PubMed Health⌕ Search

Biomedical subjects

J D Sandy

Publications and source records attributed to J D Sandy.

At least 19 recordsLinked to original sources
PubMed2006

ADAMTS5-mediated aggrecanolysis in murine epiphyseal chondrocyte cultures.

OBJECTIVE: Aggrecan degradation by aggrecanases [a disintegrin and metalloproteinase with thrombospondin-like motifs (ADAMTS) 1, 4, 5, 8, 9, 15] is considered to initiate much of the cartilage pathology seen in human arthritis, however, the proteinase responsible and its mode of control is unclear. The present work was done to examine mechanisms of aggrecanase control in a novel murine epiphyseal cell system and to determine whether ADAMTS5 alone is responsible for aggrecanolysis by these cells. METHODS: Epiphyseal cells from 4-day-old mice (wild type, TS-5 (-/-), CD44(-/-), syndecan-1(-/-), membrane type-4 matrix metalloproteinase [MT4MMP(-/-)]) were maintained in non-adherent aggregate cultures and aggrecanolysis studied by biochemical and histochemical methods. Confocal immunolocalization analyses were done with specific probes for ADAMTS5, hyaluronan (HA) and aggrecanase-generated fragments of aggrecan. RESULTS: Aggrecanolysis by these cells was specifically aggrecanase-mediated and it occurred spontaneously without the need for addition of catabolic stimulators. Chondrocytes from ADAMTS5-null mice were aggrecanase-inactive whereas all other mutant cells behaved as wild type in this regard suggesting that ADAMTS5 activity is not controlled by CD44, syndecan-1 or MT4MMP in this system. Immunohistochemical analysis supported the central role for ADAMTS5 in the degradative pathway and indicated that aggrecanolysis occurs primarily in the HA-poor pericellular region in these cultures. CONCLUSION: These findings are consistent with published in vivo studies showing that single-gene ADAMTS5 ablation confers significant protection on cartilage in murine arthritis. We propose that this culture system and the analytical approaches described provide a valuable framework to further delineate the expression, activity and control of ADAMTS-mediated aggrecanolysis in human arthritis.

ADAM Proteins↗
PubMed2005

A contentious issue finds some clarity: on the independent and complementary roles of aggrecanase activity and MMP activity in human joint aggrecanolysis.

Our understanding of aggrecanolysis in the human joint has recently been clarified by detailed analysis of naturally occurring intermediates in cartilage and synovial fluids. The most studied aspect has been the proteolysis of the interglobular domain (IGD) of aggrecan with release of the glycosaminoglycan (GAG)-attachment regions, because this appears to be most destructive to tissue function. In this Editorial review, a working model is presented which supports the view that one or more aggrecanases (ADAMTS 1, 4, 5, 8, 9, 15) are responsible for cleavage of the IGD with destructive loss of tissue GAG. In contrast, one or more metalloproteinases (MMPs) (MMP 1, 2, 3, 7, 8, 9, 10, 13, 14, 19, 20) are responsible for cleavage of the IGD (at Asn360-Phe361) within a separate pool of aggrecan, which does not bear GAG, because it has previously been C-terminally truncated in a separate slow turnover process. These findings, along with recent gene deletion studies in mice, suggest that ADAMTS-mediated aggrecanolysis is destructive to cartilage function whereas MMP-mediated aggrecanolysis may actually be beneficial.

Aggrecans↗
PubMed2005

Analysis of ADAMTS4 and MT4-MMP indicates that both are involved in aggrecanolysis in interleukin-1-treated bovine cartilage.

