Extracellular matrix proteins interact with cell-signaling pathways in modifying risk of achilles tendinopathy.

The aim of this study was to investigate interactions between variants within genes encoding components of the collagen fibril and components of cell-signaling pathways within the extracellular matrix, and determine the relative contribution of these variants to Achilles tendinopathy risk in a polygenic model. A total of 339 asymptomatic control participants and 179 participants clinically diagnosed with Achilles tendinopathy were genotyped for variants within six genes encoding components of the collagen fibril and three genes encoding components of cell-signaling pathways. Logistic regression, stepwise selection, and receiver operating characteristic curve (ROC) analysis was used to select and evaluate genetic interactions and determine the relative contribution of these variants to overall genetic risk. The strongest, best fit polygenic risk model included the variables sex, three COL27A1 variants (rs4143245; rs1249744; rs946053), COL5A1 rs12722, CASP8 rs1045485, and CASP8 rs2824129 with an area under the ROC curve of 0.737 and the maximum sum of sensitivity and specificity indicators equal to 134%. Significant interactions between genes encoding components of the collagen fibril and genes encoding components of the cell-signaling pathways modify risk of Achilles tendinopathy.

Variants within the COMP and THBS2 genes are not associated with Achilles tendinopathy in a case-control study of South African and Australian populations.

Cartilage oligomeric matrix protein is a structural protein of the extracellular matrix, while thrombospondin-2 is a matricellular protein involved in cell-matrix interactions. Recent studies have shown that genetic variation is a significant risk factor for Achilles tendinopathy, and the genes encoding cartilage oligomeric matrix protein (COMP) and thrombospondin-2 (THBS2) were identified as good candidate genes for association with Achilles tendinopathy. This study aimed to test the association of sequence variants within these candidate genes with the risk of Achilles tendinopathy in participants from South Africa (SA) and Australia (AUS). Three-hundred and forty (133 SA; 207 AUS) control participants with no history of Achilles tendinopathy and 178 (94 SA; 84 AUS) participants clinically diagnosed with Achilles tendinopathy were genotyped for five single nucleotide polymorphisms within the COMP and THBS2 genes in this case-control study. There was no difference in genotype distributions between control and tendinopathy groups for either the THBS2 variants rs9505888, rs6422747 and rs9283850, or the COMP variants rs730079 and rs28494505 in the SA and AUS populations. As the selection of COMP and THBS2 as candidate genes was hypothesis driven, based on biological function, the possibility that other variants within these genes are associated with Achilles tendinopathy cannot be excluded.

Association of type XI collagen genes with chronic Achilles tendinopathy in independent populations from South Africa and Australia.

BACKGROUND: Type XI collagen, which is expressed in developing tendons and is encoded by the COL11A1, COL11A2 and COL2A1 genes, shares structural and functional homology with type V collagen, which plays an important role in collagen fibril assembly. We investigated the association of these three polymorphisms with Achilles tendinopathy (AT) and whether these polymorphisms interact with COL5A1 to modulate the risk of AT. METHODS: 184 participants diagnosed with chronic AT (TEN) and 338 appropriately matched asymptomatic controls (CON) were genotyped for the three polymorphisms. RESULTS: Although there were no independent associations with AT, the TCT pseudohaplotype constructed from rs3753841 (T/C), rs1676486 (C/T) and rs1799907 (T/A) was significantly over-represented (p=0.006) in the TEN (25.9%) compared with the CON (17.1%) group. The TCT(AGGG) pseudohaplotypes constructed using these type XI collagen polymorphisms and the functional COL5A1 rs71746744 (-/AGGG) polymorphism were also significantly over-represented (p<0.001) in the TEN (25.2%) compared with the CON (9.1%) group. DISCUSSION: The genes encoding structural and functionally related type XI (COL11A1 and COL11A2) and type V (COL5A1) collagens interact with one another to collectively modulate the risk for AT. Although there are no immediate clinical applications, the results of this study provide additional evidence that interindividual variations in collagen fibril assembly might be an important molecular mechanism in the aetiology of chronic AT.

