Short link N acts as a disease modifying osteoarthritis drug.

Osteoarthritis (OA) is a degenerative joint disease characterised by a progressive degradation of articular cartilage and underlaying bone and is associated with pain and disability. Currently, there is no medical treatment to reverse or even retard OA. Based on our previous reports, where we establish the repair potential of short Link N (sLN) in the intervertebral disc, a cartilage-like tissue, we hypothesise that sLN may hold similar promises in the repair of articular cartilage. This study aimed to determine if sLN, could prevent OA disease progression. Skeletally mature New Zealand white rabbits underwent unilateral anterior cruciate ligament transection (ACLT) of their left femorotibial joints to induce joint degeneration typical of OA. Beginning 3 weeks post-operatively, and every three weeks thereafter for 12 weeks, either saline (1 mL) or sLN (100 µg in 1 mL saline) was injected intraarticularly into the operated knee. Six additional rabbits underwent sham surgery but without ACLT or post-operative injections. The effects on gross joint morphology and cartilage histologic changes were evaluated. In the Saline group, prominent erosion of articular cartilage occurred in both femoral condyle compartments and the lateral compartment of the tibial plateau while, sLN treatment reduced the severity of the cartilage damage in these compartments of the knee showing erosion. Furthermore, statistically significant differences were detected between the joint OA score of the saline and sLN treated groups (p = 0.0118). Therefore, periodic intraarticular injection of sLN is a promising nonsurgical treatment for preventing or retarding OA progression, by reducing cartilage degradation.

Dynamic loading, matrix maintenance and cell injection therapy of human intervertebral discs cultured in a bioreactor.

Low back pain originating from intervertebral disc (IVD) degeneration affects the quality of life for millions of people, and it is a major contributor to global healthcare costs. Long-term culture of intact IVDs is necessary to develop ex vivo models of human IVD degeneration and repair, where the relationship between mechanobiology, disc matrix composition and metabolism can be better understood. A bioreactor was developed that facilitates culture of intact human IVDs in a controlled, dynamically loaded environment. Tissue integrity and cell viability was evaluated under 3 different loading conditions: low 0.1-0.3, medium 0.1-0.3 and high 0.1-1.2 MPa. Cell viability was maintained > 80 % throughout the disc at low and medium loads, whereas it dropped to approximately 70 % (NP) and 50 % (AF) under high loads. Although cell viability was affected at high loads, there was no evidence of sGAG loss, changes in newly synthesised collagen type II or chondroadherin fragmentation. Sulphated GAG content remained at a stable level of approximately 50 µg sGAG/mg tissue in all loading protocols. To evaluate the feasibility of tissue repair strategies with cell supplementation, human NP cells were transplanted into discs within a thermoreversible hyaluronan hydrogel. The discs were loaded under medium loads, and the injected cells remained largely localised to the NP region. This study demonstrates the feasibility of culturing human IVDs for 14 days under cyclic dynamic loading conditions. The system allows the determination a safe range-of-loading and presents a platform to evaluate cell therapies and help to elucidate the effect of load following cell-based therapies.

The non-aggregated aggrecan in the human intervertebral disc can arise by a non-proteolytic mechanism.

Analysis of both the aggregated and non-aggregated fractions of aggrecan isolated from adult human intervertebral disc using immunoblotting with antibodies specific for the different domains constituting the aggrecan core protein or atomic force microscopy revealed that many components contained the G1 domain. However, little of the disc aggrecan was able to reform aggregates with hyaluronan, as determined by gel filtration chromatography, suggesting that the G1 domains had been rendered non-functional. Since previous studies have shown that disc aggrecan undergoes non-enzymatic glycation with age, the functional effect of such modification was investigated in vitro using bovine aggrecan isolated from young animals. Incubation of monomeric aggrecan with ribose to induce glycation rendered it unable to form complexes with hyaluronan stable to agarose gel electrophoresis or gel filtration chromatography. Similarly, extended treatment of intact proteoglycan aggregate with ribose resulted in destabilisation of the complex with separation of the aggrecan from the hyaluronan. Although it is clear that proteolysis occurs in the intervertebral disc and gives rise to some non-aggregating molecules, a different mechanism is required to explain the presence of many non-aggregating molecules bearing the G1 domain. The products of non-enzymatic glycation of the globular domains of aggrecan would account for this phenomenon and explain why some of the non-aggregating molecules are still large proteoglycans. While such molecules may be retained in the nucleus pulposus, they may be able to diffuse within it, reducing the ability of the tissue to resist compression under asymmetric loading such as bending and ultimately contributing to disc degeneration.

