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ims: Keratan sulphate (KS) is the predominant glycosaminoglycan (GAG) present in the corneal stroma where it is thought to regulate collagen fibril diameter. In this study we investigated the distribution of KS in normal and keratoconic corneas. Methods: Four normal, one mild, and four severe keratoconic corneas were used for the study. Distribution of keratan sulphate proteoglycans (KS-PG) was investigated using a primary monoclonal antibody (5-D-4) that recognizes disulphated disaccharides in the poly- N-acetyllactosamine repeats of KS. The immuno-reactivity of 5-D-4 was analyzed by immunohistochemistry and immuno-electron microscopy. Results: Immuno-histochemistry showed diffuse 5-D-4 staining in keratoconic cornea compared to the punctuate staining in normal corneas. In the single cornea with mild keratoconus, immunogold microscopy revealed a very high density of KS-PG staining, especially in the posterior stroma, compared to severe keratoconic and normal cornea. The amount of KS-PG in the stroma in severe keratoconus was slightly less compared to the normal cornea. In the mild keratoconic cornea, a higher quantity of KS-PG was present around the keratocytes. In severe keratoconic corneas, a higher quantity of KS-PG was present within the keratocytes compared to normal cornea. Conclusions: The finding of an altered expression of KS in our keratoconic corneas, in particular the strong expression of KS in keratocytes, is in keeping with reports of an altered expression of proteoglycan metabolism in keratoconus. KS-PG plays an important role in stromal collagen fibril assembly and a dysregulation of KS-PG synthesis or catabolism could explain changes in collagen fibril spacing and diameter, which we have reported elsewhere. [ABSTRACT FROM AUTHOR]

Tendons are dense, fibrous connective tissues that are responsible for transmitting mechanical forces from skeletal muscle to bone. From a clinical perspective, tendinopathy (defined as a syndrome of tendon pain, tenderness and swelling that affects function) is very common, both within the sporting arena and in the workplace. Importantly, proteoglycans are essential components of the tendon extracellular matrix (ECM) and changes in their expression and metabolism/turnover have been associated with tendinopathy. Within tendons, the small leucine-rich proteoglycans (SLPRs), decorin, fibromodulin, lumican and keratocan predominate within tensional regions, while in tendon fibrocartilage, increased concentrations of proteoglycans common to the articular cartilage phenotype are present, including aggrecan, biglycan and proteoglycan 4. However, the rate of proteoglycan turnover within tendon is markedly higher than that of cartilage, mediated via the “aggrecanases,” which are constitutively active in the tendon matrix. Data suggest that this increased proteoglycan turnover is likely to be required to maintain normal tendon homeostasis, with perturbations in proteoglycan metabolism contributing to tissue dysfunction. Thus, future studies aimed at furthering our fundamental knowledge of tendon proteoglycan metabolism in health and disease are important in the development of improved treatments for tendon disorders. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies that specifically recognize epitopes on keratan sulfate glycosaminoglycans were used in this study to identify carbohydrate epitopes associated with many, but not all, types of epithelial cells. Immunoreactive cells included: keratinocytes, sebaceous gland cells, eccrine sweat gland duct cells, salivary gland excretory duct cells, colon adenocarcinoma cells, embryonic chick lung epithelial cells, embryonic chick mesonephric and metanephric kidney epithelial cells, and selected embryonic chick neural tube cells. Depending upon the type of epithelium, epitopes were located either within the cytoplasm or were located on cell surfaces. These epitopes were shared by cells from both human and chick tissues, indicating the absence of species specificity. Not all anti-keratan sulfate antibodies were equally effective in identifying epithelial-associated epitopes. One of the seven antibodies employed in this study failed to detect epitopes in almost all epithelial tissues studied. Of the remaining six antibodies, three were more effective than the others in recognizing epithelial-associated epitopes. These data indicate that carbohydrates that are typically associated with extracellular matrix can also be associated with epithelial cells, but in a form that is not necessarily related to extracellular matrix. These antibodies should prove to be useful in studies of the development of epithelial cells and tissues. [ABSTRACT FROM AUTHOR]

Presents several abstracts related to matrix molecules presented during the British Connective Tissue Society Meeting at the University of York in York, England. 'An Investigation of Hyaluroan Synthesis and Hyaluroan Binding Proteins During Rat Palatogenesis,' by S. Thomas and R.C. Hall.

