Mirna Z. Ilic

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.

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.

Effects of long-term exposure to glucosamine and mannosamine on aggrecan degradation in articular cartilage ☆

OBJECTIVE To investigate the effect of long-term exposure to glucosamine or mannosamine on the catabolism of aggrecan by explant cultures of bovine articular cartilage maintained in the presence of retinoic acid. DESIGN The kinetics of loss of 35S-labeled and total aggrecan from explant cultures of bovine articular cartilage maintained in the presence of 1 micro M retinoic acid and exposed to varying concentrations of glucosamine or mannosamine was investigated over a 9-day culture period. In other experiments, the reversibility of the inhibition of aggrecan catabolism by glucosamine or mannosamine was investigated in cultures exposed to these amino sugars for the first 5 days of a 15-day culture period. The metabolism of chondrocytes exposed to these amino sugars was evaluated by measurement of lactate production or 3H-serine and 35S-sulfate incorporation into protein and glycosaminoglycans, respectively. The direct effect of these amino sugars on soluble aggrecanase activity was determined from immunoblots of aggrecan digests. RESULTS Glucosamine at 5mM concentration and mannosamine at 2mM concentration inhibited degradation of radiolabeled and chemical levels of aggrecan. At concentrations of up to 10mM amino sugars, the metabolism of chondrocytes was not impaired, as determined by lactate production, protein synthesis and the incorporation of 35S-sulfate into proteoglycans. These amino sugars did not inhibit soluble aggrecanase activity. The exposure of articular cartilage explants to 5mM glucosamine or mannosamine for 5 days in culture in the presence or absence of retinoic acid did not provide long-term suppression of stimulated aggrecan loss. CONCLUSIONS This study indicates that continuous presence of amino sugars is required to protect cartilage from stimulated loss of aggrecan.

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.

Highly sulfated glycosaminoglycans inhibit aggrecanase degradation of aggrecan by bovine articular cartilage explant cultures

The catabolism of 35S-labeled aggrecan and loss of tissue glycosaminoglycans was investigated using bovine articular cartilage explant cultures maintained in medium containing 10(-6) M retinoic acid or 40 ng/ml recombinant human interleukin-1alpha (rHuIL-1alpha) and varying concentrations (1-1000 microg/ml) of sulfated glycosaminoglycans (heparin, heparan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate and keratan sulfate) and calcium pentosan polysulfate (10 microg/ml). In addition, the effect of the sulfated glycosaminoglycans and calcium pentosan polysulfate on the degradation of aggrecan by soluble aggrecanase activity present in conditioned medium was investigated. The degradation of 35S-labeled aggrecan and reduction in tissue levels of aggrecan by articular cartilage explant cultures stimulated with retinoic acid or rHuIL-1alpha was inhibited by heparin and heparan sulfate in a dose-dependent manner and by calcium pentosan polysulfate. In contrast, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate and keratan sulfate did not inhibit the degradation of 35S-labeled aggrecan nor suppress the reduction in tissue levels of aggrecan by explant cultures of articular cartilage. Heparin, heparan sulfate and calcium pentosan polysulfate did not adversely affect chondrocyte metabolism as measured by lactate production, incorporation of [35S]-sulfate or [3H]-serine into macromolecules by articular cartilage explant cultures. Furthermore, heparin, heparan sulfate and calcium pentosan polysulfate inhibited the proteolytic degradation of aggrecan by soluble aggrecanase activity. These results suggest that highly sulfated glycosaminoglycans have the potential to influence aggrecan catabolism in articular cartilage and this effect occurs in part through direct inhibition of aggrecanase activity.

Cathepsin D cleaves aggrecan at unique sites within the interglobular domain and chondroitin sulfate attachment regions that are also cleaved when cartilage is maintained at acid pH.

Bovine aggrecan was digested with bovine cathepsin D at pH 5.2 under conditions of partial digestion and the resulting aggrecan core protein fragments were separated by electrophoresis on gradient polyacrylamide gels. The fragments were characterized by their reactivity to specific antibodies and by N-terminal amino acid sequencing. It was also demonstrated that cathepsin D cleaved bovine aggrecan at five sites within the core protein, between residues Phe(342)-Phe(343) in the interglobular domain, Leu(1462)-Val(1463) between the chondroitin sulfate attachment regions 1 and 2 and Leu(1654)-Val(1655), Phe(1754)-Val(1755) and Leu(1854)-Ile(1855) that are located within the chondroitin sulfate attachment region 2 of the core protein. The time course of digestion showed that there was a continued degradation of aggrecan and there was no preferential cleavage of the core protein at any one site. It was shown that cathepsin D digested aggrecan over the pH range 5.2-6.5 resulting in the same products. When bovine cartilage was maintained in explant culture at pH 5.2 there was a rapid loss of both radiolabeled and chemical pools of sulfated glycosaminoglycans into the culture medium and this loss was inhibited by the inclusion in the medium of the aspartic proteinase inhibitor, pepstatin A. The aggrecan core protein fragments appearing in the medium of cultures maintained at pH 5.2 were characterized and it was shown that the fragments had N-terminal sequences starting at Phe(343), Ile(1855), and Val(1755) or Val(1463). This work demonstrates that cathepsin D present within the extracellular matrix of articular cartilage has the potential to contribute to the proteolytic processing of the core protein of aggrecan in this tissue.

