortic endothelial cells (AEC) and smooth muscle cells (SMC) resulted in a marked increase in hyaluronic acid (HA) levels, a smaller but significant increase in sulphated glycosaminoglycans (GAG), and an increase in the HA/sulphated GAG ratio, compared with the separate culture of the two cell types. Culture of SMC in AEC-conditioned medium produced similar changes in GAG levels, whereas SMC-conditioned medium had no effect on AEC GAG levels. These results add further support for the concept that epithelial cells in general can modulate the GAG composition of adjacent connective tissue and thereby influence its morphological and physiological properties. It is suggested that the normal amounts, types and distribution of GAG in the arterial wall, and especially in the intima, may be partly dependent on interaction between the endothelium and SMC. It is further suggested that injury to endothelium, with a consequent failure in this interaction, could lead directly to changes in intimal GAG composition that contribute to lesion development.

Glycosaminoglycan synthesis by glomeruli in vivo and in vitro

The distribution of glycosaminoglycans in disrupted glomerular fractions was studied using 35SO 4-labeling in vivo and in vitro. The majority of 35S of isolated glomerular basement membrane was found in heparan sulfate after in vivo and in vitro pulses, although the absolute proportion and the degrees of N-sulfation and N-acetylation varied with the conditions of exposure. Varying amounts of chondroitin sulfate and dermatan sulfate were found in the glomerular basement membrane fraction and larger proportions of both of these glycosaminoglycans as well as of heparan sulfate were found in various glomerular fractions. Glomerular glycosaminoglycans distribution studies must take into account the experimental conditions. Basement membrane-like components of the glomerulus such as the mesangial matrix may have varying glycosaminoglycan composition which may be found in association with glomerular basement membrane fractions.

Aqueous outflow pathway glycosaminoglycans

The acidic glycosaminoglycan distribution patterns of the aqueous outflow pathway, the iris-ciliary body, and sclera of the New Zealand Red rabbit were identified by analysis of the glycosaminoglycan moieties and by the use of zone electorphoresis. Alcian blue positive bands on cellulose acetate membranes were characterized by a determination of their electrophoretic mobility in two electrolyte systems and by a comparison to the electrophoretic mobility of standard reference glycosaminoglycans. To verify the identity of each band, specific glycosaminoglycan degrading enzymes were used to remove the glycosaminoglycans. Glycosaminoglycan samples equivalent to 5 μg uronic acid were treated with hyaluronate lyase, testicular hyaluronidase, chondroitin AC lyase and chondroitin ABC lyase. The glycosaminoglycans resistant to chondroitin ABC lyase were treated with nitrous acid and tested for solubility in cetyl pyridinium chloride. The scleral distribution pattern was hyaluronic acid, chondroitin sulfate and hybrid dermatan sulfate-chondroitin sulfate. The aqueous outflow pathway and iris-ciliary body distribution patterns were hyaluronic acid, keratan sulfate, heparan sulfate and hybrid dermatan sulfate-chondroitin sulfate.

Acidic glycosaminoglycans in liver from five patients with mucopolysaccharidosis and mucolipidosis.

Content, composition and molecular weight distribution of acidic glycosaminoglycans (GAGs) were determined in liver from five patients with genetic lysosomal storage diseases (Hurler syndrome, Hunter syndrome of severe type, Morquio syndrome, GM1-gangliosidosis type II and I-cell disease). There was a 30- to 40-fold increase in GAGs content of liver from patients with Hurler and Hunter syndromes. The GAGs accumulated in the livers consisted mainly of heparan sulfate and dermatan sulfate, and had a much lower molecular weight than those from control liver. The major GAG accumulated in liver from Morquio syndrome was keratan sulfate, which was not found in the livers from control and other patients, and chondroitin-6-sulfate was also increased. The content and the composition of liver GAGs from GM1-gangliosidosis and I-cell disease were similar to those of control liver. However, there was about a 33-fold increase in the amount of hexose on the liver GAGs from GM1-gangliosidosis. A molar ratio of sialic acid to hexosamine was 1.35 for the liver GAGs from I-cell disease and that of hexose to hexosamine was 8.47, while they were 0.46 and 2.32, respectively, for the control liver.

Sulfated glycoproteins and extracellular matrix of cultured human pulmonary endothelial cells.

Endothelial cells derived from human pulmonary arteries incorporate (3H)-glucosamine and 35SO4 into glycosaminoglycans and into the carbohydrate side chains of glycoproteins. These 3H/35S-carbohydrate chains were isolated from cells and culture medium after Pronase digestion. The 3H/35S-glycosaminoglycans were separated from the 3H/35S glycopeptides by chromatography on Sephadex G-50. The distribution of cellular glycosaminoglycans and glycopeptides indicated that 30-60% of the cellular 35S-glycopeptides may be associated with the matrix components that are synthesized by the cell and attached to a plastic substratum. Human pulmonary arterial endothelial cells were grown on collagen or on a matrix derived from vascular smooth muscle cells in order to investigate how smooth muscle cell extracellular matrix components may regulate the synthesis of endothelial cell glycoconjugates. Endothelial cells grown on plastic release various proportions of the glycoconjugates they synthesize into the culture medium. However, these same cells, when grown on substratum composed of extracellular matrix materials, synthesized altered proportions of cell-associated glycosaminoglycans and reduced the levels of total glycosaminoglycans they released into the culture medium. Thus the growth of endothelial cells on a matrix of smooth muscle cell components indicates that the glycosaminoglycan materials released into the culture medium by cells grown on a plastic substratum may not be an accurate reflection of the levels or composition of extracellular matrix materials made by endothelial cells in vivo.

