The MMP3 gene in musculoskeletal soft tissue injury risk profiling: A study in two independent sample groups.

Matrix metalloproteinase-3 (MMP3) is a mediator of matrix remodelling and a proposed susceptibility locus in the genetic profile of musculoskeletal soft tissue injuries. Therefore, this study aimed to validate the MMP3 gene as a risk marker for these injuries by conducting a case control genetic association study in two independent samples groups. Three previously investigated MMP3 variants (rs679620, rs591058 and rs650108) in addition to the functional promoter variant (rs3025058) were genotyped in 195 Australian control participants and 79 Australian individuals with chronic Achilles tendinopathy. Similarly, 234 South African individuals with acute anterior cruciate ligament ruptures and 232 matched control participants were also analysed. Based on high linkage with the previously associated MMP3 variant rs679620, rs3025058 was inferred and found to be associated with increased risk for Achilles tendinopathy within the South African group (P = 0.012; OR: 2.88; 95% CI: 1.4 to 6.1). Lastly, the 6A-G-C-G haplotype, constructed from the investigated variants, was significantly associated with reduced risk for Achilles tendinopathy (29% CON vs. 20% TEN, P = 0.037) in the Australian group. In conclusion, a signal surrounding MMP3 is apparent with respect to Achilles tendinopathy. However, whether the investigated variants are contributing to injury susceptibility or whether they are merely linked to the risk conferring variants mapping elsewhere within the MMP gene cluster on chromosome 11, still requires refining.

Involvement of proteoglycans in tendinopathy.

A major feature of chronic tendinopathy is a change in the nature and organisation of the extracellular matrix of tendon. Increased levels of proteoglycans have been shown in the extracellular matrix of tendinopathic tendons and these appear to influence the increased hydration and swelling of the tissue that is a feature of this condition. There is a paucity of knowledge about proteoglycans in normal and tendinopathic tendons. This review sets out to describe the nature, function and metabolism of proteoglycans present in normal tendon and in tendinopathy and outlines how changes in proteoglycan metabolism may contribute to the development and progression of this disease.

Variants within the COL5A1 gene are associated with Achilles tendinopathy in two populations.

OBJECTIVES: A COL5A1 gene variant was shown to be associated with chronic Achilles tendinopathy in a South African population. The aim of this case-control genetic association study was to investigate the BstUI and DpnII restriction fragment length polymorphisms (RFLP) in a second population from Australia and to identify a predisposing haplotype for Achilles tendinopathy in both populations. METHODS: 85 Australian and 93 South African patients with tendinopathy, as well as 210 Australian and 132 white South African control subjects were genotyped for the BstUI (rs12722) and DpnII (rs13946) RFLP, as well as markers rs10858286, rs3196378, rs11103544, rs4504708 and rs3128575. RESULTS: The BstUI RFLP (p<0.001) and marker rs3196378 (p = 0.016) were associated with chronic Achilles tendinopathy in Australian subjects. Individuals within both populations with a CC genotype for the BstUI RFLP had a significantly decreased risk of developing tendinopathy versus any other genotypes (Australian odds ratio 0.42, 95% CI 0.20 to 0.86, p = 0.017). The TC inferred haplotype (rs12722, rs3196378) was found to be overrepresented (global p = 0.008) in the South African tendinopathy group compared with all other haplotypes. CONCLUSION: The BstUI RFLP is associated with chronic Achilles tendinopathy in a second population and a region within the COL5A1 3' untranslated region may predispose individuals to an increased risk of developing chronic Achilles tendinopathy.

Effects of glucosamine on proteoglycan loss by tendon, ligament and joint capsule explant cultures.

OBJECTIVE: To investigate the effect of glucosamine on the loss of newly synthesized radiolabeled large and small proteoglycans by bovine tendon, ligament and joint capsule. DESIGN: The kinetics of loss of (35)S-labeled large and small proteoglycans from explant cultures of tendon, ligament and joint capsule treated with 10mM glucosamine was investigated over a 10-day culture period. The kinetics of loss of (35)S-labeled small proteoglycans and the formation of free [(35)S]sulfate were determined for the last 10 days of a 15-day culture period. The proteoglycan core proteins were analyzed by gel electrophoresis followed by fluorography. The metabolism of tendon, ligament and joint capsule explants exposed to 10mM glucosamine was evaluated by incorporation of [(3)H]serine and [(35)S]sulfate into protein and glycosaminoglycans, respectively. RESULTS: Glucosamine at 10mM stimulated the loss of small proteoglycans from ligament explant cultures. This was due to the increased loss of both macromolecular and free [(35)S]sulfate to the medium indicating that glucosamine affected the release of small proteoglycans as well as their intracellular degradation. The degradation pattern of small proteoglycans in ligament was not affected by glucosamine. In contrast, glucosamine did not have an effect on the loss of large or small proteoglycans from tendon and joint capsule or large proteoglycans from ligament explant cultures. The metabolism of cells in tendon, ligament and joint capsule was not impaired by the presence of 10mM glucosamine. CONCLUSIONS: Glucosamine stimulated the loss of small proteoglycans from ligament but did not have an effect on small proteoglycan catabolism in joint capsule and tendon or large proteoglycan catabolism in ligament, tendon or synovial capsule. The consequences of glucosamine therapy at clinically relevant concentrations on proteoglycan catabolism in joint fibrous connective tissues need to be further assessed in an animal model.

