Collagen, proteoglycan and hyaluronidase activity in cultures from normal and scoliotic chicken fibroblasts

Connective tissue matrix components were investigated using skin fibroblasts from normal or inbred scoliotic lines of chickens. Specifically, the fibroblasts were obtained from either an isogenic line or a backcross, derived by crossing the isogenic line with a pure line of scoliotic birds. From the backcross, both affected (35-45%) and non-affected (55-65%) progeny were produced. The affected birds had spinal curves greater than 20 degrees. Several abnormalities of connective tissue were observed when cells from scoliotic chicks were grown in culture: increased collagen extractability, decreased aggregatability of proteoglycans under associative conditions and lower than normal levels of hyaluronic acid. There was also less collagen deposited in the cell layer with proportionately increased amounts of collagen secreted into the culture media by cells from scoliotic versus normal chick fibroblasts. Values for collagen matrix stability, as estimated by extractability and net deposition, were intermediate for cells from the backcrossed, but non-affected, birds. Moreover, hyaluronidase, an enzyme that degrades hyaluronic acid, was abnormally elevated in the fibroblast cultures from scoliotic chicks. It is proposed that the increase in hyaluronidase contributes to the abnormalities observed in extracellular matrix components and may be a factor in the expression of scoliosis in susceptible birds.     

Collagen proteins of human hyaline cartilage in normal states and in various bone dysplasias

Using SDS electrophoresis and subsequent densitometry, isolated collagen proteins of infantile rib and knee joint hyaline cartilage were characterized. Both the normal samples and hyaline cartilages of children with osteochondrodysplasias were shown to contain collagens type I and II as well as collagen proteins with Mr 160 (A), 150 (B), 140 (C), 120 (D), 110 (E) and 39 kD (F), whose content in normal samples varied, depending on the donor age. An analysis of normal and pathological samples revealed the following biochemical markers of intensive chondrocyte proliferation: an increased content of collagen proteins A--F and a decreased number of intramolecular cross-links of collagen type II. Conversely, the increased number of intramolecular cross-links in collagen type II and the elevation of the relative content of collagen type I in lethal forms of osteochondrodysplasias and funnel chest may testify to chondrocyte dedifferentiation. It was assumed that collagen proteins D and E correspond to proteins 1 alpha and 2 alpha, whereas proteins A, B, C and F are the products of hydrolysis by pepsin type M of collagen detected previously only in animal cartilages. Mapping of collagen type II CNBr-peptides and electron microscopic analysis of its SLS-form were carried out. The experimental results are suggestive of the involvement of collagen proteins in the pathogenesis of human osteochondridysplasias as well as of the pronounced biochemical heterogeneity of the disease.     

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.     

Collagen composition of normal and myxomatous human mitral heart valves.

The collagens were studied in 13 normal and 19 myxomatous human mitral valves. The collagens of the valve were completely solubilized by using a method consisting of guanidinium chloride extraction, limited pepsin digestions and CNBr cleavage of the residue. The normal valves contained 74% type I, 24% type III and 2% type V collagen. The type I and type III collagens had similar solubility patterns, although only type I collagen was detected in the guanidinium chloride extract. Type V collagen was only detected in the first pepsin extract. The type I and III collagens had higher contents of hydroxylysine than did the same collagens from age-matched dermis. The two-dimensional electrophoretic 'maps' of CNBr-cleavage peptides showed low recoveries of the C-terminal alpha 1(I) CB6 and alpha 1(III) CB9 peptides, which are involved in forming intermolecular cross-linkages. Most of the reducible cross-linkages were present in large-Mr peptide complexes, and these complexes were shown by labelling with 125I to include the tyrosine-containing alpha 1(I) CB6 peptide. The myxomatous valves contained 67% type I, 31% type III and 2% type V collagens. There was a significant increase in the concentration of each type of collagen, which consisted of a 9% increase of type I collagen, a 53% increase of type III collagen and a 25% increase of type V collagen. The contents of hydroxylysine in type I and III collagens and the electrophoretic 'maps' of the CNBr-cleavage peptides involved in cross-linkages did not differ significantly from the results obtained from the normal valves. The biochemical findings suggest that there is an increased production of collagen, in particular type III collagen, and glycosaminoglycan as well as a proliferation of cells as part of a repair process in the myxomatous valves.     