The dynamics of changes in the cellularity and extracellular matrix composition of dental pulp varies considerably during tooth development and maturation. In this paper, we studied matrix proteoglycans where we hypothesized that they played important roles in structural, spatial, and transport aspects of pulpal development and maintenance. The pulpal tissue was collected from partially erupted bovine incisors, pulverized, and then extracted with 6 M guanidine-HC1. The extract was subjected to anion column chromatography (DEAE-8HR), and the fractions collected were screened by dot-blot immunoassay by means of monoclonal antibodies generated against 4- and 6-sulfated chondroitin sulfate isomers, and keratan sulfate, 2-B-6, 3-B-3, and 5-D-4, respectively. The chondroitin-6-sulfate was the major glycosaminoglycan species and occurred as a large-molecular-weight proteoglycan (> 500 kDa). After further purification, it was subjected to agarose/acrylamide composite gel electrophoresis, and it migrated as a single band stained with Stains-All. The band was immunopositive against antibody 3-B-3 by Western blot analysis. The partial amino acid sequence analyses of the core protein clearly indicated this molecule to be versican. The presence of link protein was also confirmed by Western blot analysis with an anti-link protein monoclonal antibody, 8-A-4. Furthermore, immunohistochemical study indicated that the distributions of versican and link protein coincide in the dental pulp and are enriched in the peripheral area of the tissue just beneath the odontoblast layer. Since the dental pulp contains hyaluronan, versican may bind to hyaluronan via its hyaluronan-binding domain, where this association is stabilized by link protein. This complex, then, could form large hydrated proteoglycan aggregates that fill the extracellular space, support odontoblasts, and/or facilitate the transport function of metabolites and nutrients within the tissue. [ABSTRACT FROM AUTHOR]

A calcium ion precipitable, trypsin-generated proteoglycan fragment has been isolated from the demineralized, EDTA-insoluble matrices of bone. The demineralized matrix was completely digested with trypsin, increasing concentrations of CaCl2 were added to the supernatant, and the resulting precipitates were analyzed. The amount of precipitate gradually increased with higher concentrations of calcium and was reversibly solubilized by EDTA. After molecular sieve and anion exchange chromatography, a proteoglycan-containing peak was obtained. Immunochemical analysis showed that this peak contained chondroitin 4-sulfate and possibly keratan sulfate. Amino acid analysis showed that this proteoglycan contained high amounts of aspartic acid/asparagine (Asx), serine (Ser), glutamic acid/glutamine (Glx), proline (Pro), and glycine (Gly); however, it contained little leucine (Leu) which suggests that it is not a member of the leucine-rich small proteoglycan family. In addition, significant amounts of phosphoserine (P-Ser) and hydroxyproline (Hyp) were identified in hydrolysates of this fraction. A single band (M(r) 59 kDa) was obtained on SDS-PAGE that stained with Stains-all but not with Coomassie Brilliant Blue R-250. If bone powder was trypsinized prior to demineralization, this proteoglycan-containing fraction was not liberated. Collectively, these results indicate that a proteoglycan occurs in the demineralized matrix that is precipitated with CaCl2 and is closely associated with both mineral and collagen matrices. Such a molecule might facilitate the structural network for the induction of mineralization in bone. [ABSTRACT FROM AUTHOR]

Examines the in situ regulation of cartilage adamts/aggrecanase expression. Role of articular chondrocytes during the turnover of cartilage extracellular matrix molecules; Causes of cartilage matrix degradation; Agents involved in the upregulation of aggrecanase activity.

Compares the metabolism of proteoglycans between young and mature tendons. Structural characteristics of tendons; Role of matrix metalloproteinases in aggrecan interglobular domain catabolism; Accumulation of aggrecan metabolites in tissues of deep digital flexor tendons.

