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Biomedical subjects

T E Hardingham

Publications and source records attributed to T E Hardingham.

At least 19 recordsLinked to original sources
PubMed2001

Chondrocyte phenotype and cell survival are regulated by culture conditions and by specific cytokines through the expression of Sox-9 transcription factor.

OBJECTIVE: To investigate the effects of culture conditions, serum and specific cytokines such as insulin-like growth factor (IGF) 1 and interleukin (IL) 1alpha on phenotype and cell survival in cultures of Syrian hamster embryonic chondrocyte-like cells (DES4(+).2). METHODS: Proteins and RNA extracted from subconfluent and confluent early- and late-passage DES4(+).2 cells cultured in the presence or absence of serum and IL-1alpha or IGF-1 or both cytokines together were analysed for the expression of chondrocyte-specific genes and for the chondrogenic transcription factor Sox-9 by Western and Northern blotting. Apoptosis was assessed by agarose gel electrophoresis of labelled low-molecular weight DNA extracted from DES4(+).2 cells and another Syrian hamster embryonic chondrocyte-like cell line, 10W(+).1, cultured under the different conditions and treatments. RESULTS: Early passage DES4(+).2 cells expressed chondrocyte-specific molecules such as collagen types alpha1(II) and alpha1(IX), aggrecan, biglycan and link protein and collagen types alpha1(I) and alpha1(X) mRNAs, suggesting a prehypertrophic chondrocyte-like phenotype. The expression of all genes investigated was cell density- and serum-dependent and was low to undetectable in cell populations from later passages. Early-passage DES4(+).2 and 10W(+).1 cells survived when cultured at low cell density, but died by apoptosis when cultured at high cell density in the absence of serum or IGF-1. IGF-1 and IL-1alpha had opposite and antagonistic effects on the chondrocyte phenotype and survival. Whereas IL-1alpha acting alone suppressed cartilage-specific gene expression without significantly affecting cell survival, IGF-1 increased the steady-state mRNA levels and relieved the IL-1alpha-induced suppression of all the chondrocyte-specific genes investigated; it also enhanced chondrocyte survival. Suppression of the chondrocyte phenotype by the inflammatory cytokine IL-1alpha correlated with marked down-regulation of the transcription factor Sox-9, which was relieved by IGF-1. The expression of the Sox9 gene was closely correlated with the expression of the chondrocyte-specific genes under all conditions and treatments. CONCLUSIONS: The results suggest that the effects of cartilage anabolic and catabolic cytokines IGF-1 and IL-1alpha on the expression of the chondrocyte phenotype are mediated by Sox-9. As Sox-9 appears to be essential for matrix production, the potent effect of IL-1alpha in suppressing Sox-9 expression may limit the ability of cartilage to repair during inflammatory joint diseases.

Aggrecans↗
PubMed2000

Generation and novel distribution of matrix metalloproteinase-derived aggrecan fragments in porcine cartilage explants.

We have studied aggrecan catabolism mediated by matrix metalloproteinases (MMPs) in a porcine cartilage culture system. Using antibodies specific for DIPEN(341) and (342)FFGVG neoepitopes, we have detected MMP-derived fragments in conditioned medium and cultured cartilage, by radioimmunoassay, Western blotting, and immunolocalization. Radioimmunoassay revealed that the amount (pmol of epitope/mg of total glycosaminoglycan) of (342)FFGVG epitope released from cartilage remained constant over a 5-day culture period and was not increased by IL-1alpha or retinoate. However, the proportion (pmol of epitope/mg of released glycosaminoglycan) of (342)FFGVG epitope released was decreased upon stimulation, consistent with the involvement of a non-MMP proteinase, such as aggrecanase. The data suggest that in vitro MMPs may be involved in the base-line catabolism of aggrecan. Immunolocalization experiments showed that DIPEN(341) and ITEGE(373) epitopes were increased by treatment with IL-1alpha and retinoate. Confocal microscopy revealed that ITEGE(373) epitope was largely intracellular but with matrix staining in the superficial zone, whereas DIPEN(341) epitope was cell-associated and widely distributed in the matrix. Surprisingly, the majority of (342)FFGVG epitope, determined by radioimmunoassay and Western blotting, was retained in the tissue despite the absence of a G1 domain anchor. Interleukin-1alpha stimulation caused a marked increase in tissue DIPEN(341) and (342)FFGVG epitope, and the (342)FFGVG fragments retained in the tissue were larger than those released into the medium. Active porcine aggrecanase was unable to cleave (342)FFGVG fragments at the downward arrowGlu(373) downward arrowAla(374) bond but cleaved intact aggrecan at this site, suggesting that (342)FFGVG fragments are not substrates for aggrecanase. The apparent retention of large (342)FFGVG fragments within cartilage, and their resistance to N-terminal cleavage by aggrecanase suggests that (342)FF6V6 fragments may have a role in cartilage homeostasis.

