Ultrastructural demonstration of cartilage acid glycosaminoglycans

Synopsis A method for the demonstration of cartilage acid glycosaminoglycans by light and electron microscopy is described. Rabbit ear cartilage was fixed in cacodylate buffered 2.5% methanol-free formaldehyde with 0.001 M Ruthenium Red andp-chloromercuribenzoate (PCMB). Dehydration was carried out in ethylene glycol followed by embedding in the water-soluble glycol methacrylate (GMA). In some experiments unfixed cartilage was rapidly dehydrated. Sections, 1 thick, and ultrathin sections from the same blocks were stained with 0.001 M Ruthenium Red. Semi-thin sections from cartilage fixed without heavy metal additives were, in addition, stained with the acidophilic fluorochrome Berberine sulphate. It was found that Ruthenium Red intensely stained the same pericellular zone that stained metachromatically with Toluidine Blue or fluoresced after staining with Berberine sulphate. Prior treatment with 0.05% cetylpyridinium chloride entirely blocked the three reactions. Previous digestion with 0.2 mg hyaluronidase/ml for 30 min at 37°C led to the abolition of the fluorescence reaction with Berberine sulphate. It is concluded that Ruthenium Red selectively stains cartilage acid glycosaminoglycans. With the electron microscope the pericellular zones were found to be built up of a three-dimensional branched meshwork of fibrils covered with a mantle of electron-dense material, presumably acid glycosaminoglycans bound to Ruthenium Red.     

Histochemistry of glycosaminoglycans in cartilage ground substance

Summary The critical-electrolyte-concentration staining method using Alcian blue (AB) was applied to etched semithin Epon-embedded sections. The distribution of various glycosaminoglycans (GAGs) was studied in hyaline, elastic, cellular and fibrous cartilage obtained from humans and rodents. The staining patterns in semithin sections were found to correspond to those obtained using paraffin-embedded material. Lectin histochemistry was performed on consecutive sections. The following peroxidase-labelled lectins were used: Ricinus communis A I, Arachis hypogaea, Ulex curopaeus A I, Triticum vulgaris, Helix pomatia, Limax flavus, and concanavalin A. The lectin-binding capacity of cartilaginous ground substance was found to be low, as was expected on account of the few free sugar residues of GAGs. Chondroitin sulphate, the most widely distributed GAG, did not exhibit lectin staining. The lectin-binding site (positive staining for all lectins tested except H. pomatia) observed corresponded to areas positive forkeratan sulphate, as shown by AB staining in preceding or following sections. The pronounced lectin binding seen in cellular structures and the inner territorial matrix regions is considered to be due to higher concentrations of oligosaccharides involved in the metabolism of GAGs.Supported by the Hochschuljubiläumsstiftung der Stadt Wien     

Age changes in the localization and distribution of glycosaminoglycans in human hyaline cartilage

Summary The localization and distribution of acid glycosaminoglycans, glycoproteins and basic proteins have been studied in human costal cartilage, trachea and primary bronchi from the fetal period through old age. Alcian blue and PAS were used for the staining of acid glycosaminoglycans and glycoprotein and/or sugar-containing compounds respectively. Bromphenol blue and/or bromsulfalein were utilized for the detection of basic proteins. In a further attempt to histochemically identify the various polyanions staining was carried out with AB containing different concentrations of electrolytes. Mild acid hydrolysis, hyaluronidase and proteolytic digestions were also employed. It appeared that the distribution, localization and concentration of the different macromolecules vary according to age, to the type of hyaline cartilage and to the different areas of the same segment. The results also suggested that in developing tissues the areas of appositional growth synthesize mainly chondroitinsulphate while in the territory and interterritory this acidic polyanion is found with keratansulphate. With advancing age the latter slowly disappears from the interterritorial matrix and is nearly exclusively found in the matrix adjacent to the chondrocytes.The susceptibility of these tissues to enzyme digestion also varied according to age and to the type of hyaline cartilage. Proteolytic and hyaluronidase treatment, which were extremely effective in the prenatal period and in young subjects, had practically no effect on mature and old ribs. In the bronchi, instead, the substrates were easily extracted even at an old age. The problems related to the different chemical state of the various macromolecules, their localization and distribution and their possible effect on senescence of hyaline cartilage are discussed.Supported by Research Grant DE-01952(03) of the National Institutes of Public Health, Bethesda, Md.     

