Total Proteoglycan Research Articles

Regulation of the atherogenic properties of vascular smooth muscle proteoglycans by oral anti-hyperglycemic agents

The present study aimed to investigate the actions of several classes of oral hypoglycemic agents [e.g., sulfonylureas (SUs), biguanides (BGs) and thiazolidinediones (TZDs)] in an in vitro model of lipid binding based on the “response to retention” hypothesis of atherogenesis. The incorporation of [ 35S]-SO 4 into proteoglycans synthesized by human vascular smooth muscle cells (VSMCs) was assessed by cetylpyridinium chloride (CPC) precipitation method, proteoglycan electrophoretic mobility was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and binding to low-density lipoprotein (LDL) was assessed by gel mobility shift assay (GMSA). The SUs evaluated showed no effect on [ 35S]-SO 4 incorporation into proteoglycans. Only one BG, phenformin, caused a concentration-related inhibition of proteoglycan synthesis under basal conditions and in the presence of transforming growth factor-β1 (TGF-β1), caused by an inhibition of proteoglycan core protein synthesis secondary to a reduction in total protein synthesis. However, neither metformin nor phenformin (30–300 μmol/l) had any effect on the electrophoretic mobility of proteoglycans. The TZDs—troglitazone (TRO), rosiglitazone (ROS), and pioglitazone (PIO) (10, 30, and 30 μmol/l, respectively)—inhibited proteoglycan biosynthesis and stimulated total proteoglycan core protein synthesis, while TRO alone inhibited overall protein synthesis. All three TZDs moderately reduced the electrophoretic mobility of synthesized proteoglycans assessed by SDS-PAGE, reduced the sizes of cleaved glycosaminoglycan (GAG) chains assessed by size exclusion chromatography, and significantly reduced binding to LDL. The data indicate that TZDs show anti-atherogenic actions through the modification of proteoglycan structure, leading to a possible reduction in lipid retention in the vessel wall.

Decorin synthesized by arterial smooth muscle cells is retained in fibrin gels and modulates fibrin contraction

Fibrin serves as a provisional extracellular matrix (ECM) for arterial smooth muscle cells (ASMC) after vascular injury, yet little is known about the effect of fibrin on ECM remodeling by these cells. To address this question, monkey ASMC were grown on fibrin gels and tissue culture (TC) plastic, and proteoglycan synthesis and accumulation were assessed by radiolabeling. Initial rates of (35)S-sulfate incorporation into proteoglycans were identical for both groups, but increased proteoglycan accumulation was observed in cultures grown for 48 h on fibrin. This increased accumulation on fibrin was due to reduced proteoglycan turnover and retention within the fibrin gel. Decorin and biglycan constituted 40 and 14% of the total proteoglycan in the fibrin gels, whereas their combined contribution was only 12% in control matrices. To explore whether the retention of decorin in fibrin had any influence on the properties of the fibrin gel, ASMC-mediated fibrin contraction assays were performed. Both de novo synthesis of decorin as well as decorin added during polymerization inhibited the ability of the cells to contract fibrin. In contrast, decorin added exogenously to mature fibrin matrices had no effect on fibrin gel contraction. This study illustrates that decorin derived from ASMC selectively accumulates in fibrin and modifies fibrin architecture and mechanical properties. Such an accumulation may influence wound healing and the thrombotic properties of this provisional pro-atherosclerotic ECM.

Changes in perlecan during chondrocyte differentiation in the fetal bovine rib growth plate

Perlecan is a heparan sulfate proteoglycan present in the growth plate and essential for endochondral ossification. We evaluated the synthesis and structure of perlecan in the different zones of the growth plate. The growth plates from fetal bovine ribs were isolated and sequentially sliced into 1-mm sections containing the hypertrophic zone, lower proliferative zone, upper proliferative zone, intermediate zone, and resting zone, respectively. The slices were then either incubated in culture medium with 35SO4 to measure total sulfated proteoglycan synthesis and perlecan synthesis, extracted for perlecan core protein analysis by Western blot, or extracted for perlecan isolation and subsequent characterization of glycosaminoglycan size and disaccharide composition. 35SO4 incorporation into perlecan was three-fourfold higher in the proliferating/hypertrophic zone than the resting zone. Western blot showed perlecan content was greatest in the lower and upper proliferating zones and that a perlecan fragment lacking portions of the N- and C-terminal domains containing heparan sulfate was also present in all zones. Purified perlecan from the hypertrophic/lower proliferative zone had larger chondroitin sulfate chains and a different composition of CS and HS disaccharides than the perlecan isolated from the resting zone. These results indicate perlecan deposition is increased and is turned over during proliferation to be replaced by a perlecan with a different sulfation pattern.