Articular cartilage and changes in Arthritis: Collagen of articular cartilage

The extracellular framework and two-thirds of the dry mass of adult articular cartilage are polymeric collagen. Type II collagen is the principal molecular component in mammals, but collagens III, VI, IX, X, XI, XII and XIV all contribute to the mature matrix. In developing cartilage, the core fibrillar network is a cross-linked copolymer of collagens II, IX and XI. The functions of collagens IX and XI in this heteropolymer are not yet fully defined but, evidently, they are critically important since mutations in COLIX and COLXI genes result in chondrodysplasia phenotypes that feature precocious osteoarthritis. Collagens XII and XIV are thought also to be bound to fibril surfaces but not covalently attached. Collagen VI polymerizes into its own type of filamentous network that has multiple adhesion domains for cells and other matrix components. Collagen X is normally restricted to the thin layer of calcified cartilage that interfaces articular cartilage with bone.     

Articular cartilage and changes in Arthritis: Collagen of articular cartilage

The extracellular framework and two-thirds of the dry mass of adult articular cartilage are polymeric collagen. Type II collagen is the principal molecular component in mammals, but collagens III, VI, IX, X, XI, XII and XIV all contribute to the mature matrix. In developing cartilage, the core fibrillar network is a cross-linked copolymer of collagens II, IX and XI. The functions of collagens IX and XI in this heteropolymer are not yet fully defined but, evidently, they are critically important since mutations in COLIX and COLXI genes result in chondrodysplasia phenotypes that feature precocious osteoarthritis. Collagens XII and XIV are thought also to be bound to fibril surfaces but not covalently attached. Collagen VI polymerizes into its own type of filamentous network that has multiple adhesion domains for cells and other matrix components. Collagen X is normally restricted to the thin layer of calcified cartilage that interfaces articular cartilage with bone.     

Isolation and characterization of matrix proteoglycans from human nasal cartilage: Compositional and structural comparison between normal and scoliotic tissues

The content, types and the fine structures of proteoglycans (PGs) present in human normal nasal cartilage (HNNC) were investigated and compared with those in human scoliotic nasal cartilage (HSNC). Three PG types were identified in both HNNC and HSNC; the large-sized high buoyant density aggrecan, which is the predominant PG population, and the small-sized low buoyant density biglycan and decorin. HSNC contained a significantly higher amount of keratan sulfate (KS)-rich aggrecan (30%) of smaller hydrodynamic size as compared to HNNC. The average molecular sizes (M_rs) of aggecan-derived chondroitin sulfate (CS) chains in both HNNC and HSNC were identical (18 kDa), but they significantly differ in disaccharide composition, since CS isolated from HSNC contained higher proportions of 6-sulfated disaccharides as compared to those from HNNC. Scoliotic tissue contained also higher amounts (67%) of the small PGs, biglycan and decorin as compared to HNNC. It is worth noticing that both normal and scoliotic human nasal cartilage contain also non-glycanated forms of decorin and biglycan. Dermatan sulfate (DS) was the predominant glycosaminoglycan (GAG) present on biglycan and decorin in both tissues. The small PGs-derived CS chains in both normal and scoliotic cartilage had the same M_r (20 kDa), whereas DS chains from scoliotic cartilage were of greater M_r (32 kDa) than those from normal cartilage (24 kDa). Furthermore, scoliotic tissue-derived DS chains contained higher amounts of iduronate (20%) as compared to those of normal cartilage (12%). Disaccharide analysis of small PGs showed that both HNNC and HSNC were rich in 4-sulfated disaccharides and in each case, the small size PGs contained a considerably higher proportion of 4-sulfated disaccharides than the aggrecan of the same tissue. The higher amounts of matrix PGs identified in scoliotic tissue as well as the differences in fine chemical composition of their GAG chains may reflect the modified architecture and functional failure of scoliotic tissue.     

Isolation and characterization of matrix proteoglycans from human nasal cartilage. Compositional and structural comparison between normal and scoliotic tissues