Studies on glycosaminoglycans of regenerating rabbit ear cartilage

Cartilage regeneration in the adult rabbit ear was examined with respect to glycosaminoglycan (GAG) synthesis at various stages of the regeneration process. Increased hyaluronic acid and chondroitin sulfate synthesis was first seen 31 days after wounding, when a metachromatic cartilage matrix could be distinguished from blastemal cells. Analysis of cartilage and the overlying skin separately showed that 90% of the labeled chondroitin sulfate was found in the cartilage being regenerated. DEAE-cellulose chromatography of GAG preparations from 35-day regenerating cartilages showed hyaluronic acid and chondroitin sulfate peaks eluting in the same position as those isolated from normal cartilages. The identity of the hyaluronic acid and chondroitin sulfate peaks was confirmed by their susceptibility to Streptomyces hyaluronidase and chondroitinase ABC, respectively. Although the degree of sulfation in normal and regenerated cartilages was similar, the ratio of chondroitin 6-sulfate to chondroitin 4-sulfate was increased in regenerated cartilages. GAG preparations from unlabeled cartilages were digested with chondroitinase ABC and the disaccharide digestive products were identified and quantitiated. Normal cartilage had a $\text{Δ}\text{Di-6S}\text{Δ}\text{Di-4S}$ ratio of 0.27; the same ratio for the regenerated cartilage was 1.58.     

The tensile properties of the cartilage of human femoral condyles related to the content of collagen and glycosaminoglycans

A method is described of measuring the tensile stiffness and fracture stress of human femoral condylar cartilage in planes parallel to and at increasing depth below the articular surface. The axis of tension was either parallel or perpendicular to the predominant collagen fibre direction in the superficial zone. Specimens were analysed for their collagen and glycosaminoglycan contents and partial correlation coefficients were determined between the tensile properties and each of the chemical constituents.The correlations between the tensile properties and the collagen content of specimens oriented parallel to the collagen fibre direction was statistically significant in the superficial zone but the significance level decreased with increasing depth. In specimens which were oriented perpendicularly to the collagen fibre direction the correlations between the above variables were less significant.There was no significant correlation between the tensile properties and the glycosaminoglycans in cartilage.Visibly normal specimens from the superficial layer which were situated adjacent to visibly degenerate cartilage were weaker and less stiff than specimens situated on normal joints or remote from visibly degenerate cartilage. Such differences decreased with depth below articular surface and were greater in parallel-oriented specimens.     

The effect of scurvy on glycosaminoglycans of granulation tissue and costal cartilage

1. The effect of ascorbic acid deficiency on glycosaminoglycans of granulation tissue and cartilage of guinea pigs was investigated by determination of the changes in the glucosamine and galactosamine contents 12 days after tendonectomy. 2. In normal granulation tissue, the glucosamine and galactosamine contents rose to a peak at 5 and 10 days respectively, whereas the hydroxyproline and proline contents continued to rise throughout the 20 days after tendonectomy. 3. The galactosamine in scorbutic granulation tissue, but not in that of pair-fed controls, decreased significantly in absolute amount and relatively to glucosamine, which remained practically unchanged; the cartilage galactosamine did not decrease during the 22 days of deficiency owing to the presence of excess of preformed galactosaminoglycans, which masked the small amount of newly formed glycosaminoglycans. 4. The chemical results were confirmed by radioactivity studies in vivo of incorporation of [U-(14)C]glucose into galactosamine and glucosamine of scorbutic granulation tissue and cartilage. The incorporation of (14)C into galactosamine decreased significantly in scurvy in both tissues. 5. The results indicated in both tissues a decreased formation of galactosamine during scurvy, although an increased degradation of polymerized glycosaminoglycans could not be entirely ruled out. It is concluded that, if lack of ascorbic acid causes an impaired galactosamine formation, the most likely position for the block may be in the UDP-N-acetylglucosamine 4-epimerase reaction.     