P199 DISPARATE EFFICACY OF COLLAGEN HYDROLYSATE AND GLUCOSAMINE ON THE EXTRACELLULAR MATRIX METABOLISM OF ARTICULAR CHONDROCYTES

Purpose: Glucosamine has been used in the treatment of osteoarthritis (OA) for several years. More recently, collagen fragments were shown to provide a positive effect on joint health. The objective of this study was to investigate the efficacy of a specific type of collagen hydrolysate (CH) on the biosynthesis of ECM macromolecules in comparison with glucosamine sulfate (GS) and glucosamine hydrochloride (GH) in a chondrocyte culture model. Methods: Primary porcine articular chondrocytes and human femoral head chondrocytes were cultured under reduced oxygen conditions. The culture medium was supplemented with various concentrations of CH. In parallel experiments chondrocytes were treated with either GS or GH in various concentrations up to 2.5mM. At the end of the culture period the amount of cellassociated proteoglycans (PGs) and the extent of PG synthesis were quantified by measuring 35S-sulfate incorporation and by colorimetric assay. The amount of aggrecan accumulated in the ECM was determined by Western Blotting. In addition, type II collagen biosynthesis was quantified by means of ELISA technique. The results were confirmed by immunocytochemical detection of type II collagen and by analyzing the incorporation of 14C-proline into matrix proteins. Results: Supplementation of the culture medium with CH resulted in a statistically significant (p<0.05) increase of PG synthesis. The amount of cell-associated PGs was almost doubled after CH treatment, compared with the control cells. Administration of CH was also associated with increased aggrecan expression and a statistically significant (p<0.05) 1.5-fold increase of type II collagen biosynthesis. In contrast, the administration of GS or GH had no stimulatory effect on the type II collagen biosynthesis of the chondrocytes. Moreover, although slight differences could be observed between GS and GH, supplementation of glucosamine had no significant effect on the amount of cell-associated PGs, or total PG synthesis, compared to the controls. Conclusions: These results indicate a stimulatory effect of CH on the synthesis of PG and type II collagen. In contrast, GS and GH failed to stimulate the synthesis extracellular matrix (ECM) macromolecules by chondrocytes. These data suggest that CH may help reduce degenerative changes of the ECM by stimulating anabolic processes in cartilage tissue.

Basic fibroblast growth factor inhibits the anabolic activity of insulin‐like growth factor 1 and osteogenic protein 1 in adult human articular chondrocytes

To determine the effects of basic fibroblast growth factor (bFGF) on the chondrocyte anabolic activity promoted by insulin-like growth factor 1 (IGF-1) and osteogenic protein 1 (OP-1). Human articular chondrocytes were cultured in alginate beads or as cartilage explants in serum-free medium with or without IGF-1 (100 ng/ml), OP-1 (100 ng/ml), or bFGF (0-100 ng/ml). Cell survival, proliferation, proteoglycan synthesis, and total proteoglycan accumulation were measured after 21 days of culture in alginate beads, and proteoglycan synthesis was measured in explants. Cell survival was not altered by bFGF at any dose, and chondrocyte proliferation was stimulated only at doses above 1 ng/ml. When combined with IGF-1, 1 ng/ml of bFGF stimulated proliferation to 170% of control, but when combined with IGF-1 and OP-1, proliferation increased to 373% of control. Doses of bFGF of 100 ng/ml decreased total proteoglycan levels accumulated per cell by 60% compared with control and also inhibited the ability of IGF-1 or OP-1 to increase proteoglycan production. Likewise, sulfate incorporation in response to IGF-1 and OP-1 alone or together was completely inhibited by 50 ng/ml bFGF in both alginate and explant cultures. The anabolic activity of IGF-1 and OP-1, alone and in combination, is significantly inhibited by bFGF. The results suggest that excessive release of bFGF from the cartilage matrix during injury, with loading, or in arthritis could contribute to increased proliferation and reduced anabolic activity in articular cartilage.