OBJECTIVE: To investigate the mechanism of aggrecanolysis in interleukin-1 (IL-1)-treated cartilage tissue by examining the time course of aggrecan cleavages and the tissue and medium content of membrane type 4-matrix metalloproteinases (MT4-MMP) and a disintegrin and metalloproteinase with thrombospondin type I motifs (ADAMTS)4. METHODS: Articular cartilage explants were harvested from newborn bovine femoropatellar groove. The effects of IL-1 treatment with or without aggrecanase blockade were investigated by Western analysis of aggrecan fragment generation, ADAMTS4 species (p68 and p53), and MT4-MMP, as well as by realtime PCR (polymerase chain reaction) for ADAMTS4 and 5. Aggrecanase was blocked with mannosamine (ManN), an inhibitor of glycosylphosphatidylinositol anchor synthesis, and esculetin (EST), an inhibitor of MMP-1, MMP-3, and MMP-13 gene expression. RESULTS: IL-1 treatment caused a major increase in MT4-MMP abundance in the tissue and medium. ADAMTS4 (p68) was abundant in fresh cartilage and this was retained in the tissue in untreated cartilage. IL-1 treatment for 6 days caused a marked loss of p68 from the cartilage and the appearance of p53 in the medium. Addition of either 1.35 mM ManN or 31-500 microM EST blocked IL-1-mediated aggrecanolysis and this was accompanied by nearly complete inhibition of the MT4-MMP increase, the p68 loss and the formation of p53. IL-1 treatment increased mRNA abundance for ADAMTS4 ( approximately 3-fold) and ADAMTS5 ( approximately 10-fold) but this was not accompanied by a marked change in enzyme protein abundance. CONCLUSION: These studies support a central role for MT4-MMP in IL-1-induced cartilage aggrecanolysis and are consistent with the identification of p68 as the aggrecanase that cleaves within the CS2 domain, and of p53 as the aggrecanase that generates G1-NITEGE. Since the induction by IL-1 was not accompanied by marked changes in total ADAMTS4 protein, but rather in partial conversion of p68 to p53 and release of both from the tissue, we conclude that aggrecanolysis in this model system results from MT4-MMP-mediated processing of a resident pool of ADAMTS4 and release of the p68 and p53 from their normal association with the cell surface.

ADAM Proteins↗
PubMed2002

Association between protease-specific proteolytic cleavage of brevican and synaptic loss in the dentate gyrus of kainate-treated rats.

Proteolytic fragments generated by ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs)-mediated cleavage of the aggregating chondroitin sulfate proteoglycan, brevican, have been identified, but not localized in the CNS. The purpose of this study, using kainate-induced CNS lesion, was to examine the spatial and quantitative relationship between ADAMTS1 and 4 mRNA expression and ADAMTS-mediated cleavage of brevican (as determined by the abundance of the neo-epitope QEAVESE at the C-terminal of the cleaved brevican G1 domain). In untreated rats, in situ hybridization and reverse transcriptase polymerase chain reaction indicated that ADAMTS4 expression was higher than ADAMTS1 and was localized to hippocampus, temporal lobe and other areas of cortex, striatum and hypothalamus. ADAMTS4 mRNA expression in these regions correlated with the presence of the QEAVESE neo-epitope, which was concentrated in perineuronal nets and in neuropil. In rats that seized after kainate, there was a dramatic elevation in ADAMTS1 and ADAMTS4 transcript that correlated and co-localized with a robust elevation in an extractable, 55-kDa fragment of brevican in temporal lobe and hippocampus. This fragment consisted, at least in part, of the ADAMTS-cleaved epitope G1-QEAVESE. The kainate-induced elevation in this ADAMTS-cleaved fragment was localized to amygdaloid and thalamic nuclei, hippocampus, caudate-putamen, cingulate cortex, and the outer molecular layer of the dentate gyrus where it was accompanied by a robust elevation in ADAMTS1 and 4 mRNA and a 28% decline in synaptic density 5 days after kainate.Thus, complexes of extracellular matrix proteins that exist in perineuronal nets and in the neuropil are cleaved by specific matrix-degrading proteases at early time points during excitotoxic neurodegeneration. The observed ADAMTS-induced cleavage of brevican in the dentate outer molecular layer is closely associated with diminished synaptic density, and may, therefore, contribute to synaptic loss and/or reorganization in this region.

ADAM Proteins↗
PubMed2001

Analysis of aggrecan in human knee cartilage and synovial fluid indicates that aggrecanase (ADAMTS) activity is responsible for the catabolic turnover and loss of whole aggrecan whereas other protease activity is required for C-terminal processing in vivo.