Polymorphic variation within the ADAMTS2, ADAMTS14, ADAMTS5, ADAM12 and TIMP2 genes and the risk of Achilles tendon pathology: a genetic association study.

OBJECTIVES: Achilles tendon pathology (ATP) is a multifactorial condition for which genetic risk factors have been identified. The ADAMTS, ADAM12 and TIMP2 genes encode enzymes that are important regulators of tendon homeostasis. ADAMTS2 and ADAMTS14 proteins are procollagen N-propeptidases for pro-collagen type I, type II, and type III. ADAMTS2, like COL5A1, has been linked to Ehlers-Danlos syndrome. Variants within ADAMTS5 and ADAM12 have been associated with osteoarthritis. TIMP2, a metalloprotease inhibitor, maintains homeostasis in the ECM by inhibiting ADAM, ADAMTS and MMP functions. We sought to determine whether single nucleotide polymorphisms (SNPs) within the ADAMTS2, ADAMTS5, ADAMTS14, ADAM12 and TIMP2 genes were associated with the risk of ATP in two independent populations. DESIGN: 213 (115 ATP cases and 98 asymptomatic controls) South African Caucasian participants and 209 (60 ATP cases and 149 asymptomatic controls) Australian Caucasian participants were recruited for this case-control genetic association study. METHODS: All participants were genotyped using TaqMan technology for the ADAMTS2 rs1054480, ADAMTS5 rs226794, ADAMTS14 rs4747096, ADAM12 rs3740199, and TIMP2 rs4789932 SNPs. RESULTS: We report for the first time a significant (p=0.016) genotypic association between the TIMP2 rs4789932 variant and ATP in a combined Caucasian cohort. We also identify an interaction between the ADAMTS14 rs4747096 variant and age of onset of ATP (p=0.024). CONCLUSIONS: Our data show that DNA sequence variation within the TIMP2 gene is a risk factor for ATP in Caucasians. Furthermore, carriage of the ADAMTS14 rs4747096 GG variant appears to delay onset of the injury in the ATP group.

Investigation of variants within the COL27A1 and TNC genes and Achilles tendinopathy in two populations.

The TNC gene has previously been associated with Achilles tendinopathy (AT) in a South African population. The aims of this study were (i) to investigate the association of single nucleotide polymorphisms within the TNC gene, and the additional candidate gene, COL27A1, with AT in two populations, and (ii) to identify if there is a risk haplotype for AT in both populations. Three hundred and thirty nine healthy control participants (CON) and 179 participants clinically diagnosed with AT (TEN) from South Africa and Australia, were genotyped for variants: rs4143245, rs1249744, rs753085, rs946053 (COL27A1) and rs13321, rs2104772, rs1330363 (TNC). Haplotypes were inferred using the genotype data. The rs2104772 (p = 0.017) and rs1330363 (p = 0.020) variants within TNC showed a significant allele association with AT. The GCA haplotype (rs946053-rs13321-rs2104772) occurred significantly more frequently in TEN participants compared to CON (27% vs. 18%; p = 0.019). This study further implicates the genomic region containing the TNC and COL27A1 genes in influencing risk of AT, and maps the potential risk allele to a genetic interval flanked by rs946053 and rs2104772. This region may have functional effects on the transcription, structure and properties of tenascin-C and the alpha-1 chain of type XXVII collagen.

The apoptosis pathway and the genetic predisposition to Achilles tendinopathy.