The effect of sonication on simulated osteoarthritis. Part II: Alleviation of osteoarthritis pathogenesis by 1 MHz ultrasound with simultaneous hyaluronate injection

In our previous study, we demonstrated the effects of ultrasound (US) on the delivery of hyaluronan (HA) into the synovium, even at molecular sizes as high as 3000 kDA. We hypothesized that a combined therapy with US and HA would have synergistic effects on alleviating the pathogenesis of osteoarthritis (OA). In the present study, we evaluated the effectiveness of sonication on the progress of induced OA in rabbits. We measured the cartilage degradation and inflammation, synovial fluid amount along with protein content and, finally, performed histologic analyses of the integrity of the cartilage and synovium. Low-intensity continuous US at 1 MHz, 400 mW/cm 2 was applied to the knees for 10 min bid. Combined treatment with US and HA most strikingly reduced total synovial fluid volume and also significantly alleviated the OA-induced accretion of total protein, proteoglycan and prostaglandin E 2 in the synovial fluid. It also attenuated the release of collagen type II and matrix metalloproteinase-3 in the OA-induced joint to normal levels. Histopathology revealed that combined HA and US treatment also reduced the severity of OA-induced structural damages in the cartilage and synovium. The effectiveness of HA with regard to the alleviation of OA pathogenic changes can be greatly enhanced by the simultaneous treatment with HA and US. (E-mail: bhmin@ajou.ac.kr)

Differentiation of Mesenchymal Stem Cells Transplanted to a Rabbit Degenerative Disc Model

An in vivo study to assess the differentiation status of mesenchymal stem cells (MSCs) transplanted to the nucleus pulposus of degenerative discs in a rabbit model. To evaluate the fate of MSCs transplanted to the nucleus pulposus of degenerative discs in a rabbit and to determine whether they are a suitable alternative for cell transplantation therapy for disc degeneration. Although MSCs have been proposed as candidate donor cells for transplantation to treat intervertebral disc degeneration, their differentiation after transplantation has not been adequately investigated. Autologous MSCs, labeled with green fluorescent protein, were transplanted into mature rabbits. Consecutive counts of transplanted MSCs in the nucleus area were performed for 48 weeks after transplantation. Differentiation of transplanted cells was determined by immunohistochemical analysis. The proteoglycan content of discs was measured quantitatively using a dimethylmethylene blue assay, and mRNA expression of Type I and II collagen, aggrecan and versican was measured semi-quantitatively using reverse transcription polymerase chain reaction. Many cells that were positive for green fluorescent protein were observed in the nucleus pulposus of cell-transplanted rabbit discs 2 weeks after transplantation. Their number increased significantly by 48 weeks. Some GFP-positive cells were positive for cell-associated matrix molecules, such as Type II collagen, keratan sulfate, chondroitin sulfate, aggrecan, and the nucleus pulposus phenotypic markers, hypoxia inducible factor 1 alpha, glutamine transporter 1, and matrix metalloproteinase 2. MSCs did not show significant expression of these molecules before transplantation. Biochemical and gene expression analyses showed significant restoration of total proteoglycan content and matrix-related genes compared with nontransplanted discs. MSCs transplanted to degenerative discs in rabbits proliferated and differentiated into cells expressing some of the major phenotypic characteristics of nucleus pulposus cells, suggesting that these MSCs may have undergone site-dependent differentiation. Further studies are needed to evaluate their functional role.

Lung Fibroblast Proteoglycan Production Induced by Serum Is Inhibited by Budesonide and Formoterol

Proteoglycans contribute to extracellular matrix remodeling in asthmatic airways. We investigated the effects of budesonide, a glucocorticoid, and formoterol, a long-acting beta2-adrenergic agonist, on serum-induced proteoglycan production by human lung fibroblasts. In 10% serum, total proteoglycan production was increased 1.5-fold (P < 0.01) compared with basal production in 0.4% serum. Budesonide (10(-8) M) reduced this increase by 44% (P < 0.01) and, whereas formoterol (10(-10)-10(-8) M) had no inhibitory effects, the drug combination abolished the increase (P < 0.01) without affecting fibroblast proliferation. This synergistic effect required functional glucocorticoid and beta-adrenergic receptors. The production of the proteoglycans decorin, biglycan, perlecan, and versican was increased 2.5- to 5-fold (P < 0.01) in 10% serum. Combination treatment with budesonide (10(-8) M) and formoterol (10(-10) M) abolished this increase to a significantly greater extent than either drug alone. In 10% serum, only versican mRNA was increased 1.4-fold (P < 0.05), whereas decorin mRNA was reduced to 39% (P < 0.01) of basal expression. These serum effects were counteracted by the drug combination, but there were no significant differences between the combination and either drug alone. Thus, the budesonide and formoterol combination seems to synergistically control serum-induced proteoglycan production, primarily at the post-transcriptional level. In conclusion, the proteoglycan upregulation characteristic of asthmatic airways may be limited by combination therapy with budesonide and formoterol.