Studies of aggrecan proteolysis in human joints have implicated both the aggrecanase [ADAMTS, a disintegrin-like and metalloprotease (reprolysin-type) with thrombospondin type 1 motif] and matrix metalloproteinase (MMP) families. We have analysed the aggrecan core protein species present in vivo in both articular cartilage and synovial fluids from normal, acutely injured and osteoarthritic joints. Normal cartilage contains at least seven major G1 domain (the N-terminal globular domain of aggrecan)-bearing species, of which three (full-length core, G1-NITEGE(373) and G1-VDIPEN(341)) have been identified. The C-terminals of the others are unknown but digestion of fetal human aggrecan with MMP-3 and crude aggrecanase suggests that they are products of MMP-like activity in vivo. Normal synovial fluids contain at least 10 species, of which nine result from ADAMTS-dependent cleavage, and this cleavage occurs at all of the five known aggrecanase sites. Aggrecan fragments in the cartilage and synovial fluids of acutely injured joints are generally similar to normal, but all contain a markedly increased ratio of G1-NITEGE to G1-VDIPEN. Aggrecan from the cartilage of late-stage osteoarthritis patients is remarkably similar to normal, whereas the synovial fluid aggrecan is more fragmented than that from normal or injured knees. The analyses suggest that the role of the ADAMTS and these MMP-like activities in human cartilage are distinctly different. Excessive ADAMTS activity in vivo is destructive to cartilage matrix, since the bulk of the glycosaminoglycan (GAG)-bearing products are released from the tissue into the synovial fluid following cleavage of the Glu(373)-Ala(374) bond. In contrast, the MMP-like activity appears to be essentially non-destructive, since much of the GAG-bearing product is retained in the tissue following cleavages that are in the more C-terminal regions of the molecule.

Aggrecans↗
PubMed2001

Intact aggrecan and fragments generated by both aggrecanse and metalloproteinase-like activities are present in the developing and adult rat spinal cord and their relative abundance is altered by injury.

Aggrecan is a large proteoglycan (PG) that has been grouped with different PG families on the basis of its physical characteristics. These families include the chondroitin sulfate PGs, which appear to inhibit the migration of cells and axons during development. Although aggrecan has been studied primarily in cartilage, in the present study, tissue samples from developing, mature, and injured-adult rat spinal cords were used to determine whether aggrecan is present in the mammalian spinal cord. By the use of Western blot analysis, tissues were probed with aggrecan-specific antibodies (ATEGQV, TYKHRL, and LEC-7) and aggrecan-specific neoepitope antibodies (NITEGE, FVDIPEN, and TFKEEE) to identify full-length aggrecan and several fragments. Unlike many other aggrecan gene family members, aggrecan species were similar in embryonic day 14, postnatal day 1, and adult spinal cords. Spinal cord injury caused significant decreases in aggrecan. Partial recovery in some aggrecan species was evident by 2 weeks after injury. The presence of specific aggrecan neoepitopes suggested that aggrecan is cleaved in the spinal cord by both a disintegrin and metalloproteinase thrombospondin (also known as aggrecanase) and metalloproteinase-like activities. Many aggrecan species found in the spinal cord were similar to species in cartilage. Additional antibodies were used to identify two other aggrecan gene family members, neurocan and brevican, in the adult spinal cord. These studies present novel information on the aggrecan core protein species and enzymes involved in aggrecan cleavage in vivo in the rat spinal cord throughout development and after injury. They also provide the basis for investigating the function of aggrecan in the spinal cord.

Aggrecans↗
PubMed2001

Versican V1 proteolysis in human aorta in vivo occurs at the Glu441-Ala442 bond, a site that is cleaved by recombinant ADAMTS-1 and ADAMTS-4.