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

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

Synthesis of aberrant glycosaminoglycans during cartilage culture in 'sulfate free' medium

Incorporation of radiolabeled sulfate into glycosaminoglycans is a widely accepted assay to measure the rate of proteoglycan synthesis. Although glycosaminoglycan synthesis is dependent on the quantity of inorganic sulfate available to proteoglycan synthesizing cells, 'sulfate free' medium is regularly used in studies regarding proteoglycan synthesis. In this study murine patellar cartilage glycosaminoglycans synthesized under 'sulfate free' conditions were compared with those synthesized at physiological sulfate concentration. Under 'sulfate free' conditions synthesis was not only decreased but low sulfated glycosaminoglycans were made that were not synthesized during incubation at physiological sulfate concentration. The use of 'sulfate free' medium should be avoided in proteoglycan synthesis studies.     

人血清脂蛋白与糖胺聚糖及人主动脉蛋白聚糖的相互作用

本文报道了在[Ca~(2+)]=30mmol/L时,人血清或人血清脂蛋白与各种糖胺聚糖(GAG)及人主动脉两种蛋白聚糖(PG)的相互作用。GAG与血清的作用能力为6—硫酸软骨素(C6—S)>肝素(Hep)>4—硫酸软骨素(C4—S)>透明质酸(HA)>硫酸皮肤素(DS)。极低密度脂蛋白(VLDL)及低密度脂蛋白(LDL)可与肝素作用形成不溶性复合物,而高密度脂蛋白(HDL)则不能。人主动脉硫酸软骨素—PG(CS—PG)、硫酸皮肤素—硫酸软骨素—PG(DS—CS—PG)与血清形成不溶性复合物的曲线类型不同,后者的类型似有利于DS—CS—PG与血清脂蛋白结合从而使之在动脉壁沉积。     

The nature of the non-ultrafilterable glycosaminoglycans of normal human urine

1. The non-ultrafilterable acidic glycosaminoglycans from pooled urine of normal men, aged about 20, were isolated and characterized. The isolation procedure included digestion with sialidase and pronase, and fractionation by stepwise elution from an ECTEOLA-cellulose column. The glycosaminoglycans in each fraction were separated from each other by preparative electrophoresis in sodium barbital buffer and in barium acetate. 2. Approximate relative amounts of the different glycosaminoglycans were: chondroitin sulphate 60%, chondroitin 2%, hyaluronic acid 4%, dermatan sulphate 1%, heparan sulphate 15% and keratan sulphate 18%. Chondroitin sulphate-dermatan sulphate hybrids seemed to occur in trace amounts. 3. Chondroitin sulphate, heparan sulphate and keratan sulphate were heterogeneous with respect to degree of sulphation. Two distinct groups of chondroitin sulphate fractions were found, with sulphate/hexosamine molar ratios of about 0.5 and 1 respectively. The sulphate/hexosamine molar ratios in the heparan sulphate fractions varied from 0.5 to 0.9; the N-sulphate/hexosamine ratio was about 0.5 in all fractions. The sulphate/hexosamine molar ratios in the keratan sulphate fractions varied from 0.2 to 0.7.     

Observations on the glycosaminoglycans of aging bronchial cartilage studied with Alcian Blue

Synopsis The changes in the distribution of acidic glycosaminoglycans in the extracellular matrix of aging human bronchial cartilage have been studied with Alcian Blue using the critical electrolyte technique described by Scott & Dorling (1965). Keratan sulphate was detected in the interterritorial matrix early in the first decade. The factors initiating the synthesis of keratan sulphate by chondrocytes are discussed and a hypothesis is proposed to explain the subsequent localization of this glycosaminoglycan in the extracellular matrix.     

Optimized extraction of glycosaminoglycans from normal and osteoarthritic cartilage for glycomics profiling

Articular cartilage is a highly specialized smooth connective tissue whose proper functioning depends on the maintenance of an extracellular matrix consisting of an integrated assembly of collagens, glycoproteins, proteoglycans (PG), and glycosaminoglycans. Isomeric chondroitin sulfate glycoforms differing in position and degree of sulfation and uronic acid epimerization play specific and distinct functional roles during development and disease onset. This work introduces a novel glycosaminoglycan extraction method for the quantification of mixtures of chondroitin sulfate oligosaccharides from intact cartilage tissue for mass spectral analysis. Glycosaminoglycans were extracted from intact cartilage samples using a combination of ethanol precipitation and enzymatic release followed by reversed-phase and strong anion exchange solid-phase extraction steps. Extracted chondroitin sulfate glycosaminoglycans were partially depolymerized using chondroitinases, labeled with 2-anthranilic acid-d(4) (2-AA) and subjected to size exclusion chromatography with online electrospray ionization mass spectrometric detection in the negative ion mode. The method presented herein enabled simultaneous determination of sulfate position and uronic acid epimerization in juvenile bovine and adult human cartilage samples. The method was applied to a series of 13 adult human cartilage explants. Standard deviation of the mean for the measurements was 1.6 on average. Coefficients of variation were approximately 4% for all compositions of 40% or greater. These results show that the new method has sufficient accuracy to allow determination of topographical distribution of glycoforms in connective tissue.     