Gingival tissue proteoglycan and chondroitin‐4‐sulphate levels in cyclosporin A‐induced gingival overgrowth and the effects of initial periodontal treatment

Cyclosporin A (CsA) is a potent immunosuppressive drug used in organ transplant patients to prevent graft rejection. CsA-induced gingival overgrowth is one of the side effects of this drug and its pathogenesis is still unclear. The present study was planned to comparatively analyse total proteoglycan (PG) and chondroitin-4-sulphate (C4S) levels in CsA-induced overgrown gingival tissue samples obtained before and after initial periodontal treatment and to compare these findings with the situation in healthy gingiva. Gingival tissue samples were obtained from nine patients with CsA-induced gingival overgrowth before and 4 weeks after initial periodontal treatment including oral hygiene instruction and scaling and also from 10 healthy control subjects. Total PG and C4S levels were determined by biochemical techniques. PG levels were analysed using modified Bitter and Muir method. C4S assay was carried out using chondroitin sulphate lyase AC and chondroitin-6 sulphate sulphohydrolase enzymes. The results were tested statistically using non-parametric tests. All clinical measurements in the CsA-induced gingival overgrowth group demonstrated significant reductions 4 weeks after initial periodontal treatment (p<0.05). There was no significant difference between the levels of baseline total PG in CsA-induced gingival overgrowth and healthy control groups (p>0.05). The gingival tissue levels of PG in CsA-induced gingival overgrowth group decreased significantly 4 weeks after treatment (p=0.043). Gingival tissue C4S levels in the overgrowth group were significantly higher than the healthy control group at baseline (p=0.000). C4S levels of the overgrowth group were significantly reduced after treatment (p=0.033), but these levels were still significantly higher than the healthy control group (p=0.000). The observed prominent increase in gingival tissue C4S levels may be interpreted as a sign of an increase in C4S synthesis in CsA-induced gingival overgrowth. Furthermore, remission of clinical inflammation by means of initial periodontal treatment had a positive effect on tissue levels of these extracellular matrix molecules.

Differential Effects of Fibronectin Fragment on Proteoglycan Metabolism by Intervertebral Disc Cells: A Comparison With Articular Chondrocytes

This in vitro study used the alginate bead culture system to probe for differences in the effects of fibronectin fragment on cell proliferation and proteoglycan metabolism by different populations of intervertebral disc cells and articular chondrocytes. To compare the effects of fibronectin fragment on cell proliferation, and proteoglycan synthesis and degradation by cells from the nucleus pulposus, the anulus fibrosus, and articular cartilage. In articular cartilage, the administration of fibronectin fragment stimulates cartilage degeneration. Fibronectin fragment levels were increased in human intervertebral discs with increased disc degeneration. Fibronectin fragment injected into the central region of the rabbit intervertebral disc induced a progressive degeneration of that disc. Bovine tails and metacarpophalangeal joints from 14- to 18-month-old animals were used. Alginate beads containing cells isolated from intervertebral discs and articular cartilage were cultured with (1-100 nmol/L) or without (control) fibronectin fragment in the presence of 10% fetal bovine serum. In these cultures, deoxyribonucleic acid and proteoglycan contents, as well as the rate of proteoglycan synthesis were determined. Proteoglycan degradation was measured in cultures with or without 10 nmol/L fibronectin fragment. In articular chondrocytes, fibronectin fragment strongly suppressed proteoglycan synthesis and stimulated proteoglycan degradation; the total proteoglycan content was diminished in a dose-dependent manner. Compared to articular chondrocytes, nucleus pulposus cells responded to fibronectin fragments in a similar, although less pronounced manner. On the other hand, anulus fibrosus cells treated with fibronectin fragment did not show any significant effects on proteoglycan degradation. A slight but statistically significant up-regulation of proteoglycan synthesis was observed at 10 nmol/L fibronectin fragment in outer anulus fibrosus cells. However, total proteoglycan content was decreased significantly at high concentrations of fibronectin fragment. Fibronectin fragment has different effects on cell proliferation, proteoglycan synthesis, degradation, and accumulation by articular chondrocytes and intervertebral disc cells. The different effects of fibronectin fragment in those different cell types suggest metabolic differences between these cells, and may further suggest differences in pathways of fibronectin fragment signaling as well as a differential need of these cells to be involved in tissue remodeling in which both anabolic and catabolic pathways might be altered.