Mature human aorta contains a 70-kDa versican fragment, which reacts with a neoepitope antiserum to the C-terminal peptide sequence DPEAAE. This protein therefore appears to represent the G1 domain of versican V1 (G1-DPEAAE(441)), which has been generated in vivo by proteolytic cleavage at the Glu(441)-Ala(442) bond, within the sequence DPEAAE(441)-A(442)RRGQ. Because the equivalent aggrecan product (G1-NITEGE(341)) and brevican product (G1-EAVESE(395)) are generated by ADAMTS-mediated cleavage of the respective proteoglycans, we tested the capacity of recombinant ADAMTS-1 and ADAMTS-4 to cleave versican at Glu(441)-Ala(442). Both enzymes cleaved a recombinant versican substrate and native human versican at the Glu(441)-Ala(442) bond and the mature form of ADAMTS-4 was detected by Western analysis of extracts of aortic intima. We conclude that versican V1 proteolysis in vivo can be catalyzed by one or more members of the ADAMTS family of metalloproteinases.

ADAM Proteins↗
PubMed2000

Chondrocyte extracellular matrix synthesis and turnover are influenced by static compression in a new alginate disk culture system.

The goal of this study was to examine the effects of mechanical compression on chondrocyte biosynthesis of extracellular matrix (ECM) components during culture in a new alginate disk culture system. Specifically, we have examined chondrocyte biosynthesis rates, and the structure of aggrecan core protein species present in the cell-associated matrix (CM), in the further removed matrix (FRM) and in the surrounding culture medium. In this alginate disk culture system, chondrocytes can be subjected to mechanical deformations similar to those experienced in vivo. Our results show that over an 8-week culture period, chondrocytes synthesize a functional ECM and can respond to mechanical forces similarly to chondrocytes maintained in native cartilage. In the alginate disk system, static compression was shown to decrease and dynamic compression to increase synthesis of aggrecan of bovine chondrocytes. Western blot analysis of the core proteins of aggrecan molecules identified a number of different species that were present in different relative amounts in the CM, FRM, and medium. Over 21 days of culture, the predominant form of aggrecan found in the ECM was a full-length link-stabilized species. In addition, our data show that the application of 40 h of static compression caused an increase in the proportion of newly synthesized aggrecan molecules released into the medium. However, this was not accompanied by a significant change in the size and composition of aggrecan and aggrecan fragments in the different compartments, suggesting that mechanical compression did not alter the catabolic pathways. Together, these data show that chondrocyte function is maintained in an alginate disk culture system and that this culture system is a useful model to examine chondrocyte ECM assembly and some aspects of catabolism normally found in vivo.

Aggrecans↗
PubMed2000

The intermediates of aggrecanase-dependent cleavage of aggrecan in rat chondrosarcoma cells treated with interleukin-1.

We have examined the abundance and structure of intermediates in the chondrocyte-mediated degradation of aggrecan by aggrecanase(s). Degradation products were identified by Western-blot analysis with antibodies to cleavage-site neoepitopes and to peptides within the globular domains. Rat chondrosarcoma tumour contained full-length aggrecan and all of the individual peptides expected from single independent cleavages at each of the four aggrecanase sites in the chondroitin sulphate (CS) domain. Kinetic analysis of the products present in rat chondrosarcoma cell cultures treated with interleukin-1b showed that the first aggrecanase-mediated cleavages occurred at the four sites within the CS attachment region to generate two stable intermediates, Val(1)-Glu(1459) and Val(1)-Glu(1274). These species were subsequently cleaved at the Glu(373) site in the interglobular domain to form the terminal products, Val(1)-Glu(373), Ala(374)-Glu(1274) and Ala(374)-Glu(1459). It therefore appears that the aggrecanase-mediated processing of native aggrecan by chondrocytes in situ is initiated within the CS-attachment region and completed by cleavage within the interglobular domain. Since it has been shown that digestion of aggrecan monomer in solution with recombinant ADAMTS-4 [Tortorella, Pratta, Liu, Austin, Ross, Abbaszade, Burn and Arner (2000) Sites of aggrecan cleavage by recombinant human aggrecanase-1 (ADAMTS-4). J. Biol. Chem. 275, 18566-18573] exhibits similar kinetics, it appears that preferential proteinase cleavage in the CS-rich region is determined by properties inherent in the aggrecan monomer itself, such as preferred peptide sequences for enzyme binding or enhanced accessibility to the core protein at these sites.

Aggrecans↗
PubMed2000

Mannosamine inhibits aggrecanase-mediated changes in the physical properties and biochemical composition of articular cartilage.