Aggrecans↗
PubMed2000

The analysis of intermolecular interactions in concentrated hyaluronan solutions suggest no evidence for chain-chain association.

Confocal fluorescence recovery after photobleaching (confocal-FRAP) was used to examine the influence of electrolytes (NaCl, KCl, MgCl(2), MnCl(2) and CaCl(2)) on the network and hydrodynamic properties of fluoresceinamine-labelled hyaluronan (FA-HA) at concentrations up to 10 mg/ml. Self and tracer lateral diffusion coefficients showed that in Ca(2+) and Mn(2+), FA-HA (830 kDa) was more compact than in Mg(2+), Na(+) or K(+). These results were correlated with changes in the hydrodynamic radius of HA, determined by multi-angle laser-light-scattering analysis in dilute solution, which was smaller in CaCl(2) (36 nm) than in NaCl (43 nm). The permeability of more concentrated solutions of HA (<10 mg/ml) to FITC-dextran tracers (2000 kDa) was higher in CaCl(2). The properties of HA in urea (up to 6 M) were investigated to test for hydrophobic interactions and also in ethanol/water (up to 62%, v/v). In both, there was reduced hydrodynamic size and increased permeability to FITC-dextran, suggesting increased chain flexibility, but it did not show the changes predicted if chain-chain association was disrupted by urea, or enhanced by ethanol. Oligosaccharides of HA (HA(20-26)) also had no effect on the self diffusion of high-molecular-mass FA-HA (830 kDa) solutions, or on dextran tracer diffusion, showing that there were no chain-chain interactions open to competition by short-chain segments. The results suggest that the effects of electrolytes and solvent are determined primarily by their effect on HA chain flexibility, with no evidence for association between chain segments contributing significantly to the major properties.

Fluorescent Dyes↗
PubMed1999

The folded protein modules of the C-terminal G3 domain of aggrecan can each facilitate the translocation and secretion of the extended chondroitin sulfate attachment sequence.

Aggrecan is a multidomain proteoglycan containing both extended and folded protein modules. The C-terminal G3 domain contains a lectin-like, complement regulatory protein-like, and two alternatively spliced epidermal growth factor-like modules. It has been proposed that the lectin module alone has a necessary role in the intracellular translocation and secretion of proteins expressed containing G3. Constructs containing human aggrecan G3 together with 1155 bases of the adjacent chondroitin sulfate attachment region (CS-2) were prepared with different combinations and deletions of the protein modules and transfected into mammalian cells of monkey or hamster origin. The results showed that the products containing only the unfolded protein sequences (CS-2 with or without the C-terminal tail sequence) were translated and accumulated intracellularly but were not secreted. In contrast the constructs containing any of the folded protein modules and the extended CS-2 region were translated and secreted from the cells. The results show that the lectin module was not unique in facilitating the intracellular translocation and secretion of the G3 domain. The conservation of G3-like domains within the aggrecan family of proteoglycans may therefore result from their participation in other extracellular functions.

Aggrecans↗
PubMed1999

The molecular basis of the solution properties of hyaluronan investigated by confocal fluorescence recovery after photobleaching.