CALCIUM PENTOSAN POLYSULFATE INHIBITS THE CATABOLISM OF AGGRECAN IN ARTICULAR CARTILAGE EXPLANT CULTURES

OBJECTIVE The catabolism of aggrecan and loss of aggrecan fragments from articular cartilage is a key event in the pathogenesis of arthritic diseases such as osteoarthritis. The catabolism of aggrecan is mediated by the specific proteolytic activity termed aggrecanase. The aim of this study was to investigate the effect of the chondroprotective agent calcium pentosan polysulfate (CaPPS) on the aggrecanase-mediated catabolism of aggrecan. METHODS The catabolism of 35S-labeled aggrecan and loss of tissue glycosaminoglycans (GAGs) were investigated using bovine articular cartilage explant cultures maintained in medium containing varying concentrations of CaPPS (1-100 microg/ml) in the presence or absence of 10(-6)M retinoic acid or 7 ng/ml recombinant human interleukin-1alpha (rHuIL-1alpha). In addition, the effect of CaPPS on the degradation of aggrecan monomers by aggrecanase activity present in conditioned medium from joint capsule explant cultures was investigated. RESULTS CaPPS inhibited the catabolism of 35S-labeled aggrecan in a dose-dependent manner, particularly when retinoic acid or rHuIL-1alpha was used to stimulate aggrecan catabolism. These effects were reflected in the tissue levels of GAG remaining in these cultures at the end of the experiment. CaPPS inhibited the degradation of aggrecan monomers by soluble aggrecanase activity. CONCLUSION CaPPS inhibits the catabolism of aggrecan by articular cartilage in a dose-dependent manner, particularly when the processes responsible for aggrecan loss are stimulated. This effect occurs, at least in part, through direct inhibition of aggrecanase activity. CaPPS did not adversely affect overall chondrocyte metabolism, as shown by the incorporation of 35S-sulfate and 3H-leucine into macromolecules and by lactate production in cartilage explant cultures.

Bovine joint capsule and fibroblasts derived from joint capsule express aggrecanase activity

Bovine joint capsule was maintained in explant culture in the presence of bovine aggrecan monomer and it was shown that the aggrecan monomer was degraded. Amino-terminal sequence analysis of the resulting aggrecan core protein fragments revealed that the core protein was cleaved at five specific sites attributed to glutamyl endopeptidases referred to as aggrecanase activity. Fibroblast cultures were established from explant cultures of joint capsule and when these cells were exposed to aggrecan, cleavage of the core protein of aggrecan at the aggrecanase sites was observed. Inclusion of either retinoic acid or interleukin-1alpha in medium of either joint capsule explant cultures or fibroblast cultures did not increase the rate of cleavage of exogenous aggrecan present in the culture medium. When aggrecan monomer was incubated with conditioned medium from explant cultures of joint capsule maintained in medium, degradation could be detected after 10 min. After a 6-h incubation period the same fragments of aggrecan core protein were observed as those for tissue or cells incubated directly with aggrecan monomer. RT-PCR analysis of mRNA extracted from joint capsule fibroblasts showed that these cells express both aggrecanase-1 and -2 [ADAMTS-2 (Tang) and ADAMTS-5].

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 (Δcat). Cartilage explants harvested from single and double ADAMTS-4 and -5 Δcat mice were cultured with or without interleukin (IL)-1α or retinoic acid and analyzed for (i) the kinetics of 35S-labeled aggrecan loss, (ii) the pattern of 35S-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 Δcat cartilage was less than that from ADAMTS-4, ADAMTS-5, or wild-type cartilage under nonstimulated conditions. IL-1α and retinoic acid stimulated radiolabeled aggrecan loss from wild-type and ADAMTS-4 Δcat 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 Δcat 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-1α, stimulated radiolabeled aggrecan loss from ADAMTS-4/-5 Δcat cartilage explants. Even though there was a 300% increase in aggrecan loss from ADAMTS-4/-5 Δcat 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 Δcat 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.

Structure, metabolism, and tissue roles of chondroitin sulfate proteoglycans

Publisher Summary Proteoglycans (PGs) are a complex group of glycoproteins that are characterized by the presence of one or more sulfated glycosaminoglycan (GAG) chains and various oligosaccharides that are covalently attached to a core protein. The majority of chondroitin sulfate proteoglycans (CSPGs) belongs to the group of large aggregating PGs (hyalectins) and small leucine-rich PGs. The sulfated GAG chains, oligosaccharides, and domains within the core proteins of these macromolecules allow PGs to interact with a large number of macromolecules. As a result, PGs have wide‐ranging roles in tissues where they are involved in the organization and function of extracellular matrices, matrix cell interactions, and the regulation of cellular processes. The cellular and tissue location of PGs is largely dependent on the interactions of specific domains in the core proteins and the GAG chains with other molecules present in the cell membrane or extracellular structures. Proteoglycans are multifunctional macromolecules that have wide-ranging functions within the body. Not only are they involved in the organization of extracellular functions, they also have cell regulatory functions. These attributes of PGs arise from structural domains that are present on both the core proteins and the GAG chains of this group of macromolecules.