The enzymatic processes underlying the degradation of aggrecan in cartilage and the corresponding changes in the biomechanical properties of the tissue are an important part of the pathophysiology of osteoarthritis. Recent studies have demonstrated that the hexosamines glucosamine (GlcN) and mannosamine (ManN) can inhibit aggrecanase-mediated cleavage of aggrecan in IL-1-treated cartilage cultures. The term aggrecanase describes two or more members of the ADAMTS family of metalloproteinases whose glutamyl endopeptidase activity is known to be responsible for much of the aggrecan degradation seen in human arthritides. In this study we examined the effect of ManN and GlcN on aggrecanase-mediated degradation of aggrecan induced by IL-1alpha and the corresponding tissue mechanical properties in newborn bovine articular cartilage. After 6 days of culture in 10 ng/ml IL-1 plus ManN, mechanical testing of explants in confined compression demonstrated that ManN inhibited the IL-1alpha-induced degradation in tissue equilibrium modulus, dynamic stiffness, streaming potential, and hydraulic permeability, in a dose-dependent fashion, with peak inhibition ( approximately 75-100% inhibition) reached by a concentration of 1.35 mM. Aggrecan from explants cultured in IL-1 was found by Western analysis to be almost entirely processed down to the G1-NITEGE(373) end product. Addition of ManN or GlcN was found to produce 75-90% inhibition of this cleavage, but the proportion of aggrecan remaining in the tissue which was cleaved at aggrecanase sites in the chondroitin sulfate (CS)-rich region (Glu(1501) and Glu(1687)) was higher than with IL-1 alone. This result suggests that the preservation of mechanical properties by hexosamines in explants is primarily due to inhibition of cleavage at the Glu(373) site in the interglobular domain. While the precise mechanism by which hexosamines function in this system is unclear, the present analysis suggests that the mechanical properties examined may be predominantly a function of electrostatic repulsion due to the charged CS chains in the tightly packed repetitive sequences of the CS-1 region.

Aggrecans↗
PubMed1999

Chondrocyte-mediated catabolism of aggrecan: evidence for a glycosylphosphatidylinositol-linked protein in the aggrecanase response to interleukin-1 or retinoic acid.

The control of chondrocyte-mediated degradation of aggrecan has been studied in rat chondrosarcoma cells and bovine cartilage explants treated with either IL-1 or retinoic acid. The capacity of glucosamine to inhibit the aggrecanase-mediated response (J. D. Sandy, D. Gamett, V. Thompson, and C. Verscharen (1998) Biochem. J. 335, 59-66) has been extended to an investigation of the effect of other hexosamines. Mannosamine inhibits the aggrecanase response to both IL-1 and RA at about one-tenth the concentration of glucosamine in both rat cell and bovine explant systems. This effect of mannosamine appears to be due to its capacity to inhibit the synthesis of glycosylphosphatidylinositol (GPI)-linked proteins by chondrocytes since the GPI synthesis inhibitor 2-deoxyfluoroglucose (2-DFG) also inhibited the aggrecanase response to IL-1b and RA in rat cells. Moreover, phosphatidylinositol-specific phospholipase C (PIPLC) treatment of rat cells markedly inhibited the aggrecanase response to IL-1b and RA. These inhibitory effects of mannosamine, 2-DFG, and PIPLC in rat cells did not appear to be due to an interference with general biosynthetic activity of the cells as measured by [3H]proline incorporation into secreted proteins. We suggest that the aggrecanase response by chondrocytes to IL-1 and RA is dependent on the activity of a GPI-anchored protein on the chondrocyte cell surface.

Aggrecans↗
PubMed1999

Physical and biological regulation of proteoglycan turnover around chondrocytes in cartilage explants. Implications for tissue degradation and repair.