The content, types and the fine structures of proteoglycans (PGs) present in human normal nasal cartilage (HNNC) were investigated and compared with those in human scoliotic nasal cartilage (HSNC). Three PG types were identified in both HNNC and HSNC; the large-sized high buoyant density aggrecan, which is the predominant PG population, and the small-sized low buoyant density biglycan and decorin. HSNC contained a significantly higher amount of keratan sulfate (KS)-rich aggrecan (30%) of smaller hydrodynamic size as compared to HNNC. The average molecular sizes (M(r)s) of aggecan-derived chondroitin sulfate (CS) chains in both HNNC and HSNC were identical (18 kDa), but they significantly differ in disaccharide composition, since CS isolated from HSNC contained higher proportions of 6-sulfated disaccharides as compared to those from HNNC. Scoliotic tissue contained also higher amounts (67%) of the small PGs, biglycan and decorin as compared to HNNC. It is worth noticing that both normal and scoliotic human nasal cartilage contain also non-glycanated forms of decorin and biglycan. Dermatan sulfate (DS) was the predominant glycosaminoglycan (GAG) present on biglycan and decorin in both tissues. The small PGs-derived CS chains in both normal and scoliotic cartilage had the same M(r) (20 kDa), whereas DS chains from scoliotic cartilage were of greater M(r) (32 kDa) than those from normal cartilage (24 kDa). Furthermore, scoliotic tissue-derived DS chains contained higher amounts of iduronate (20%) as compared to those of normal cartilage (12%). Disaccharide analysis of small PGs showed that both HNNC and HSNC were rich in 4-sulfated disaccharides and in each case, the small size PGs contained a considerably higher proportion of 4-sulfated disaccharides than the aggrecan of the same tissue. The higher amounts of matrix PGs identified in scoliotic tissue as well as the differences in fine chemical composition of their GAG chains may reflect the modified architecture and functional failure of scoliotic tissue.     

Comparative evaluation of the glycosaminoglycan composition in plasma membranes of normal and transformed mouse liver cells

Using electrophoresis in agarose gel, a comparative study of composition of membrane-bound glycosaminoglycans (GAG) from normal mouse liver cells and from o-aminoazatoluene-induced mouse hepatoma cells was carried out. Differences in the composition and localization of GAG were revealed. The plasma membrane fraction of hepatoma cells contained no GAG; the bulk of GAG (approximately 98%) was localized in the plasma membrane free fraction. Within this fraction GAG contained no heparan sulfates with a high electrophoretic mobility that were detected in plasma membranes of normal liver cells. The possible involvement of proteoglycans bound to cell surface in transmembrane signal transfer is discussed.     

Regional distribution of water and glycosaminoglycan in immature articular cartilage

The distribution of water and glycosaminoglucan in different functional regions of bovine immature articular cartilage were studied. There was always more water in each articulating than in the corresponding growin zone, but there was less water in both zones in the areas of maximum contact. There was more hyaluronate and much more keratan sulphate in the articulating areas of maximum contact than in the minimum contact areas. In the growing zone the distribution of these two glycosaminoglycans did not vary as significantly but there was slightly more keratan sulphate in the area of maximum contact. Regional variations in chondroitin sulphate were also present although not as striking as those of keratan sulphate. The results suggest that some keratan sulphate may be synthesized as reaction to load.     

Effects of medium composition and metabolic inhibitors on glycosaminoglycan synthesis in chick embryo cartilage and its stimulation by serum and triiodothyronine

Incorporation of inorganic sulfate into glycosaminoglycans of chick embryo sternum is stimulated by serum and triiodothyronine. Variations in the amino acid content of the medium, and in particular in the concentration of glutamine, changed the incorporation in control and stimulated sterna to the same degree. Omission of Na⁺ from the medium greatly reduced incorporation in both control and stimulated sterna; incorporation, and its stimulation by triiodothyronine, was restored by raising the concentration of Na⁺. Ouabain and valinomycin inhibited incorporation more than 90%, and triiodothyronine did not stimulate under these conditions. Puromycin and cycloheximide also inhibited incorporation almost completely, and abolished the stimulation by triiodothyronine and serum. Addition of p-nitrophenyl-β-xyloside, in the presence of puromycin or cycloheximide, restored sulfation to a level of 5–10% of the control value; however, this level of incorporation was not increased by addition of serum or triiodothyronine.Actinomycin D, colchicine and vinblastine inhibited incorporation by 40% or less at the highest concentrations tested; however, these three agents completely abolished the ability of triiodothyronine to stimulate incorporation. Lumicolchicine and cytochalasin B decreased incorporation in controls slightly, but did not affect the stimulation by serum or triiodothyronine.The results indicate that thyroid hormones stimulate glycosaminoglycan synthesis only under conditions which support efficient synthesis in control incubations, and suggest that microtubule formation may be essential to the mode of action of thyroid hormones in this system.     