Hyaluronan (HA) is a highly hydrated polyanion, which is a network-forming and space-filling component in the extracellular matrix of animal tissues. Confocal fluorescence recovery after photobleaching (confocal-FRAP) was used to investigate intramolecular hydrogen bonding and electrostatic interactions in hyaluronan solutions. Self and tracer lateral diffusion coefficients within hyaluronan solutions were measured over a wide range of concentrations (c), with varying electrolyte and at neutral and alkaline pH. The free diffusion coefficient of fluoresceinamine-labeled HA of 500 kDa in PBS was 7.9 x 10(-8) cm(2) s(-1) and of 830 kDa HA was 5.6 x 10(-8) cm(2) s(-1). Reductions in self- and tracer-diffusion with c followed a stretched exponential model. Electrolyte-induced polyanion coil contraction and destiffening resulted in a 2.8-fold increase in self-diffusion between 0 and 100 mM NaCl. Disruption of hydrogen bonds by strong alkali (0.5 M NaOH) resulted in further larger increases in self- and tracer-diffusion coefficients, consistent with a more dynamic and permeable network. Concentrated hyaluronan solution properties were attributed to hydrodynamic and entanglement interactions between domains. There was no evidence of chain-chain associations. At physiological electrolyte concentration and pH, the greatest contribution to the intrinsic stiffness of hyaluronan appeared to be due to hydrogen bonds between adjacent saccharides.

Animals↗
PubMed1998

Macromolecular diffusion of biological polymers measured by confocal fluorescence recovery after photobleaching.

Fluorescence recovery after photobleaching with an unmodified confocal laser scanning microscope (confocal FRAP) was used to determine the diffusion properties of network forming biological macromolecules such as aggrecan. The technique was validated using fluorescein isothiocyanate (FITC)-labeled dextrans and proteins (molecular mass 4-2000 kDa) at 25 degrees C and with fluorescent microspheres (207 nm diameter) over a temperature range of 5-50 degrees C. Lateral diffusion coefficients (D) were independent of the focus position, and the degree and extent of bleach. The free diffusion coefficient (Do) of FITC-aggrecan determined by confocal FRAP was 4.25 +/- 0.6 x 10(-8) cm2 s-1, which is compatible with dynamic laser light scattering measurements. It appeared to be independent of concentration below 2.0 mg/ml, but at higher concentrations (2-20 mg/ml) the self-diffusion coefficient followed the function D = Do(e)(-Bc). The concentration at which the self-diffusion coefficient began to fall corresponded to the concentration predicted for domain overlap. Multimolecular aggregates of aggrecan ( approximately 30 monomers) had a much lower free diffusion coefficient (Do = 6.6 +/- 1.0 x 10(-9) cm2 s-1) but showed a decrease in mobility with concentration of a form similar to that of the monomer. The method provides a technique for investigating the macromolecular organization in glycan-rich networks at concentrations close to those found physiologically.

Aggrecans↗
PubMed1996

Age-related changes in the content of the C-terminal region of aggrecan in human articular cartilage.

The content of the C-terminal region of aggrecan was investigated in samples of articular cartilage from individuals ranging in age from newborn to 65 years. This region contains the globular G3 domain which is known to be removed from aggrecan in mature cartilage, probably by proteolytic cleavage, but the age-related changes in its abundance in human cartilage have not been described previously. The analysis was performed by immunosorbant assay using an antiserum (JD5) against recombinant amino acid residues of human aggrecan, on crude extracts of cartilage without further purification of aggrecan. The results showed that the content of the C-terminal region decreased with age relative to the G1 domain content (correlation coefficient = 0.463). This represented a 92% fall in the content of this region of the molecule from newborn to 65 years of age. furthermore, when the G1 content of the cartilage extracts was corrected to only include the G1 attached to aggrecan and to exclude the G1 fragments which accumulate as a by-product of normal aggrecan turnover (free G1), the age-related decrease in the C-terminal region remained very pronounced. Analysis by composite agarose/PAGE showed that the number of subpopulations of aggrecan resolved increased from one in newborn to three in adult cartilage. All of these reacted with an antiserum to the human G1 domain, but only the slowest migrating species reacted with the C-terminal region antiserum (JD5). Similar analysis by SDS/PAGE confirmed the presence of high-molecular-mass (200 kDa) proteins reactive with JD5, but no reactive fragments of lower electrophoretic mobility were detected. In contrast, when probed with the antiserum to the human G1 domain, the immunoblots showed protein species corresponding to the free G1 and G1-G2 fragments, which were present at high concentrations in adult cartilage. The results suggest that the loss of the C-terminal region is not directly part of the process of aggrecan turnover, but it is a slow independent matrix process that occurs more extensively with aging as turnover rates become slower. Young cartilage with the fastest turnover contains least molecules lacking the C-terminal region, whereas in old tissue with slow turnover few molecules retain this region. An increase in the cleavage of this region with age may also contribute to this change. The content of the C-terminal region may thus give a measure of the abundance of newly synthesized aggrecan.