Achilles tendinopathy (AT) is a degenerative condition for which several risk factors have been implicated including components of the inflammatory pathway. The aim was to assess functional variants within genes encoding components of the apoptosis signaling cascade and the effectiveness of a polygenic apoptosis profile to capture tendinopathy (TEN) risk. A total of 358 unaffected control (CON) participants [159 South Africa (SA CON) and 199 Australia (AUS CON)] and 166 affected AT (TEN) participants (87 SA TEN and 79 AUS TEN) were genotyped for four variants [CASP8 (rs384129), CASP8 (rs1045485), NOS3 (rs1799983), and NOS2 (rs2779249)]. Logistic regression was used to derive risk models for AT. A receiver operator characteristic (ROC) curve was plotted to determine the effectiveness of a model to capture AT risk. This study indicates the independent association of CASP8_rs1045485 and CASP8_rs3834129 as well as their haplotype with AT risk and the identification of an optimal model which included genetic loci CASP8_rs384129 and CASP8_rs1045485 together with sex to capture AT risk in both SA and AUS. Collectively, these results further implicate the apoptosis signaling cascade as one of the biological pathways involved in the development of AT.

Short- and long-term exposure of articular cartilage to curcumin or quercetin inhibits aggrecan loss.

The aim of this study was to determine if curcumin and quercetin inhibit induced aggrecan loss from bovine articular cartilage explants given that these polyphenols have been shown to suppress the expression of matrix-degrading enzymes. The kinetics of loss of ³5S-aggrecan and the loss of total aggrecan in cartilage explants maintained in catabolic medium containing either 1 µM retinoic acid or 50 ng/ml interleukin (IL)-1α were studied in the presence of either 1-25 µM curcumin or 10-50 µM quercetin. The reversibility of catabolism of ³5S-aggrecan was also studied in catabolically stimulated cultures treated with 25 µM curcumin or 50 µM quercetin for the initial 4-5 days of culture followed by 10-15 days of culture in catabolic medium in the absence of either polyphenol. Curcumin and quercetin suppressed ³5S-aggrecan and total aggrecan loss from the explants in a dose-dependent manner. When the exposure of explants to curcumin or quercetin was limited to the first 4-5 days of culture, the suppression of ³5S-aggrecan loss was maintained in the extended culture period when the tissue was stimulated with either retinoic acid or IL-1α. Quercetin suppressed IL-1α-stimulated expression of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4. Curcumin suppressed retinoic acid stimulated expression of ADAMTS-5, and both polyphenols suppressed basal expression of ADAMTS-5. The ability of curcumin and quercetin to protect cartilage from stimulated aggrecan loss and to maintain this protection posttreatment may, at least in part, be due to the suppression of gene expression of ADAMTS-4 and -5.

Effectiveness of intra-articular hyaluronan (Synvisc, hylan G-F 20) for the treatment of first metatarsophalangeal joint osteoarthritis: a randomised placebo-controlled trial.

OBJECTIVES: To evaluate the effectiveness of a single intra-articular injection of hylan G-F 20 (Synvisc) for symptomatic first metatarsophalangeal joint (MTPJ) osteoarthritis (OA). METHODS: Participants (n = 151) with symptomatic first MTPJ OA were randomly allocated to receive up to 1 ml intra-articular injection of either hylan G-F 20 or placebo (saline). Participants and assessors were blinded. Outcomes were evaluated at 1, 3 and 6 months after injection. The primary outcome measurement was the foot pain domain of the Foot Health Status Questionnaire (FHSQ) at 3 months. Secondary outcome measurements were foot function assessed via the FHSQ, first MTPJ pain and stiffness, magnitude of symptom change, global satisfaction, health-related quality of life (assessed using the Short-Form-36 version two), first MTPJ dorsiflexion range of motion, hallux plantar flexion strength, use of pain-relieving medication or co-interventions and changes in plantar pressures. RESULTS: No statistically significant differences in foot pain were found between the groups at 3 months. There were few statistically significant differences in the secondary outcome measures. Overall, the incidence of adverse effects was not significantly different between groups. CONCLUSIONS: An intra-articular injection of hylan G-F 20 is no more effective than a placebo in reducing symptoms in people with symptomatic first MTPJ OA.Australian New Zealand Clinical Trials Registry: number ACTRN12607000654459.