Antisense inhibition of osteogenic protein 1 disturbs human articular cartilage integrity

To delineate the role of endogenous osteogenic protein 1 (OP-1) in human articular cartilage homeostasis via the inhibition of OP-1 gene expression by antisense oligonucleotides. Human adult normal articular cartilage was obtained from the knee and ankle joints of 34 organ donors. Chondrocytes were cultured as tissue explants or isolated cells in alginate or high-density monolayers for 48 hours in the presence of OP-1 antisense or sense oligonucleotides. The effect of OP-1 antisense inhibition was evaluated by reverse transcription-polymerase chain reaction, (35)S incorporation, dimethylmethylene blue assay, histology with Safranin O staining, and immunohistochemistry with anti-proOP-1, anti-mature OP-1, and anti-aggrecan antibodies. Antisense treatment inhibited OP-1 gene expression by a mean +/- SD of 34 +/- 12% (P < 0.01) in chondrocytes cultured in monolayers and by 77 +/- 27% (P < 0.03) in alginate beads. The inhibition of autocrine OP-1 caused a striking decrease in aggrecan gene expression, in total proteoglycan content accumulated in cartilage matrix, and in the ability of chondrocytes to newly synthesize proteoglycans. OP-1 antisense reduced aggrecan messenger RNA expression by 42 +/- 17% (P < 0.05) and proteoglycan synthesis by 48 +/- 23% (P < 0.01). Histology and immunohistochemistry revealed a dramatic decrease in Safranin O staining and reduced anti-aggrecan staining (primarily in the superficial and middle cartilage layers) with OP-1 antisense treatment. Our results suggest that OP-1 is an important endogenous cartilage factor that regulates matrix integrity and possibly needs to be induced or up-regulated to maintain normal cartilage homeostasis. These findings confirm our hypothesis that a lack of autocrine OP-1 may lead to an elevated susceptibility of chondrocytes to the catabolic processes, thus contributing/promoting cartilage degeneration.

Low density lipoprotein stimulation of human macrophage proteoglycan secretion

Lipoprotein trapping in arterial intima increases the risk for lipoprotein oxidation, foam cell formation, and inflammatory response in surrounding cells. Modified lipoproteins increase smooth muscle cell production of proteoglycans likely to retain lipoproteins in intimal extracellular matrix. We hypothesized that macrophage proteoglycan production is regulated in a similar manner, and characterized glycosaminoglycan side chains of secreted proteoglycans. Incubation with native low density lipoproteins (LDL) strongly stimulates total proteoglycan secretion in a time and concentration dependent manner. The main secretion product is small-sized (120 kDa) with unusually long galactosaminoglycan chains, predominantly chondroitin-6-O-sulfated. The effect appears specific for native LDL; oxidized LDL, very low density lipoproteins or phospholipid liposomes have only minor effects compared to control. These observations suggest that native LDL stimulate macrophages to secrete a chondroitin sulfate-rich proteoglycan moiety likely to have high capacity for vascular extracellular trapping of apolipoprotein B-containing lipoproteins.

Proteoglycans and catabolic products of proteoglycans present in ligament.

The aim of the present study was to characterize the proteoglycans and catabolic products of proteoglycans present in the tensile region of ligament and explant cultures of this tissue, and to compare these with those observed in the tensile region of tendon. Approx. 90% of the total proteoglycans in fresh ligament was decorin, as estimated by N-terminal amino acid sequence analysis. Other species that were detected were biglycan and the large proteoglycans versican (splice variants V(0) and/or V1 and/or V2) and aggrecan. Approx. 23% of decorin detected in the matrix was degraded. Intact decorin and decorin fragments similar to those observed in the matrix that retained the N-terminus were also observed in the medium of ligament cultures. Intact biglycan core protein was detected in the matrix and medium of ligament cultures, and two fragments originating from the N-terminal region of biglycan were observed in the matrix of cultured ligament. Versican and versican fragments that retained the N-terminus of versican core protein were detected in fresh matrix and medium of tendon cultures. Approx. 42% of versican present in the fresh ligament was degraded. Aggrecan catabolites appearing in the culture medium were derived from aggrecanase cleavage of the core protein. An intact link protein and a degradation product from the N-terminal region of type XII collagen were also detected in the medium of the ligament explant.