Adolescent↗
PubMed1995

Increased proteoglycan synthesis in cartilage in experimental canine osteoarthritis does not reflect a permanent change in chondrocyte phenotype.

OBJECTIVE: To determine whether chondrocytes in early experimental osteoarthritic (OA) cartilage continue to show increased synthesis and turnover of proteoglycans (PGs) during explant culture. A comparison was also made between the responsiveness of experimental OA and control cartilage to interleukin-1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF alpha) after 1 day and 3 days in culture. METHODS: OA was induced in mature animals by sectioning of the anterior cruciate ligament followed by 3 months of normal exercise. PG synthesis in the articular cartilage was determined by measuring 35S-sulfate incorporation during explant culture over 1-3 days. Inhibition of PG synthesis was also determined with various concentrations of IL-1 beta and TNF alpha after 1 and 3 days in culture. PGs extracted from the articular cartilage over 1-3 days in culture were examined by agarose-polyacrylamide gel electrophoresis. RESULTS: Up to 24 hours after excision from the joint, PG synthesis was higher in experimental OA cartilage than in control cartilage. It was also less sensitive to inhibition by TNF alpha. These differences were no longer detected after 48-72 hours in culture. There were no changes in the relative proportions of aggrecan and decorin/biglycan extracted from and synthesized by control and experimental OA cartilage over the 3 days in culture. CONCLUSION: Previous results indicated that PG synthesis and turnover in articular cartilage was increased for many months after induction of experimental OA. Our present results show that the enhanced rate of PG synthesis and turnover were evident in freshly explanted tissue, but the differences were lost over 3 days in culture. A decreased responsiveness to TNF alpha was also lost. The hypermetabolic activity of experimental OA chondrocytes was thus reversible and not a permanent change in chondrocyte phenotype.

Aggrecans↗
PubMed1995

Changes in glycosaminoglycan epitope levels in knee joint fluid following injury.

OBJECTIVE: To measure the levels of epitope on the chondroitin sulfate (CS) and keratan sulfate (KS) chains of proteoglycan fragments in synovial fluids from injured and contralateral uninjured knees of patients with traumatic cruciate ligament and/or meniscus damage. METHODS: Enzyme-linked immunosorbent assays were used to determine the levels of monoclonal antibody epitopes 3-B-3 and 7-D-4 (CS), and 5-D-4 (KS), in paired joint fluids from the injured and uninjured knees of trauma patients. RESULTS: Levels of the CS epitopes were increased in the trauma joint fluids from most patients, with higher levels of 3-B-3 epitope in 12 of the 16 patients, but the difference did not achieve significance; however, 7-D-4 levels were higher in 15 patients, and the difference was highly significant (P = 0.0005). In contrast, the KS epitope detected by 5-D-4 was decreased in 13 of 15 patients, and the difference was significant (P = 0.0074). CONCLUSION: The increased level of 7-D-4 epitope on proteoglycans in joint fluid from injured knees may reflect the response of the articular cartilage to acute trauma resulting in altered expression of specific CS epitopes on cartilage proteoglycans. The fall in KS epitope levels may reflect the synthesis of proteoglycans that have lower KS content.

Antibodies, Monoclonal↗
PubMed1995

Antifibrotic action of interleukin-1 receptor antagonist in lapine monoarticular arthritis.