Increased versican content is associated with tendinosis pathology in the patellar tendon of athletes with jumper's knee.

Expansion of the extracellular matrix is a prominent but poorly characterized feature of tendinosis. The present study aimed to characterize the extent and distribution of the large aggregating proteoglycan versican in patients with patellar tendinosis. We obtained tendon from tendinopathy patients undergoing debridement of the patellar tendon and from controls undergoing intramedullary tibial nailing. Versican content was investigated by Western blotting and immunohistochemistry. Microvessel thickness and density were determined using computer-assisted image analysis. Markers for smooth muscle actin, endothelial cells (CD31) and proliferating cells (Ki67) were examined immunohistochemically. Western blot analysis and immunohistochemical staining revealed elevated versican content in the proximal patellar tendon of tendinosis patients (P=0.042). Versican content was enriched in regions of fibrocartilage metaplasia and fibroblast proliferation, as well as in the perivascular matrix of proliferating microvessels and within the media and intima of arterioles. Microvessel density was higher in tendinosis tissue compared with control tissue. Versican deposition is a prominent feature of patellar tendinosis. Because this molecule is not only a component of normal fibrocartilagenous matrices but also implicated in a variety of soft tissue pathologies, future studies should further detail both pathological and adaptive roles of versican in tendons.

Extracellular matrix metabolism by chondrocytes. 5. The proteoglycans and glycosaminoglycans synthesized by chondrocytes in high density cultures.

Proteoglycans were extracted from the extracellular matrix of cultures of embryonic chick chondrocytes grown at high density and were purified by CsC1 density gradient centrifugation. The chemical, physical and hyaluronate binding properties of the proteoglycans were similar to those observed in proteoglycans from other hyaline cartilages. Proteoglycans in the media were also purified and on analysis showed three populations of proteoglycans to be present. One population had the physical characteristics of a typical proteoglycan subunit and bound hyaluronate, the other two populations were unable to complex with hyaluronate but one had the physical characteristics of the proteoglycan subunit and the other was of smaller molecular weight. The small molecular weight appears to be a product of the enzymatic degradation of the larger molecular weight species.

Inhibition of proteoglycan biosynthesis by hyaluronic acid in chondrocytes in cell culture.

The depression of proteoglycan synthesis in ten-day-old high density chondrocyte cultures was shown to be dependent on both the concentration and time of exposure of the cells to hyaluronic acid. Hyaluronic acid had no effect on the overall protein synthesis by the cultured cells. Using benzyl-beta-D-xyloside an exogenous acceptor, it was shown that glycosaminoglycan biosynthesis by the chondrocytes was not affected by hyaluronic acid. It was concluded that hyaluronic acid was effecting glycosaminoglycan chain initiation, hence proteoglycan biosynthesis, either by specifically depressing the synthesis of the core protein or by repressing the activity of the xylosyltransferase.

Extracellular matrix metabolism by chondrocytes. III. Modulation of proteoglycan synthesis by extracellular levels of proteoglycan in cartilage cells in culture.

Proteoglycan biosynthesis by cultured chondrocytes was shown to be depressed by extracellular concentrations of proteoglycan and partially degraded proteoglycan. This reduction in proteoglycan synthesis was reversible on removal of the added proteoglycan. Benzyl-beta-D-xyloside, an exogenous acceptor of glycosaminoglycan synthesis, was used and it was shown that proteoglycan was inhibiting glycosaminoglycan synthesis. Proteoglycan had no effect on the overall protein synthesis by the cultured cells. It was concluded that the exogenous proteoglycan was inhibiting proteoglycan synthesis at the level of initiation or elongation of the glycosaminoglycan chains.