A pathway-based approach investigating the genes encoding interleukin-1ß, interleukin-6 and the interleukin-1 receptor antagonist provides new insight into the genetic susceptibility of Achilles tendinopathy.

OBJECTIVES: Achilles tendinopathy (AT) is a multifactorial condition for which genetic risk factors have been identified. A pathway-based approach was used to investigate genes within the inflammatory pathway. METHODS: Functional polymorphisms within IL-1ß (-31T→C and -511C→T), IL-1RN (variable number tandem repeat) and IL-6 (-172G→C) were investigated for associations with AT in a South African (SA) and Australian (AUS) case-control studies. A total of 369 (161 SA and 208 AUS) asymptomatic control participants (CON) and 175 (90 SA and 85 AUS) participants with AT (TEN) were genotyped. Allele combinations were constructed using the above polymorphisms in combination with the COL5A1 BstUI RFLP. RESULTS: Independently, no associations were observed between any of the polymorphisms tested and risk of TEN. The allele combinations of five polymorphisms were, however, found to have a highly significant relationship with AT (p=0.005), after adjusting for gender and country (SA or AUS). CONCLUSIONS: Variations within the interleukin genes and the COL5A1 BstUI CC genotype are collectively significantly associated with risk of AT. This research emphasises that a pathway-based genetic association study may be a more effective approach to capture and understand the genetic risk factors underlying the multifactorial conditions, such as AT.

Change in proteoglycan metabolism is a characteristic of human patellar tendinopathy.

OBJECTIVE: To determine differences in the metabolism of proteoglycans and the gene expression of proteinases and their inhibitors between patellar tendons exhibiting chronic overuse tendinopathy and normal patellar tendons in humans. METHODS: Rates of loss and synthesis of proteoglycans were determined. Radiolabeled and total proteoglycans retained in and lost from the tissue were analyzed by fluorography and Western blotting. Levels of messenger RNA for matrix metalloproteinase 1 (MMP-1), MMP-2, MMP-3, MMP-9, MMP-13, ADAMTS-1, ADAMTS-4, ADAMTS-5, tissue inhibitor of metalloproteinases 1 (TIMP-1), TIMP-2, TIMP-3, and TIMP-4 were determined in fresh tissue. RESULTS: The rate of loss of (35)S-labeled proteoglycans was greater in abnormal tendons, as was the rate of synthesis of proteoglycans. Fluorography and Western blotting revealed the presence of greater amounts of large proteoglycans (aggrecan and versican) in abnormal tendons, and these proteoglycans were rapidly lost from the matrix of abnormal tendons. There was no significant difference in the expression of ADAMTS-1, ADAMTS-4, ADAMTS-5, MMP-1, MMP-2, MMP-3, MMP-13, TIMP-2, TIMP-3, or TIMP-4. There was a significant increase in the expression of MMP-9 and TIMP-1 in abnormal tendons. CONCLUSION: Our findings suggest that a change in the proteoglycan content of the extracellular matrix in abnormal tendons results from the altered metabolism of the cells, reflected in the enhanced synthesis of the large proteoglycans aggrecan and versican, and does not appear to result from changes at the level of gene expression.

Components of the transforming growth factor-beta family and the pathogenesis of human Achilles tendon pathology--a genetic association study.