Altered glycosaminoglycan composition in reactive and neoplastic human liver

We have investigated the glycosaminoglycan composition of normal human liver, focal nodular hyperplasia, hepatic adenoma, and hepatocellular carcinoma. Uronic acid increased about 4 fold in the benign and reactive lesions, and greater than 7 fold in the carcinoma. Whereas in focal nodular hyperplasia and adenoma dermatan sulfate was the predominant glycosaminoglycan, in hepatocellular carcinoma chondroitin sulfate was the predominant species; it increased 24 fold over normal liver and 3-5 fold over all the other tissues. HPLC analysis of chondroitinase ABC or AC digests showed a 58 fold increase in Delta-Di-OS disaccharides in hepatocellular carcinoma, indicating significant undersulfation of chondroitin sulfate. Surprisingly, the normal-appearing liver surrounding the carcinoma showed glycosaminoglycan changes similar to adenoma and nodular hyperplasia. These results thus indicate that specific glycosaminoglycan changes occur in hepatocellular carcinoma, and suggest for the first time that proteoglycan metabolism is also altered in the non-cirrhotic, hepatic parenchyma adjacent to liver carcinoma.     

Effects of medium composition and metabolic inhibitors on glycosaminoglycan synthesis in chick embryo cartilage and its stimulation by serum and triiodothyronine.

Incorporation of inorganic sulfate into glycosaminoglycans of chick embryo sternum is stimulated by serum and triiodothyronine. Variations in the amino acid content of the medium, and in particular in the concentration of glutamine, changed the incorportion in control and stimulated sterna to the same degree. Omission of Na+ from the medium greatly reduced incorporation in both control and stimulated sterna; incorporation, and its stimulation by triiodothyronine, was restored by raising the concentration of Na+. Ouabain and valinomycin inhibited incorporation more than 90%, and triiodothyronine did not stimulate under these conditions. Puromycin and cycloheximide also inhibited incorporation almost completely, and abolished the stimulation by triiodothyronine and serum. Addition of p-nitrophenyl-beta-xyloside, in the presence of of puromycin ir cycloheximide, restored sulfation to a level of 5-10% of the control value; however, this level of incorporation was not increased by addition of serum or triiodothyronine. Actinomycin D, colchicine and vinblastine inhibited incorporation by 40% or less at the highest concentrations tested; however, these three agents completely abolished the ability of triiodothyronine to stimulate incorporation. Lumicolchicine and cytochalasin B decreased incorporation in controls slightly but did not affect the stimulation by serum or triiodothyronine. The results indicate that thyroid hormones stimulate glycosaminoglycan synthesis only under conditions which support efficient synthesis in control incubations, and suggest that microtubule formation may be essential to the mode of action of thyroid hormones in this system.     

Characterisation of collagen from normal and scoliotic human spinal ligament

Acid0soluble and pepsin-soluble collagens have been isolated from spinal ligaments of normal and scoliotic individuals. Polyacrylamide gel electrophoresis of native and cyanogen bromide-treated collagens, and amino acid analysis, showed that the ligament collagen is almost all of the Type I variety with only trace amounts of Type III present. There was no evidence for abnormal ratios of collagen α-chains, or underhydroxylation of proline and lysine in the scoliotic ligament. These results indicate that collagen biochemistry is normal with respect to type, post-translational modification and cross-linking in spinal ligaments of patients with idiopathic scoliosis. Elastin and proteoglycan were only minor components of the ligaments. The nature of the non-collagenous part of the ligament is unknown, although it contains some proteins with a hydrophobic nature.     

Collagen from human joint cartilage in deforming arthritis

Type II collagen of human cartilage has been comparatively studied in norm and at deforming arthrosis. Collagen preparations are characterized by aminoacid composition and electrophoretic mobility in polyacrylamide gel. It is supposed that at deforming arthrosis the degenerative-dystrophic processes in the articular cartilage of man are caused by appearance of procollagen.     

The effect of anti-inflammatory drugs on glycosaminoglycan sulphation in pig cartilage

Indomethacin, aspirin and hydrocortisone have been shown to inhibit glycosaminoglycan sulphation in pig costal cartilage discs. The doses at which inhibition was observed varied from pig to pig from 0.1 – 50 ug/ml for indomethacin, 40 – 150 ug/ml for aspirin and 0.5 – 1 mg/ml for/hydrocortisone, doses which are/achieved clinically in the treatment of joint diseases. In a minority of experiments a significant stimulation of glycosaminoglycan sulphation was observed at lower doses of indomethacin and hydrocortisone but not aspirin. The results suggest that in rheumatoid arthritis but more especially osteoarthritis, treatment with these drugs should aim at finding the lowest doses which have a therapeutic effect. As importantly the clinician should always consider using lower doses just as much as higher doses when the first dose regimen is unsatisfactory.