Passive loss of proteoglycan from articular cartilage explants.

The addition of proteinase inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 mM N-ethylmaleimide, 0.25 mM benzamidine hydrochloride, 6.25 mM EDTA, 12.5 mM 6-aminohexanoic acid and 2 mM iodoacetic acid) to explant cultures of adult bovine articular cartilage inhibits proteoglycan synthesis as well as the loss of the macromolecule from the tissue. Those proteoglycans lost to the medium of explant cultures treated with proteinase inhibitors were either aggregates or monomers with functional hyaluronic acid-binding regions, whereas proteoglycans lost from metabolically active tissue also included a population of monomers that were unable to aggregate with hyaluronate. Analysis of the core protein from proteoglycans lost into the medium of inhibitor-treated cultures showed the same size distribution as the core proteins of proteoglycans present in the extracellular matrix of metabolically active cultures. The core proteins of proteoglycans appearing in the medium of metabolically active cultures showed that proteolytic cleavage of these macromolecules occurred as a result of their loss from the tissue. Explant cultures of articular cartilage maintained in medium with proteinase inhibitors were used to investigate the passive loss of proteoglycan from the tissue. The rate of passive loss of proteoglycan from the tissue was dependent on surface area, but no difference in the proportion of proteoglycan aggregate to monomer appearing in the medium was observed. Furthermore, proteoglycans were lost at the same rate from the articular and cut surfaces of cartilage. Proteoglycan aggregates and monomer were lost from articular cartilage over a period of time, which indicates that proteoglycans are free to move through the extracellular matrix of cartilage. The movement of proteoglycans out of the tissue was shown to be temperature dependent, but was different from the change of the viscosity of water with temperature, which indicates that the loss of proteoglycan was not solely due to diffusion. The activation energy for the loss of proteoglycans from articular cartilage was found to be similar to the binding energies for electrostatic and hydrogen bonds.

Effect of dibutyryl cyclic AMP on the sulphation of proteoglycans by chondrocytes.

The addition of a 1 mM N6, O2'-dibutyryl cyclic AMP to incubations of foetal calf articular cartilage for a 4 h period resulted in a 13 and a 15% decrease, respectively, in the rate of incorporation of [3H]glucosamine and [14C]glucose into glycosaminoglycans. Under the same conditions, a 41% increase in the rate of incorporation of [35S]sulphate into glycosaminoglycans was observed. Dibutyryl cyclic AMP had no effect on protein synthesis over a 4 h period or on the hydrodynamic size of the proteoglycan subunits or glycosaminoglycans synthesized by the cartilage. When the glycosaminoglycans were digested with chondroitin ABC lyase and the resulting disaccharides analysed on Dowex 1 (formate form), it was observed that dibutyryl cyclic AMP increased the degree of sulphation of the disaccharides. Furthermore, an over-sulphated disaccharide corresponding to chondroitin 4,6-disulphate was identified. It was concluded that dibutyryl cyclic AMP was stimulating the process of sulphation of glycosaminoglycans by cartilage.

Extracellular matrix metabolism by chondrocytes. 4. Role of glutamine in glycosaminoglycan synthesis in vitro by chondrocytes.

The rate of synthesis of glycosaminoglycans by cartilage was shown to be dependent on an exogenous source of L-glutamine. In the absence of L-glutamine the tissue and cellular levels of this amino acid were rapidly depleted. The levels of nucleotide sugars and their precursors were measured after separation on Dowex 1 (formate form) in cartilage incubated with and without L-glutamine. It was found that the levels of N-acetylhexoamine 6-phosphate and UDP-N-acetylhexosamine were decreased by 27 and 40% respectively. This demonstrates that L-glutamine is required as the amido group donor in the synthesis of glucosamine 6-phosphate and that the decrease in glycosaminoglycan synthesis is due to the limitation in synthesis of UDP-N-acetylhexoamine.

Effect of insulin-like growth factor-I on the synthesis and distribution of link protein and hyaluronan in explant cultures of articular cartilage.