OBJECTIVES: Achilles tendon pathology is a multifactorial condition for which various risk factors, including genetic factors, have been identified. Gene transfection of two members of the TGF-ß family, TGF-ß1 and growth/differentiation factor-5 (GDF-5), have been shown to enhance tendon repair and mechanical strength within animal Achilles tendon injury models. The objective of this study was to investigate whether two functional 5' untranslated region (UTR) single nucleotide polymorphisms (SNPs), the TGFB1 rs1800469 variant and the GDF5 rs143383 variant, were associated with ATP within an Australian ('AUS') and a South African ('SA') case-control cohort. METHODS: One hundred and seventy-one subjects (58 AUS and 112 SA) with Achilles tendon pathology (ATP group) and 235 (142 AUS and 96 SA) asymptomatic control (CON group) subjects were genotyped for the selected SNPs using custom-designed Taqman assays. A χ(2)-analysis or Fisher's exact test was used to analyse any differences in the genotype and allele frequencies. Significance was accepted when P < 0.05. RESULTS: There were no significant TGFB1 rs1800469 genotype (P = 0.491) or allele (P = 0.400) frequency differences between the ATP and CON groups. The TT genotype of the GDF5 rs143383 variant was significantly over-represented in the ATP group of the AUS cohort [P = 0.011; odds ratio (OR) = 2.24; 95% CI 1.21, 4.16], and when the AUS and SA cohorts were combined (P = 0.004; OR = 1.82; 95% CI 1.23, 2.74). CONCLUSIONS: In conclusion, this study suggests that individuals with a TT genotype of the functional GDF5 rs143383 variant have twice the risk of developing ATP. This finding highlights a role of GDF-5 in the pathogenesis of Achilles tendon pathology.

Changes in the composition of the extracellular matrix in patellar tendinopathy.

OBJECTIVE: To compare the chemical levels and mRNA expression of proteoglycan and collagen in normal human patellar tendons and tendons exhibiting chronic overuse tendinopathy. METHODS: Sulfated glycosaminoglycan and hydroxyproline content were investigated by spectrophotometric measurement using papain-digested samples. Deglycosylated proteoglycan core proteins were analysed by Western blot using specific antibodies. Total mRNA isolated from samples of frozen tendons was assayed by relative quantitative RT-PCR for decorin, biglycan, fibromodulin, versican, aggrecan, and collagens Type I, II and III and normalised to glyceraldehyde-3-phosphate dehydrogenase. RESULTS: There was a significant increase in sulfated glycosaminoglycan content in pathologic tendons compared to normal. This was attributed to an increased deposition of the large aggregating proteoglycans versican and aggrecan and the small proteoglycans biglycan and fibromodulin, but not decorin. Aggrecan and versican were extensively degraded in both normal and pathologic tendons, biglycan was more fragmented in the pathologic tendons while predominantly intact fibromodulin and decorin were present in normal and pathologic tendons. There was a greater range in total collagen content but no change in the level of total collagen in pathologic tendons. There were no significant differences between the pathologic and normal tendon for all genes, however p values close to 0.05 indicated a trend in downregulation of Type I collagen and fibromodulin, and upregulation in versican and Type III genes in pathologic tissue. CONCLUSION: The changes in proteoglycan and collagen levels observed in patellar tendinopathy appear to be primarily due to changes in the metabolic turnover of these macromolecules. Changes in the expression of these macromolecules may not play a major role in this process.

Efficacy of intra-articular hyaluronan (Synvisc(R)) for the treatment of osteoarthritis affecting the first metatarsophalangeal joint of the foot (hallux limitus): study protocol for a randomised placebo controlled trial.