OBJECTIVE: To determine if the administration of interleukin-1 receptor antagonist (IL-1ra) to animals with established antigen induced arthritis had any beneficial effects on the synovitis and cartilage destruction. METHODS: Antigen induced arthritis was induced in New Zealand White rabbits, and after two weeks IL-1ra was administered every six hours over a 72 hour period. Animals were then killed and joint tissues examined for the degree of synovitis, synovial fibrosis, and cartilage damage. RESULTS: The response of the arthritis to the treatment was minor in terms of joint swelling, leucocyte accumulation, or cartilage proteoglycan loss. However, the synovial fibrosis was not only halted by administration of IL-1ra, but reversed. The inflamed synovial linings of IL-1ra treated animals showed a significant loss of synovial collagen content and a reappearance of the synovial fat spaces which are prominent in the normal synovial lining. CONCLUSION: This study shows that IL-1ra has potent antifibrotic activity and suggests the use of this agent for the reversal of the fibroproliferative process which is so important in the pathology of rheumatoid arthritis.

Animals↗
PubMed1995

The structure of aggrecan and its turnover in cartilage.

Aggrecan is the major proteoglycan in cartilage. It has a multidomain structure with 3 globular and 2 extended segments. It forms large aggregates by binding to hyaluronan via the G1 domain, and link protein stabilizes the aggrecan-hyaluronan bond. The extended interglobular domain joining G1 and G2 domains is the main site of proteolytic attack in aggrecan turnover. One site of cleavage is reported to predominate, but the enzyme responsible for this cleavage has not been identified. A metalloproteinase, neutrophil collagenase, has been shown to cleave at this "aggrecanase" site in vitro; however, it has yet to be shown if metalloproteinases are responsible for this activity in cartilage.

Aggrecans↗
PubMed1994

Neutrophil collagenase (MMP-8) cleaves at the aggrecanase site E373-A374 in the interglobular domain of cartilage aggrecan.

Native and recombinant neutrophil collagenase (MMP-8) was shown to cleave at the E373-A374 'aggrecanase' site in the interglobular domain of aggrecan. The time course of digestion in vitro showed that MMP-8 cleaved initially at N341-F342, the predominant metalloproteinase site, before cleaving at the E373-A374 site. A synthetic peptide, IPENFFG, inhibited cleavage at E373-A374 but not N341-F342 in vitro, indicating that the E373-A374 sequence was a less preferred site for MMP-8 cleavage than N341-F342. IPENFFG also inhibited release of A374 RGSVI fragments from cartilage in explant culture, suggesting that a metalloproteinase cleaved at the aggrecanase site in situ. The possibility remains that 'aggrecanase' may be a metalloproteinase in cartilage.

Aggrecans↗
PubMed1994

[Regulation of cartilage matrix synthesis by chondrocytes].

Articular cartilage has important load bearing properties. These depend on the integrity of its matrix which is formed by a dense network of collagen fibres (mainly type II) and a high concentration of proteglycan (mainly aggrecan). The matrix is maintained by the chondrocytes, which control the production and turnover of matrix components and are greatly affected by cytokines (such as IL-1 alpha,beta and TNF alpha) and growth factors (such as IGF-1 alpha, beta TGF beta). They have strongly antagonistic effects. IL-1 alpha, beta and TNF alpha inhibit proteoglycan synthesis and at slightly higher concentration they enhance the rates of matrix degradation. In contrast the growth factor IGF-1 stimulates proteoglycan biosynthesis and reduces matrix proteinase action. TGB beta has less direct anabolic effect on matrix biosynthesis in normal cartilage, but does not induce an anabolic response in isolated chondrocytes or in explants following IL-1 treatment. We have also investigated the action of IL-1 in inflammatory arthritis in vivo by treatment with IL-1 receptor antagonist, the natural IL-1 inhibitor. In antigen-induced arthritis the effects of the injection of rh IL-1 ra was measured over 3 days. There was little effect on the induction of joint swelling, cellular infiltration into synovium or cartilage proteoglycan depletion, but when given over a similar time in more chronic arthritis, 14 days after induction it had a major effect on suppressing synovial fibrosis although it still did not affect parameters of joint inflammation. The results suggested that IL-1 was not the major factor inducing inflammation in the joint, but was responsible for the excessive collagen deposition in the synovium in this experimental model of arthritis.