Addition of 20% (v/v) fetal calf serum or insulin-like growth factor-I (IGF-I; 20 ng/ml) to the medium of explant cultures of adult articular cartilage resulted in an increased rate of synthesis of the three components of the proteoglycan aggregate-namely link protein, hyaluronan and aggrecan. Fetal calf serum also stimulated the synthesis of other matrix proteins by articular cartilage compared with tissue maintained in medium alone or medium containing IGF-I. Although addition of fetal calf serum or IGF-I to the culture medium of cartilage explant cultures stimulated both hyaluronan and aggrecan synthesis, no change in the distribution of these two macromolecules between tissue and medium was observed. Approx. 50% of the newly synthesized hyaluronan was retained by the tissue compared to 93% of the labelled aggrecan. Culture conditions had some influence on the distribution of link protein, in cultures maintained in medium alone or in medium containing IGF-I, less than 12% of the newly synthesized link protein was lost to the medium of the cultures. However, in cultures maintained with fetal calf serum between 25% and 19% of the radiolabelled link protein was lost from the matrix of the explants. This work suggests that the chondrocyte synthesizes the macromolecules that make up the proteoglycan aggregate in a co-ordinated manner, thereby retaining the relative amounts of each component of this functionally important complex.

Characterization and synthesis of macromolecules by adult collateral ligament.

Bovine collateral ligament was found to have a water content of 67.5 +/- 2.5%, the tissue was highly collagenous containing 100.3 +/- 15.1 micrograms hydroxyproline/mg dry weight. Type I collagen was the major collagen present with small amounts of Type III and V. The hexuronate content of the tissue was found to be 2.62 +/- 0.40 micrograms hexuronate/mg dry weight of tissue. On incubation in vitro collateral ligament incorporated [35S]sulfate and [3H]acetate into proteoglycans and [3H]acetate into hyaluronate and glycoproteins. The rate of synthesis of proteoglycans by collateral ligament was shown on a weight basis to be greater than that of tendon but lower than that of articular cartilage. Analysis of the proteoglycans present in collateral ligament showed two populations of proteoglycans to be present. Approx. 20% of the total proteoglycans present were large chondroitin- and keratan sulfate-containing proteoglycans capable of forming aggregates with hyaluronate. The major species of proteoglycan present were small dermatan sulfate proteoglycans made up of a core protein with a molecular mass of 45,000 daltons with one dermatan/chondroitin sulfate glycosaminoglycan chain of 30,000 daltons attached. The N-terminal amino acid sequence of the core protein of this proteoglycan showed it to be analogous to the core protein of dermatan sulfate proteoglycan II.

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

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

Extracellular matrix metabolism by chondrocytes. VI. Concomitant depression by exogenous levels of proteoglycan of collagen and proteoglycan synthesis by chondrocytes.

The synthesis of collagen and proteoglycans by cultured chondrocytes, as measured by the incorporation of L-[3H]proline into hydroxyproline and [3H]acetate into glycosaminoglycans, was shown to be depressed by 58% and 39%, respectively, by the addition of exogenous proteoglycan at a concentration of 10 mg/ml growth media. The incorporation of L-[3H]proline into acid-insoluble protein remained unaltered in the presence of the proteoglycan. It was concluded that the effect was depressing the activity on the enzymatic steps, associated with the endoplasmic reticulum, which are responsible for the post-translational modification of collagen and proteoglycan.

Extracellular matrix metabolism by chondrocytes. 7. Evidence that L-glutamine is an essential amino acid for chondrocytes and other connective tissue cells.

The incorporation of [3H]glycine into acid-insoluble protein and of [3H]acetate into glycosaminoglycans by cultured chick chondrocytes was stimulated by the addition of L-glutamine to the incubation medium. The effect of exogenous L-glutamine on protein synthesis was studied further by examining changes in the sedimentation patterns on sucrose gradients of ribosomes isolated from chondrocytes incubated in presence and absence of L-glutamine. It was found that the absence of L-glutamine caused a disaggregation of polyribosomes that was revered by the addition of this amino acid to the culture medium. No detectable glutamine synthetase activity could be measured in avian articular cartilage. These results indicate that L-glutamine is an essential amino acid for cartilage in that an extracellular supply of this amino acid is required for the maintenance of protein and glycosaminoglycan synthesis. A dependence of L-glutamine was also demonstrated for other avain connective tissues.

Characterization of the collagen synthesized by cultured cartilage cells.

Cartilage cells from embryonic chick cartilage were grown in primary cultures. The cell layer was sequentially extracted with neutral saline, mercaptoethylamine and pepsin which revealed that these cells produced salt-soluble and salt-insoluble collagen. The alpha1- to alpha2-chain ratio was determined for the collagen extracted from the cultured cells and was found to be 13 to 1. Further analysis of the molecule was carried out by CNBr cleavage of the salt-extracted collagen and separation of resulting peptides by ion-exchange chromatography. It was shown that the cultured cartilage cells synthesize collagen of the type (alpha1[II])3.

The synthesis of elastin, collagen, and glycosaminoglycans by high density primary cultures of neonatal rat aortic smooth muscle. An ultrastructural and biochemical study.