Evaluates the in situ regulation of cartilage adamts/aggrecanase expression. Role of articular chondrocytes during the turnover of cartilage extracellular matrix molecules; Causes of cartilage matrix degradation; Agents involved in the upregulation of aggrecanase activity.

Examines biochemical characteristics of cartilage superficial zone protein using monoclonal antibody affinity columns. Similarities between superficial zone proteoglycans and megakaryocyte stimulating factors; Accumulation of proteoglycans at the articular cartilage interface.

Examines biochemical characteristics of cartilage superficial zone proteins using monoclonal antibody affinity column. Similarities between superficial zone proteoglycan and megakaryocyte stimulating factors; Accumulation of preoteoglycans at the articular cartilage interface.

Examines the effect of n-3 fatty acid supplementation to chondrocytes on the expression of aggrecanase activity, cyclooxygenase-2 and autocrine cytokine production. Role of polyunsaturated fatty acid in cell metabolism within the joint; Conditions important in the synthesis of inflammatory cytokines.

The development of ‘discogenic’ low back pain has been associated with increased nerve growth into degenerated intervertebral discs, particularly in neovascularized areas. A marked loss of proteoglycans, particularly from the disc's inner regions, is also a marked feature of the degenerative process. As proteoglycans from other tissues can have inhibitory effects on nerve growth, we have hypothesized that the alterations in proteoglycan content seen in disc degeneration may affect the growth of nerves and blood vessels into the disc. To test our hypothesis, we have established cell culture assays to determine the effects of human intervertebral disc aggrecan, which forms the major proteoglyan found in the disc, on neuronal and endothelial cell growth. Aggrecan (A1D1 preparations) isolated from the outer (anulus fibrosus, AF) and inner (nucleus pulposus, NP) regions of human lumbar intervertebral discs was incorporated into culture substrata, using methods previously described ( ). Chick dorsal root ganglia (DRG) and the cell line SH-SY5Y were used as models of nerve growth. HMEC-1 and Eahy-926 cell lines were used as models of endothelial cell growth. Human disc aggrecan inhibited SHSY-5Y cell attachment, SHSY5Y neurite outgrowth and induced sensory DRG neurite growth cone turning in a concentration-dependent manner. Sensory neurites emanating from DRG across permissive substrates (collagen/laminin) were repelled by high (1 mg/ml), but not low (0.01 mg/ml) concentrations of disc aggrecan and became aligned to the aggrecan border. Disc aggrecan similarly inhibited endothelial cell adhesion, cell spreading and migration. HMEC-1 and Eahy926 cells migrated over collagen substrates (type I) until they encountered disc aggrecan, where they either stopped migrating or, more commonly, changed their direction of movement and aligned to the aggrecan border. In general, aggrecan isolated from the AF was more inhibitory than that isolated from the NP. The inhibitory effects were partially abrogated following enzymic deglycosylation of the aggrecan, or if aggrecan was added in solution (i.e. was nonsubstrate bound). This study provides evidence that disc aggrecan inhibits neuronal and endothelial growth and migration and therefore supports a hypothesis that a loss of aggrecan from degenerated discs predispose the tissue to vascular and neuronal invasion. [ABSTRACT FROM AUTHOR]