The development of clinical strategies for cartilage repair and inhibition of matrix degradation may be facilitated by a better understanding of (1) the chondrocyte phenotype in the context of a damaged extracellular matrix, and (2) the roles of biochemical and biomechanical pathways by which matrix metabolism is mediated. Using methods of quantitative autoradiography, we examined the cell-length scale patterns of proteoglycan deposition and turnover in the cell-associated matrices of chondrocytes in adult bovine and calf cartilage explants. Results highlight a rapid turnover in the pericellular matrix, which may indicate spatial organization of PG metabolic pools, and specific biomechanical roles for different matrix regions. Subsequent to injurious compression of calf explants, which resulted in grossly visible tissue cracks and caused a decrease in the number of viable chondrocytes within explants, cell-mediated matrix catabolic processes appeared to increase, resulting in apparently increased rates of proteoglycan turnover around active cells. Furthermore, the influences of cell-stimulatory factors such as IL-1 beta appeared to be delayed in their effects subsequent to injurious compression, suggesting interactions between biomechanical and biochemical pathways of PG degradation. These results may provide a useful reference point in the development of in vitro models for cartilage injury and disease, and hint at possible new approaches in the development of cartilage repair strategies.

Animals↗
PubMed1998

Chondrocyte-mediated catabolism of aggrecan: aggrecanase-dependent cleavage induced by interleukin-1 or retinoic acid can be inhibited by glucosamine.

A rat chondrosarcoma cell line and bovine cartilage explants have been used to study the control of aggrecan degradation by chondrocytes treated with interleukin-1 (IL-1) or retinoic acid (RA). Aggrecan fragment analysis with anti-neo-epitope antibodies suggests that aggrecanase (an as yet unidentified enzyme) is the only aggrecan-degrading proteinase active in these cultures. With rat cells, aggrecanase converts the aggrecan core protein into two major G1-domain-bearing products (60 kDa with a C-terminal Glu-373, and 220 kDa with a C-terminal Glu-1459). Both products were quantified on a standardized Western analysis system with a G1-specific antibody. Immunoblots were analysed by scanning densitometry and the sensitivity, linearity and reproducibility of the assay were established. With rat cells the aggrecanase response to IL-1 was optimal at about 2 mM glutamine, but was progressively inhibited at higher concentrations, with about 90% inhibition at 10 mM glutamine. Such inhibition by glutamine was not, however, observed with bovine explants. On the other hand, marked inhibition of aggrecanase-dependent cleavage was observed with both rat cells and bovine explants when d(+)-glucosamine was included at concentrations above 2 mM. Inhibition was apparently not due to cytotoxicity or interference with IL-1 signalling, since biosynthetic activity was not inhibited and inhibition of the aggrecanase response was also obtained when RA was used as the catabolic stimulator. Possible mechanisms for the inhibition of the aggrecanase response by glucosamine in chondrocytes treated with IL-1 or RA are discussed.

Aggrecans↗
PubMed1998

Effects of injurious compression on matrix turnover around individual cells in calf articular cartilage explants.

The effects of mechanical injury on the metabolism of cartilage matrix are of interest for understanding the pathogenesis of osteoarthrosis and the development of strategies for cartilage repair. The purpose of the present study was to examine the effects of injury on matrix turnover in a calf articular cartilage explant system for which the effects of mechanical loading on cell activity and the cell-mediated pathways of matrix metabolism are already well characterized. New methods of quantitative autoradiography were used in combination with established biochemical and biomechanical techniques for the analysis of cell and matrix responses to acute mechanical injury, with particular attention to the processes of localized matrix turnover in the cell-associated matrices of individual chondrocytes. Matrix deposition and turnover around cells in control explants was spatially dependent, with the highest rates of proteoglycan deposition and turnover and the lowest rates of collagen deposition (as indicated by [3H]proline autoradiography) occurring in the pericellular matrix. Injurious compression was associated with (a) an abrupt decrease in the tensile load-carrying capacity of the collagen matrix, apparently associated with mechanical failure of the tissue, (b) a considerable but subtotal decrease in cell viability, marked by the emergence of an apparently inactive cell population interspersed within catabolically active but abnormally large cells, and (c) sustained, elevated rates of proteoglycan turnover, particularly in the cell-associated matrices of apparently viable cells, which involved the increased release of aggregating species in addition to a spectrum of degradation fragments that were also in controls. These results may represent an in vitro model for the responses of chondrocytes and the cartilage extracellular matrix to mechanical injury.