Primary cultures of neonatal rat aortic smooth muscle cells inoculated at high densities (1 X 10(6) cells/25 cm2 Falcon flask) with adequate nutrient media and pH control grow rapidly and form multilayers of cells with typical "hill and valley" organization. After 10 days growth insoluble elastin formation could be visualized by phase contrast microscopy as small particles which grew rapidly to become larger irregular refractile aggregates and later coalesced to form larger aggregates and small fibres. With light and electronmicroscopy, elastin was the predominant matrix protein formed, with the "hill regions" of cultures containing abundant elastin aggregates and some collagen. In 2-week-old cultures differentiation could be observed within the cell multilayer. The older deeper cells contained more protein synthesis organelles and myofilaments and were in close association with large often coalescing elastin aggregates; compared to younger more superficial cells which contained more free polyribosomes less myofilaments, and were associated with fewer and small elastin aggregates. In older cultures this differentiation was not apparent; the cells contained many myofilaments, dense bodies, and lysosomes. Elastin aggregates and newly formed elastic fibres were abundant in the matrix. Quantitative analysis of insoluble elastin formation in the cell layer during the 4-week culture period indicated continuous biosynthesis and deposition which paralleled that of desmosine formation. Amino-acid analysis of a hot alkali insoluble residue (regarded as elastin) from 30-day-old cultures gave a profile identical with neonatal rat aortic elastin in vivo. Insoluble collagen formation in the cell layer tended to plateau after the log phase of growth was completed (10 days). Proteoglycans were found predominantly in the supernatant media. Glycosaminoglycan analysis revealed a profile of dermatan sulphate (32%), chondroitin 4-sulphate (43%), keratan and heparan sulphate (30%), with only a trace of hyaluronic acid. This study indicates that primary cultures of neonatal rat aortic smooth muscle cells remain differentiated in culture and have the unique capacity to continue to synthesize and deposit large amounts (mg) of insoluble elastin which aggregate and from elastic fibres in vitro.

Metabolic processing of newly synthesized link protein in bovine articular cartilage explant cultures.

In explant cultures of articular cartilage from cattle of different ages radiolabeled leucine was shown to be incorporated into link proteins 1, 2 and 3. The newly synthesized link proteins were incorporated into and lost from the cartilage extracellular matrix with time. The levels of radiolabeled link proteins 1 and 2 remaining in the matrix declined over the culture period, but there was an initial increase in the amount of radiolabeled link protein 3, before its level declined. The turnover time of the radiolabeled link proteins 1 and 2 were similar, indicating that neither link protein was preferentially processed to generate link protein 3, nor lost from the extracellular matrix. The majority of the radiolabeled link protein lost from the cartilage matrix could not be recovered from the culture medium, suggesting that turnover of the radiolabeled aggrecan complexes involves the newly synthesized link protein being internalized by the chondrocytes. Inclusion of cytotoxic proteinase inhibitors to the culture medium resulted in a marked decrease in the rate of loss of link protein from the cartilage, suggesting that the catabolism of link protein is cell-mediated and dependent on metabolically active cells.

Catabolism of newly synthesized decorin by explant cultures of bovine ligament.

The catabolism of newly synthesized decorin by explant cultures of bovine collateral ligament was investigated. The tissue was placed in explant culture for 6 days then incubated with radiolabeled sulfate for 6 h and replaced in culture for 5 days to allow for the loss of the radiolabeled large proteoglycan. The metabolic fate of the remaining radiolabeled decorin present in the matrix of the tissue over the next 9-day period was determined. It was shown that this pool of decorin was lost from ligament explant cultures either directly into the culture medium or taken up and degraded within the cells of the tissue. The intracellular degradation of the radiolabeled pool of decorin by ligament explant cultures was shown to result in the generation of [35S]sulfate. This process required metabolically active cells and involved the lysosomal system since sulfate generation was inhibited when cultures were maintained at 4 degrees C or in the presence of either 10 mM ammonium chloride or 0. 05 mM chloroquine. The inhibition of intracellular processing of decorin resulted in an increase in the rate of loss of this proteoglycan into the medium of the cultures. The inhibition of intracellular degradation of decorin was reversible on incubation of the explant cultures at 37 degrees C or removal of ammonium chloride from the culture medium. After removal of the ammonium chloride from the culture medium the rate of intracellular catabolism was greater than that observed in cultures maintained in medium alone, which suggested that there was an intracellular accumulation of native and/or partially degraded material within the cells.