Although the function of proteoglycans (PGs) within the tendon extracellular matrix are not fully understood, changes in their turnover have been associated with tendinopathies ( ). In contrast to cartilage, aggrecanases are constitutively expressed and active in tendon ( ), indicative of a high rate of aggrecan turnover. Clinical trials investigating the use of active site MMP inhibitors have been confounded by side effects, which involve tendonitis and ‘musculoskeletal syndrome’. Such side effects may relate to nonspecific inhibition of tendon aggrecanases required to maintain normal metabolic homeostasis. The purpose of this study, therefore, was to compare the rate of turnover of tendon and cartilage PGs derived from the same joint and to determine the effect of MMP inhibitors (actinonin and marimastat) on aggrecan catabolism. Deep digital flexor tendon explants from compressed and tensional regions were dissected from young and mature bovine. Explants were precultured and then cultured for a further 4 days with or without marimastat (0–2 µ m) or actinonin (0–200 µ m). PG and lactate quantification, Western blot analysis of degradation products and RT-PCR analyses were performed. In a separate experiment for measurement of PG turnover, explants were set up as described above and then pulse chase labelled with [35S] sulfate. The rate of turnover of 35S-labelled PGs from the matrix of tendon (and articular cartilage obtained from the same animal) was subsequently calculated from the amount of 35S-labelled macromolecules appearing in the medium each day and that remaining in the matrix of explants at the termination of culture. PG turnover (presumably predominantly aggrecan) was markedly higher in tendon vs. cartilage. This difference was apparent in tendons from all regions and ages. Both marimastat and actinonin inhibited aggrecanase-mediated PG catabolism in both tendon and cartilage explants. As expected, mRNA expression for the aggrecanases, MMPs and TIMPs was unaffected by the addition of these inhibitors to the culture medium. Aggrecan turnover in tendon is higher than that of articular cartilage, which may be attributed to distinct physiological properties of this PG in tendon. Importantly, immunohistochemical staining for aggrecan in tendon indicates its presence in between collagen fibres and fibril bundles ( ), and thus aggrecan aggregates may dissipate resultant compressive loads by resisting the flow of water in these locations. In addition, aggrecan may facilitate the sliding of fibrils during the small amount of elongation of the tendon whilst under tension. Thus, the half-life of tendon aggrecan is significantly reduced because it constantly participates in repeated resistance to compression. Our data also demonstrates that both marimastat and actinonin can inhibit aggrecanase-mediated PG catabolism in tendon cultures. This suggests that the occurrence of ‘musculoskeletal syndrome’ in clinical trial patients may be due to the fact that these inhibitors affect the activity of aggrecanases in tendon, thus preventing them from playing their normal role in tendon aggrecan turnover and consequently perturbing normal physiological function. [ABSTRACT FROM AUTHOR]

Autologous chondrocyte implantation (ACI) is routinely used for the repair of articular cartilage defects. A similar method may be employed to treat degenerate intervertebral discs or other connective tissues. A system in which cells could not only be delivered, but also retained would offer advantages compared to ACI. Such a vehicle would also allow a homogeneous distribution of cells throughout the defect and enhance nutrient penetration to the seeded cells. Bovine nucleus cells were isolated via enzyme digestion and expanded in number to passage 3. The cells were resuspended in 0.8% alginate and loaded into polyvinyl alcohol (PVA) cubes. These constructs were placed into a solution of calcium chloride to ‘gel’ the alginate. Constructs were cultured in DMEM + 10% FBS within 15-ml conical tubes rotated at 37 °C for up to 28 days. Cell distribution/morphology and proliferation were assessed on H&E and Ki-67 stained sections, respectively. The re-expression of a disc cell phenotype was assessed using toluidine blue staining and immunohistochemistry (with antibodies to collagen types I, II, IIA, VI and X and to the glycosaminoglycans, chondroitin-4- and -6-sulfate and keratan sulfate. RT-PCR was performed using oligonucleotide primers to collagen types I, II and X, aggrecan, link protein and small leucine-rich PGs. H&E staining of 10-µm thick cryosections revealed an even distribution of loaded cells throughout the scaffold at day 1 being maintained through to day 28. Toluidine blue staining showed the presence of GAGs, increasing with time. Ki-67 staining indicated that approximately 5% of cells were proliferating at all time points. Immunohistochemistry demonstrated the production of collagen types I, II, IIA, VI and X, and the glycosaminoglycans, chondroitin-4-, -6-sulfates and keratan sulfate. RT-PCR results showed mRNA expression of fibromodulin throughout the experiment, lumican at days 14, 21 and 28. Types II and X collagen were present at days 21 and 28. Combining 0.8% alginate with PVA retained 100% of the seeded cells and allowed an even distribution of cells throughout the scaffold. The immunohistochemistry and RT-PCR demonstrated that the system allowed the bovine nucleus cells to express phenotypic markers expressed by disc cells in vivo. These preliminary results indicate that the PVA/alginate system could act as a suitable delivery device for cells during autologous repair of the intervertebral disc or other connective tissues such as meniscus. [ABSTRACT FROM AUTHOR]