Molecular size distribution of proteoglycan subunits and glycosaminoglycans synthesized by chondrocytes under conditions of reduced proteoglycan synthesis

The rate of proteoglycan synthesis by chondrocytes in vitro was depressed by either omitting l-glutamine from the incubation medium or by addition of proteoglycan subunit to the medium. The molecular size distribution on Sepharose 2B of the proteoglycan subunits synthesized by the chondrocytes under these conditions of reduced proteoglycan synthesis was found to be the same as those synthesized by the control cells. Likewise, the molecular size distribution on Sepharose 6B CL of the glycosaminoglycan chains synthesized by the depressed cells was found to be similar to that observed in untreated chondrocytes. This work demonstrates that, under conditions of reduced proteoglycan synthesis, fewer proteoglycan subunits are synthesized by chondrocytes and that the molecular size distribution of these macromolecules is similar to those synthesized by untreated cells.

Distribution of glycosaminoglycans across the normal and the scoliotic disc.

This paper reports on our investigations of the glycosaminoglycans (GAGs) (hyaluronic acid, chondroitin-6-sulfate, and keratan sulfate) of 13 adjacent zones of normal and scoliotic discs obtained at necropsy. The isolation and separation of these GAGs on the microgram scale was achieved using the different solubilities of the calcium and cetyl pyridinium chloride complexes. In normal discs the distribution of these GAGs is symmetric about the nucleus pulposus where their concentration is highest, and lowest in the outer annulus regions. A shift in this distribution profile was observed for the GAGs of the scoliotic discs, particularly at the apex of the curve. On the concave side of this disc the chondroitin-6-sulfate was elevated relative to discs at a lower level. In those annulus zones on the convex side of the curve, the keratan sulfate and hyaluronic acid levels were elevated and chondroitin sulfate depressed. These results suggest the presence in these respective regions of at least two different species of proteoglycans accompanied by elevated hyaluronate levels.

Differential histochemical staining of glycosaminoglycans in the matrix of osteoarthritic cartilage.

The distribution of chondroitin sulphate and keratan sulphate in osteoarthritic cartilage is analysed in a histochemical study, using Safranin O, Alcian Blue‐CEC and Toluidin Blue O at different pH values. The material consisted of six femoral heads obtained during replacement surgery from patients with osteoarthritis and six control femoral heads from patients who were operated on because of either fracture or tumour. The heterogeneous distribution of glycosaminoglycans in the matrix of cartilage from the osteoarthritic and control patients is described. In addition to the depletion of glycosaminoglycans, which first was seen at the surface, osteoarthritic cartilage was characterized by increasing territorial staining of chondroitin sulphate, especially around the cell clusters. This was a constant finding, even in cartilage with only slight osteoarthritic changes. In the control patients, this phenomenon was found only in the weightbearing area in very old cartilage, or in cartilage with morphological signs of osteoarthritis. Finally, the cartilage of osteophytes contained practically only chondroitin sulphate. These histochemical findings are in accordance with the increase in the rate of 35SO4 incorporation despite the loss of glycosaminoglycans in osteoarthritis, as described by others, and they reflect probably an accelerated synthesis of glycosaminoglycans.

Variations in the distribution of glycosaminoglycans in the uterine cervix of the pregnant woman

A study was made of the modifications of glycosaminoglycans in the uterine cervix and the relationship to gestation. These substances are essential constituents of connective tissue, and a modification of their concentration could affect the physical and chemical characteristics of the cervix. Glycosaminoglycans were extracted from cervical biopsies obtained from pregnant and non-pregnant women. This study showed dermatan sulfate to be quantitatively the most important glycosaminoglycan in the cervix of both the groups studied, and that a significant decrease in the concentration of both dermatan sulfate and chondroitin sulfates occurred in the biopsies obtained just after delivery. This was related to a decrease of collagen in the cervix at the end of gestation, as the proteoglycans containing dermatan sulfate are principally associated with collagen.

Determination of molecular-weight distribution of aorta glycosaminoglycans by automated gel filtration

This report describes an automated gel filtration procedure for estimation of molecular weights of glycosaminoglycans using Technicon sugar analyzer. A Sepharose CL-6B column was used for filtration. The method has been applied to determining the distribution of molecular weights of glycosaminoglycans from human aorta. The glycosaminoglycans were fractionated on a Dowex 1 (Cl-) column prior to gel filtration. On gel filtration, hyaluronic acid resolved into two components with estimated molecular weights of 38,000 and 8000. A molecular weight of 22,000 was estimated for heparan sulfate. Chondroitin 6-sulfate and dermatan sulfate eluted from the column in one peak, suggesting these two glycosaminoglycans have a similar molecular weight, 18,000. The procedure was found suitable for studying the gel filtration behavior of proteoglycans from bovine aorta and lung.