Characterization of recombinant amino-terminal NC4 domain of human collagen IX: interaction with glycosaminoglycans and cartilage oligomeric matrix protein

The N-terminal NC4 domain of collagen IX is a globular structure projecting away from the surface of the cartilage collagen fibril. Several interactions have been suggested for this domain, reflecting its location and its characteristic high isoelectric point. In an attempt to characterize the NC4 domain in more detail, we set up a prokaryotic expression system to produce the domain. The purified 27.5-kDa product was analyzed for its glycosaminoglycan-binding potential by surface plasmon resonance and solid-state assays. The results show that the NC4 domain of collagen IX specifically binds heparin with a K(d) of 0.6 microm, and the full-length recombinant collagen IX has an even stronger interaction with heparin, with an apparent K(d) of 3.6 nm. The heparin-binding site of the NC4 domain was located in the extreme N terminus, containing a heparin-binding consensus sequence, whereas electron microscopy suggested the presence of at least three additional heparin-binding sites on full-length collagen IX. The NC4 domain was also shown to bind cartilage oligomeric matrix protein. This interaction and the association of cartilage oligomeric matrix protein with other regions of collagen IX were found to be heparin-competitive. Circular dichroism analyses of the NC4 domain indicated the presence of stabilizing disulfide bonds and a thermal denaturation point of about 80 degrees C. The pattern of disulfide bond formation within the NC4 domain was identified by tryptic peptide mass mapping of the NC4 in native and reduced states. A similar pattern was demonstrated for the NC4 domain of full-length recombinant collagen IX.     

Structural analysis of purified human tracheobronchial mucins

Light scattering has been used to investigate the structure of human tracheobronchial mucin glycoproteins (HTBM) from the sputum of cystic fibrosis patients. The specimen was extracted using 6M guanidinium hydrochloride solution and fractionated by gel exclusion chromatography on Sephacryl S-1000. The fractionated HTBM was purified by density gradient ultracentrifugation. Purity of the resulting material was confirmed by SDS polyacrylamide gel electrophoresis and uv spectroscopy. Light scattering measurements on the fractionated mucins yield weight-average molecular weights M_w, and z-average radii of gyration R_{g, z}. The native cystic fibrosis HTBM consisted of a high molecular weight fraction with M_w = 9.3 × 10⁶ daltons and a lower molecular weight fraction contanining partly degraded mucins. After reduction and carboxymethylation of the high molecular weight native fraction, the resulting material was separated into three pools with M_w values of 5.1 × 10⁶, 1.6 × 10⁶, and 400,000. The derived molecular weights for the protein cores M_p,w, and the experimental radii of gyration are found to be consistent with the M_p,w – R_g relation established previously for submaxillary, cervical, and gastric mucins. These results imply that HTBM has the same extended-coil conformation reported for other mucins and has a molecular structure consisting of subunits, linked into linear chains via covalent (disulfide) bonds.     

Analyses of glycosaminoglycans in human lung cancer

Studies were conducted on the total amount of glycosaminoglycans and glycosaminoglycan composition in adenocarcinoma tissue of human lung. The glycosaminoglycans were prepared by exhaustive proteinase digestion of adenocarcinoma tissue from human lungs and of lung tissue without pulmonary diseases taken at autopsy as a control. The glycosaminoglycan classes were characterized by biochemical, enzymatic, and electrophoretic methods. The presence of heparin, which has until now not been found in lung cancer tissue, was demonstrated on both carcinoma and control tissues. The levels of whole glycosaminoglycans were markedly increased in cancer tissue compared to the controls. The classes of glycosaminoglycans which increased in lung carcinoma tissue were predominantly chondroitin-4-sulfate and chondroitin-6-sulfate. Both hyaluronic acid and heparin were slightly increased in cancer tissue.