Musculoskeletal Differentiation of Cells Derived from Human Embryonic Germ Cells

Stem cells have the potential to significantly improve cell and tissue regeneration therapies, but little is understood about how to control their behavior. We investigated the potential differentiation capability of cells derived from human embryonic germ (EG) cells into musculoskeletal lineages by providing a three-dimensional environment with increased cell-cell contact and growth factors. Cells were clustered into pellets to mimic the mesenchyme condensation process during limb development. LVEC cells, an embryoid body-derived (EBD) cell culture generated from EG cells, were cultured in micromass pellets for 21 days in the presence of bone morphogenetic protein 2 (BMP2) and/or transforming growth factor beta-3 (TGFbeta3). Gene expression for cartilage-, bone-, and muscle-specific matrix proteins--including collagen types I, II, III, IX, X; aggrecan; cartilage proteoglycan link protein; cartilage oligomeric protein; chondroitin sulfate-4-S; and myf5--was upregulated in the pellets treated with TGFbeta3, while mRNAs for neurofilament heavy (NFH), a neuron marker, and flk-1, a hematopoietic marker, decreased. Total collagen and proteoglycan production exhibited a time-dependent increase in the pellets treated with TGFbeta3, further confirming the expression of characteristic musculoskeletal markers. Furthermore, our results indicate the ability to select or differentiate stem cells toward a musculoskeletal lineage from a heterogenous EBD cell line.

Changes in nuclear composition following cyclic compression of the intervertebral disc in an in vivo rat-tail model

While in vitro studies have shown that mechanical loading can result in changes in the composition of intervertebral disc matrix, the effects of cyclic loading in vivo have not been considered. The objective of this study was to assess the effect of static and cyclic compression of different frequencies on the nuclear composition of the intervertebral disc. Thirty-six Sprague–Dawley rats were randomly divided into a control group (no pin insertion, no loading), a sham group (pins inserted in sixth and seventh caudal vertebrae, no loading), a static loading group (compression applied via pins) and cyclic loading groups (loading at 0.5, 1.5 or 2.5 Hz). Loading was applied for 1 h each day from the third to 17th day following pin insertion, and the caudal 5–6, 6–7 and 7–8 discs harvested to quantify proteoglycan content, collagen content and chondrocyte density in the nucleus pulposus. Static compression resulted in a significant reduction in total proteoglycan content as compared with the adjacent control disc, but this effect was not seen in any of the cyclic loading groups. However, comparison with the sham group appears to indicate an overall decrease in total proteoglycan content at the targeted and adjacent levels following cyclic loading. The 0.5 Hz loading group showed a significantly greater total proteoglycan content than all other compression groups, and also showed a lower total collagen content than the sham group. Results suggest that frequency dependent changes in composition occur in response to cyclic loading, but are not limited to the directly loaded disc alone. Further studies are required to verify this, but the choice of control appears to need careful consideration in all studies of this nature.

Characterisation of proteoglycans and their catabolic products in tendon and explant cultures of tendon.