BACKGROUND: Osteoarthritis of the first metatarsophalangeal joint (MPJ) of the foot, termed hallux limitus, is common and painful. Numerous non-surgical interventions have been proposed for this disorder, however there is limited evidence for their efficacy. Intra-articular injections of hyaluronan have shown beneficial effects in case-series and clinical trials for the treatment of osteoarthritis of the first metatarsophalangeal joint. However, no study has evaluated the efficacy of this form of treatment using a randomised placebo controlled trial. This article describes the design of a randomised placebo controlled trial to evaluate the efficacy of intra-articular hyaluronan (Synvisc(R)) to reduce pain and improve function in people with hallux limitus. METHODS: One hundred and fifty community-dwelling men and women aged 18 years and over with hallux limitus (who satisfy inclusion and exclusion criteria) will be recruited.Participants will be randomised, using a computer-generated random number sequence, to receive a single intra-articular injection of up to 1 ml hyaluronan (Synvisc(R)) or sterile saline (placebo) into the first MPJ. The injections will be performed by an interventional radiologist using fluoroscopy to ensure accurate deposition of the hyaluronan in the joint. Participants will be given the option of a second and final intra-articular injection (of Synvisc(R) or sterile saline according to the treatment group they are in) either 1 or 3 months post-treatment if there is no improvement in pain and the participant has not experienced severe adverse effects after the first injection. The primary outcome measures will be the pain and function subscales of the Foot Health Status Questionnaire. The secondary outcome measures will be pain at the first MPJ (during walking and at rest), stiffness at the first MPJ, passive non-weightbearing dorsiflexion of the first MPJ, plantar flexion strength of the toe-flexors of the hallux, global satisfaction with the treatment, health-related quality of life (assessed using the Short-Form-36 version two questionnaire), magnitude of symptom change, use of pain-relieving medication and changes in dynamic plantar pressure distribution (maximum force and peak pressure) during walking. Data will be collected at baseline, then 1, 3 and 6 months post-treatment. Data will be analysed using the intention to treat principle. DISCUSSION: This study is the first randomised placebo controlled trial to evaluate the efficacy of intra-articular hyaluronan (Synvisc(R)) for the treatment of osteoarthritis of the first MPJ (hallux limitus). The study has been pragmatically designed to ensure that the study findings can be implemented into clinical practice if this form of treatment is found to be an effective treatment strategy. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry: ACTRN12607000654459.

Distinguishing aggrecan loss from aggrecan proteolysis in ADAMTS-4 and ADAMTS-5 single and double deficient mice.

Aggrecan loss from mouse cartilage is predominantly because of ADAMTS-5 activity; however, the relative contribution of other proteolytic and nonproteolytic processes to this loss is not clear. This is the first study to compare aggrecan loss with aggrecan processing in mice with single and double deletions of ADAMTS-4 and -5 activity (Deltacat). Cartilage explants harvested from single and double ADAMTS-4 and -5 Deltacat mice were cultured with or without interleukin (IL)-1alpha or retinoic acid and analyzed for (i) the kinetics of (35)S-labeled aggrecan loss, (ii) the pattern of (35)S-labeled aggrecan fragments released into the media and retained in the matrix, (iii) the pattern of total aggrecan fragments released into the media and retained in the matrix, and (iv) specific cleavage sites within the interglobular and chondroitin sulfate-2 domains. The loss of radiolabeled aggrecan from ADAMTS-4/-5 Deltacat cartilage was less than that from ADAMTS-4, ADAMTS-5, or wild-type cartilage under nonstimulated conditions. IL-1alpha and retinoic acid stimulated radiolabeled aggrecan loss from wild-type and ADAMTS-4 Deltacat cartilage, but there was little effect on ADAMTS-5 cartilage. Proteolysis of aggrecan contributed most to its loss in wild-type, ADAMTS-4, and ADAMTS-5 Deltacat cartilage explants. The pattern of proteolytic processing of aggrecan in these cultures was consistent with that occurring in cartilage pathologies. Retinoic acid, but not IL-1alpha, stimulated radiolabeled aggrecan loss from ADAMTS-4/-5 Deltacat cartilage explants. Even though there was a 300% increase in aggrecan loss from ADAMTS-4/-5 Deltacat cartilage stimulated with retinoic acid, the loss was not associated with aggrecanase cleavage but with the release of predominantly intact aggrecan consistent with the phenotype of the ADAMTS-4/-5 Deltacat mouse. Our results show that chondrocytes have additional mechanism for the turnover of aggrecan and that when proteolytic mechanisms are blocked by ablation of aggrecanase activity, nonproteolytic mechanisms compensate to maintain cartilage homeostasis.