Animals↗
PubMed1997

Inhibition of cartilage degradation and changes in physical properties induced by IL-1beta and retinoic acid using matrix metalloproteinase inhibitors.

Bovine cartilage explants were treated with 100 ng/ml recombinant human interleukin-1beta (IL-1beta) or 1 microM all-trans retinoic acid (RA) and changes in biochemical, biomechanical, and physicochemical properties were assessed. Additionally, samples cultured with IL-1beta or RA were treated with 4 microM recombinant human tissue inhibitor of metalloproteinases-1 (TIMP-1) or a synthetic metalloproteinase inhibitor (L-758,354) to inhibit this degradation. Treatment with IL-1beta or RA each resulted in >90% GAG loss after 8 days in culture. Addition of TIMP or L-758,354 to the culture media inhibited IL-1beta-induced loss of tissue GAG by 40 and 65%, respectively, and inhibited RA-induced GAG loss by 35 and 65%, respectively. Analysis of degradation products in the culture media using a G1 antibody indicated that IL-1beta- and RA-treated plugs released 68-kDa fragments of aggrecan, corresponding to a segment of the aggrecan core protein from the G1 domain to the C-terminus NITEGE, consistent with "aggrecanase" activity. Release of the G1 fragment was inhibited by treatment with L-758,354. Both IL-1beta and RA induced significant loss of hyaluronan from cartilage explants after 8 days of exposure and HA loss was also inhibited by addition of L-756,354 to the culture media. IL-1beta, but not RA, induced a significant increase in swelling ratio (wet weight in 0.01 M NaCl normalized to wet weight in DMEM) after 8 days in culture, consistent with degradation of the collagen network, and the increase in tissue swelling was inhibited by treatment with TIMP-1 or L-758,354. Exposure to IL-1beta or RA resulted in significant changes in cartilage physical properties including streaming potential, equilibrium modulus, hydraulic permeability, and electrokinetic coupling coefficient after 8 days in culture, and these changes were inhibited by 40-90% by exposure to TIMP and by 50-90% by exposure to L-758,354. Measurement of dynamic streaming potential showed that changes due to treatment with IL-1beta alone were highly dependent in compression frequency, with dramatic changes seen at high frequency prior to changes in mechanical properties, and little initial change seen at low frequency. Streaming potential and equilibrium modulus of explants treated with RA decreased to 10% of their initial values after 8 days in culture, but decreased to only 40 and 90%, respectively, when treated with RA plus TIMP-1.

Aggrecans↗
PubMed1996

Activation and inhibition of endogenous matrix metalloproteinases in articular cartilage: effects on composition and biophysical properties.

Bovine cartilage explants were cultured with 1 mM 4-aminophenylmercuric acetate (APMA) to activate endogenous matrix metalloproteinases (MMPs) and changes in biochemical, biomechanical, and physicochemical properties were assessed. Additionally, graded levels of either rhTIMP-1 (recombinant human tissue inhibitor of metalloproteinases-1) or L-696-418 (a synthetic metalloproteinase inhibitor) were used to inhibit degradation induced by APMA. Treatment with APMA resulted in as much as 80% loss in tissue GAG content, a greater than threefold increase in denatured type II collagen as determined by the presence of CB11B epitope, and complete loss of biosynthetic activity after 3 days in culture. Physicochemical studies revealed that APMA treatment resulted in a significant increase in tissue swelling response, consistent with damage to the collagen network. Activation of MMPs by APMA also resulted in > 80% decrease in equilibrium modulus, dynamic stiffness, and streaming potential and > 50% decrease in electrokinetic coupling coefficient. The addition of 4 microM, 400 nM, and 40 nM TIMP inhibited PG loss by 95, 50, and 20%, respectively, and all doses effectively inhibited swelling response. The addition of 4 microM and 400 nM L-696-418 inhibited PG loss by 95% while 40 nM L-696-418 inhibited PG loss by 60%, and all doses effectively inhibited swelling response. The inhibition of APMA-induced GAG loss by 4 microM TIMP was accompanied by maintenance of streaming potential, electrokinetic coupling coefficient, dynamic stiffness, and equilibrium modulus.

Animals↗