Animals↗
PubMed1994

Human link protein gene: structure and transcription pattern in chondrocytes.

We have examined the genomic organization and the transcription unit for the human link protein gene from genomic clones and RNA prepared from human cartilage over a wide age range. Five exons cover the gene which is greater than 60 kbp. Primer extension and S1 nuclease protection analysis revealed transcription initiation to be 315 bases upstream from the translation initiation codon in RNA derived from cartilage samples ranging from fetal to 53 years of age. The first exon size therefore is 289 bp and examination of the 5' flanking sequence indicated a lack of a TATA box in close proximity to the transcription start, although a TATAA-like motif (TCTAA) was present at -75 bp. Such a sequence at a similar distance can serve as a promoter in the chicken link protein gene. The large first exon of 289 bp is similar to that of the chicken but contrasts with that described previously for human (96 bp) and rat (62 bp). We also analysed human link protein mRNA by PCR for the presence of an alternatively spliced exon that is present in rat mRNA in low abundance, but could not detect such transcripts. Equine and porcine mRNA contained this spliced form but the results suggested that this was expressed as a rare transcript.

Adult↗
PubMed1994

The sulphation pattern in chondroitin sulphate chains investigated by chondroitinase ABC and ACII digestion and reactivity with monoclonal antibodies.

We have used progressive chondroitinase digestion of pig aggrecan in conjunction with ELISA assays and disaccharide analysis to derive information about the pattern of 4- and 6-sulphation in chondroitin sulphate chains. Digestion with chondroitinase ABC resulted in the release of mainly disaccharides from the nonreducing terminal of chondroitin sulphate chains but there was also the release of some tetra- and hexa-saccharides which were degraded to disaccharides with more extensive digestion. Chondroitinase ACII, in contrast, released only disaccharides. Analysis of the disaccharide composition of the intact and digested products at different stages of digestion showed that there was a slight increase in 6-sulphate content of the chains as they were shortened. Reaction of the partially digested proteoglycans with monoclonal antibodies 3-B-3 and 3-D-5 which recognise chains terminating in 6- or 4-sulphated disaccharides, respectively, showed major differences between chondroitinase ABC and ACII products. The results suggested that chondroitinase ABC preferentially cleaved next to 4-sulphated, rather than 6-sulphated disaccharides and this resulted in some oligosaccharides as well as disaccharide being released. Chondroitinase ACII also cleaved an additional disaccharide next to the linkage to protein of chondroitin sulphate, which was not removed by chondroitinase ABC and this disaccharide was mainly nonsulphated.

Aggrecans↗
PubMed1994

The effect of recombinant human interleukin 1 receptor antagonist on the induction phase of antigen induced arthritis in the rabbit.

OBJECTIVE: To determine if systemic administration of human interleukin 1 receptor antagonist (IL-1ra) to rabbits during the induction phase of antigen induced arthritis (AIA) could block inflammation and cartilage proteoglycan loss. METHODS: Recombinant human IL-1ra was administered every 6 h to rabbits beginning 1 h before induction of arthritis. Joint swelling was monitored for 72 h and then animals were killed 6 h after the last dose of IL-1ra. Leukocyte accumulation in the joint space and synovial lining was determined and the proteoglycan content and capacity for synthesis was assessed in the articular cartilage of the control and arthritic joints. RESULTS: Administration of IL-1ra had no detectable effect on the induction of arthritis. Swelling proceeded with a similar time course to untreated AIA animals and at 3 days the cellular infiltrate into synovial fluid (SF) was similarly high, the proteoglycan content of SF was also high and cartilage proteoglycan content was depleted. The biosynthesis of proteoglycan in cartilage was also similarly inhibited. No changes were detected in the cartilage and synovium or SF of the contralateral joints of animals receiving IL-1ra. CONCLUSION: IL-1ra given at a dose shown to block synovitis and proteoglycan loss induced by a bolus injection of recombinant IL-1 in rabbits was unable to inhibit the induction of AIA. Our results suggest that the action of IL-1 is not the major factor responsible for the induction of arthritis in this animal model of inflammatory joint disease.

Animals↗