Sulfated polysaccharides inhibit the catabolism and loss of both large and small proteoglycans in explant cultures of tendon.

This study investigated the effects of two highly sulfated polysaccharides, calcium pentosan polysulfate and heparin, on the loss of newly synthesized proteoglycans from the matrix of explant cultures of bovine tendon. The tensional region of deep flexor tendon was incubated with [35S]sulfate for 6 h and then placed in culture for up to 15 days. The amount of radiolabel associated with proteoglycans lost to the medium and retained in the matrix was determined for each day in culture. It was shown that both sulfated polysaccharides at concentrations of 1000 microg x mL(-1) inhibited the loss of 35S-labeled large and small proteoglycans from the matrix and concomitant with this was a retention of chemical levels of proteoglycans in the explant cultures. In other explant cultures that were maintained in culture in the presence of both agents for more than 5 days after incubation with [35S]sulfate, inhibition of the intracellular catabolic pathway was evident, indicating that these highly sulfated polysaccharides also interfered with the intracellular uptake of small proteoglycans by tendon cells.

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.

Large aggregating and small leucine-rich proteoglycans are degraded by different pathways and at different rates in tendon.

This work investigated the kinetics of catabolism and the catabolic fate of the newly synthesized (35)S-labelled proteoglycans present in explant cultures of tendon. Tissue from the proximal region of bovine deep flexor tendon was incubated with [(35)S]sulfate for 6 h and then placed in explant cultures for periods of up to 15 days. The amount of radiolabel associated with proteoglycans and free [(35)S]sulfate lost to the medium and retained in the matrix was determined for each day in culture. It was shown that the rate of catabolism of radiolabelled small proteoglycans (decorin and biglycan) was significantly slower (T((1/2)) > 20 days) compared with the radiolabelled large proteoglycans (aggrecan and versican) that were rapidly lost from the tissue (T((1/2)) approximately 2 days). Both the small and large newly synthesized proteoglycans were lost from the matrix with either intact or proteolytically modified core proteins. When explant cultures of tendon were maintained either at 4 degrees C or in the presence of the lysosomotrophic agent ammonium chloride, inhibition of the cellular catabolic pathway for small proteoglycans was demonstrated indicating the involvement of cellular activity and lysosomes in the catabolism of small proteoglycans. It was estimated from these studies that approximately 60% of the radiolabelled small proteoglycans that were lost from the tissue were degraded by the intracellular pathway present in tendon cells. This work shows that the pathways of catabolism for large aggregating and small leucine-rich proteoglycans are different in tendon and this may reflect the roles that these two populations of proteoglycans play in the maintenance of the extracellular matrix of tendon.

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.

Selective inhibition of ADAMTS-1, -4 and -5 by catechin gallate esters.

Three mammalian ADAMTS enzymes, ADAMTS-1, -4 and -5, are known to cleave aggrecan at certain glutamyl bonds and are considered to be largely responsible for cartilage aggrecan catabolism observed during the development of arthritis. We have previously reported that certain catechins, polyphenolic compounds found in highest concentration in green tea (Camellia sinensis), are capable of inhibiting cartilage aggrecan breakdown in an in vitro model of cartilage degradation. We have now cloned and expressed recombinant human ADAMTS-1, -4 and -5 and report here that the catechin gallate esters found in green tea potently inhibit the aggrecan-degrading activity of these enzymes, with submicromolar IC50 values. Moreover, the concentration needed for total inhibition of these members of the ADAMTS group is approximately two orders of magnitude lower than that which is needed to partially inhibit collagenase or ADAM-10 activity. Catechin gallate esters therefore provide selective inhibition of certain members of the ADAMTS group of enzymes and could constitute an important nutritional aid in the prevention of arthritis as well as being part of an effective therapy in the treatment of joint disease and other pathologies involving the action of these enzymes.