Tendons are collagenous tissues made of mainly Type I collagen and it has been shown that the major proteoglycans of tendons are decorin and versican. Little is still known about the catabolism of these proteoglycans in tendon. Therefore, the aim of the study was to characterise the proteoglycans including their catabolic products present in uncultured bovine tendon and in the explant cultures of tendon. In this study, the proteoglycans were extracted from the tensile region of deep flexor tendon and isolated by ion-exchange chromatography and after deglycosylation analysed by SDS-polyacrylamide electrophoresis, Western blotting and amino-terminal amino acid sequence analysis. Based on amino acid sequence analysis, approximately 80% of the total proteoglycan core proteins in fresh tendon was decorin. Other species that were detected were biglycan and the large proteoglycans versican (splice variants V(0) and/or V(1)) and aggrecan. Approximately 35% of decorin present in the matrix showed carboxyl-terminal proteolytic processing at a number of specific sites. The analysis of small proteoglycans lost to the medium of tendon explants showed the presence of biglycan and decorin with the intact core protein as well as decorin fragments that contained the amino terminus of the core protein. In addition, two core protein peptides of decorin starting at residues K(171) and D(180) were observed in the matrix and one core protein with an amino-terminal sequence commencing at G(189) was isolated from the culture medium. The majority of the large proteoglycans present in the matrix of tendon were degraded and did not contain the G1 globular domain. Furthermore the aggrecan catabolites present in fresh tendon and lost to the medium of explants were derived from aggrecanase cleavage of the core protein at residues E(373)-A(374), E(1480)-G(1481) and E(1771)-A(1772). The analysis of versican catabolites (splice variants V(0) and/or V(1)) also showed evidence of degradation of the core protein by aggrecanase within the GAG-beta subdomain, as well as cleavage by other proteinase(s) within the GAG-alpha and GAG-beta subdomains of versican (variants V(0) and/or V(2)). Degradation products from the amino terminal region of type XII collagen were also detected in the matrix and medium of tendon explants. This work suggests a prominent role for aggrecanase enzymes in the degradation of aggrecan and to a lesser extent versican. Other unidentified proteinases are also involved in the degradation of versican and small leucine-rich proteoglycans.

Characterisation of proteoglycans and their catabolic products in tendon and explant cultures of tendon

Tendons are collagenous tissues made of mainly Type I collagen and it has been shown that the major proteoglycans of tendons are decorin and versican. Little is still known about the catabolism of these proteoglycans in tendon. Therefore, the aim of the study was to characterise the proteoglycans including their catabolic products present in uncultured bovine tendon and in the explant cultures of tendon. In this study, the proteoglycans were extracted from the tensile region of deep flexor tendon and isolated by ion-exchange chromatography and after deglycosylation analysed by SDS-polyacrylamide electrophoresis, Western blotting and amino-terminal amino acid sequence analysis. Based on amino acid sequence analysis, ∼80% of the total proteoglycan core proteins in fresh tendon was decorin. Other species that were detected were biglycan and the large proteoglycans versican (splice variants V0 and/or V1) and aggrecan. Approximately 35% of decorin present in the matrix showed carboxyl-terminal proteolytic processing at a number of specific sites. The analysis of small proteoglycans lost to the medium of tendon explants showed the presence of biglycan and decorin with the intact core protein as well as decorin fragments that contained the amino terminus of the core protein. In addition, two core protein peptides of decorin starting at residues K171 and D180 were observed in the matrix and one core protein with an amino-terminal sequence commencing at G189 was isolated from the culture medium. The majority of the large proteoglycans present in the matrix of tendon were degraded and did not contain the G1 globular domain. Furthermore the aggrecan catabolites present in fresh tendon and lost to the medium of explants were derived from aggrecanase cleavage of the core protein at residues E373–A374, E1480–G1481 and E1771–A1772. The analysis of versican catabolites (splice variants V0 and/or V1) also showed evidence of degradation of the core protein by aggrecanase within the GAG-β subdomain, as well as cleavage by other proteinase(s) within the GAG-α and GAG-β subdomains of versican (variants V0 and/or V2). Degradation products from the amino terminal region of type XII collagen were also detected in the matrix and medium of tendon explants. This work suggests a prominent role for aggrecanase enzymes in the degradation of aggrecan and to a lesser extent versican. Other unidentified proteinases are also involved in the degradation of versican and small leucine-rich proteoglycans.

The heparan sulfate-specific epitope 10E4 is NO-sensitive and partly inaccessible in glypican-1.

The monoclonal antibody 10E4, which recognizes an epitope supposed to contain N-unsubstituted glucosamine, is commonly used to trace heparan sulfate proteoglycans. It has not been fully clarified if the N-unsubstituted glucosamine is required for antibody recognition and if all heparan sulfates carry this epitope. Here we show that the epitope can contain N-unsubstituted glucosamine and that nitric oxide-generated deaminative cleavage at this residue in vivo can destroy the epitope. Studies using flow cytometry and confocal immunofluorescence microscopy of both normal and transformed cells indicated that the 10E4 epitope was partially inaccessible in the heparan sulfate chains attached to glypican-1. The 10E4 antibody recognized mainly heparan sulfate degradation products that colocalized with acidic endosomes. These sites were greatly depleted of 10E4-positive heparan sulfate on suramin inhibition of heparanase. Instead, there was increased colocalization between 10E4-positive heparan sulfate and glypican-1. When both S-nitrosylation of Gpc-1 and heparanase were inhibited, detectable 10E4 epitope colocalized entirely with glypican-1. In nitric oxide-depleted cells, there was both an increased signal from 10E4 and increased colocalization with glypican-1. In suramin-treated cells, the 10E4 epitope was destroyed by ascorbate-released nitric oxide with concomitant formation of anhydromannose-containing heparan sulfate oligosaccharides. Immunoisolation of radiolabeled 10E4-positive material from unperturbed cells yielded very little glypican-1 when compared with specifically immunoisolated glypican-1 and total proteoglycan and degradation products. The 10E4 immunoisolates were either other heparan sulfate proteoglycans or heparan sulfate degradation products.

Total proteoglycan and chondroitin-4-sulfate levels in gingiva of patients with various types of periodontitis.

The aim of the present study was to investigate the total proteoglycan (PG) and chondroitin-4-sulfate (C4S) levels in gingival tissue samples obtained from patients with aggressive periodontitis (AgP) and chronic periodontitis (CP) before therapy (baseline) and 1 month after completion of non-surgical periodontal therapy. Gingival tissue samples were obtained from 10 AgP and 10 CP patients before initiation of treatment (baseline) and 1 month after non-surgical periodontal treatment. The control group comprised 10 systemically and periodontally healthy subjects. Total PG and C4S levels were determined by biochemical techniques. PG levels were analyzed using a modified Bitter and Muir method. C4S assay was carried out using chondroitin sulphate lyase AC and chondroitin-6 sulphate sulphohydrolase enzymes. The results were tested statistically using parametric tests. The clinical periodontal parameters demonstrated significant decreases in the periodontitis groups (P<0.05) after treatment, and there was no significant difference between AgP and CP groups at baseline and after treatment (P>0.05). At baseline, total PG and C4S levels in both of the periodontitis groups were significantly lower than that of the control group (P<0.05). One month after the non-surgical periodontal treatment, total PG levels in the periodontitis groups were comparable to the control group (P>0.05), whereas C4S levels in the AgP group were significantly lower than the other study groups (P<0.05). In the CP group, total PG and C4S levels increased significantly (P = 0.001 and P = 0.006, respectively) after non-surgical periodontal treatment, but they did not increase in the AgP group (P>0.05). The significant increases observed in total proteoglycan and chondroitin-4-sulfate levels after non-surgical periodontal treatment in the CP group but not in the AgP group may suggest that healing patterns differ between the two periodontitis types in terms of PG and C4S composition of extracellular matrix.

Proteoglycans in human laryngeal cartilage. Identification of proteoglycan types in successive cartilage extracts with particular reference to aggregating proteoglycans

The content, composition and structure of proteoglycans (PGs) in adult human laryngeal cartilage (HLC) were investigated. PGs were extracted from the tissue by using two different extraction protocols. In the first protocol, PGs were extracted under dissociative conditions, 4 M guanidine HCl (GdnHCl), and in the second protocol, sequentially, with phosphate buffered saline (PBS) and solutions of increasing GdnHCl concentration (0.5, 1, 2 and 4 M). Chemical and immmunological analyses of dissociate extracts (first protocol) revealed the presence of four, at least, different types of PGs. Aggrecan was the major PG, versican, decorin and biglycan being in small amounts. Galactosaminoglycan-containing PGs (GalAGPGs) represented the vast majority of total PGs present in extracts of HLC. Differential digestion with chondroitinase ABC and AC II showed that the GalAGPGs from HLC contained a significant proportion of dermatan sulphate (DS). In addition, disaccharide analysis showed that 6-sulphated disaccharides predominated in chondroitin sulphate (CS) chains. The sequential extraction (second protocol) indicated that PBS extract contained very little amount of PGs. The 0.5, 1 and 2 M GdnHCl extracts contained 6.3%, 24.5% and 15.2% of total extracted PGs, respectively. Four molar GdnHCl extracted the larger proportion, about 53%, of total PGs. This extract contained almost only proteoglycan aggregate components i.e., G1 bearing aggrecan, hyaluronan and link protein. The characterization of the aggrecan showed that it constituted a polydisperse population of monomers with an average molecular mass of 720 kDa. The glycosaminoglycans (GAGs) present were chondroitin sulphate with a M r of 15 kDa, and keratan sulphate (KS) with a M r of 10 kDa, in proportions 84% and 16%, respectively.