Acute mechanical injury of the human intervertebral disc: link to degeneration and pain.

Excessive mechanical loading or acute trauma to intervertebral discs (IVDs) is thought to contribute to degeneration and pain. However, the exact mechanisms by which mechanical injury initiates and promotes degeneration remain unclear. This study investigates biochemical changes and extracellular matrix disruption in whole-organ human IVD cultures following acute mechanical injury. Isolated healthy human IVDs were rapidly compressed by 5% (non-injured) or 30% (injured) of disc height. 30% strain consistently cracked cartilage endplates, confirming disc trauma. Three days post-loading, conditioned media were assessed for proteoglycan content and released cytokines. Tissue extracts were assessed for proteoglycan content and for aggrecan integrity. Conditioned media were applied to PC12 cells to evaluate if factors inducing neurite growth were released. Compared to controls, IVD injury caused significant cell death. Injury also caused significantly reduced tissue proteoglycan content with a reciprocal increase of proteoglycan content in culture media. Increased aggrecan fragmentation was observed in injured tissue due to increased matrix metalloproteinase and aggrecanase activity. Injured-IVD conditioned media contained significantly elevated interleukin (IL)-5, IL-6, IL-7, IL-8, MCP-2, GROα, and MIG, and ELISA analysis showed significantly increased nerve growth factor levels compared to non-injured media. Injured-disc media caused significant neurite sprouting in PC12 cells compared to non-injured media. Acute mechanical injury of human IVDs ex vivo initiates release of factors and enzyme activity associated with degeneration and back pain. This work provides direct evidence linking acute trauma, inflammatory factors, neo-innervation and potential degeneration and discogenic pain in vivo.

A role for TNFα in intervertebral disc degeneration: a non-recoverable catabolic shift.

This study examines the effect of TNFα on whole bovine intervertebral discs in organ culture and its association with changes characteristic of intervertebral disc degeneration (IDD) in order to inform future treatments to mitigate the chronic inflammatory state commonly found with painful IDD. Pro-inflammatory cytokines such as TNFα contribute to disc pathology and are implicated in the catabolic phenotype associated with painful IDD. Whole bovine discs were cultured to examine cellular (anabolic/catabolic gene expression, cell viability and senescence using ß-galactosidase) and structural (histology and aggrecan degradation) changes in response to TNFα treatment. Control or TNFα cultures were assessed at 7 and 21 days; the 21 day group also included a recovery group with 7 days TNFα followed by 14 days in basal media. TNFα induced catabolic and anti-anabolic shifts in the nucleus pulposus (NP) and annulus fibrosus (AF) at 7 days and this persisted until 21 days however cell viability was not affected. Data indicates that TNFα increased aggrecan degradation products and suggests increased ß-galactosidase staining at 21 days without any recovery. TNFα treatment of whole bovine discs for 7 days induced changes similar to the degeneration processes that occur in human IDD: aggrecan degradation, increased catabolism, pro-inflammatory cytokines and nerve growth factor expression. TNFα significantly reduced anabolism in cultured IVDs and a possible mechanism may be associated with cell senescence. Results therefore suggest that successful treatments must promote anabolism and cell proliferation in addition to limiting inflammation.

Complex loading affects intervertebral disc mechanics and biology.

BACKGROUND: Complex loading develops in multiple spinal motions and in the case of hyperflexion is known to cause intervertebral disc (IVD) injury. Few studies have examined the interacting biologic and structural alterations associated with potentially injurious complex loading, which may be an important contributor to chronic progressive degeneration. OBJECTIVE: This study tested the hypothesis that low magnitudes of axial compression loading applied asymmetrically can induce IVD injury affecting cellular and structural responses in a large animal IVD ex-vivo model. METHODS: Bovine caudal IVDs were assigned to either a control or wedge group (15°) and placed in organ culture for 7 days under static 0.2MPa load. IVD tissue and cellular responses were assessed through confined compression, qRT-PCR, histology and structural and compositional measurements, including Western blot for aggrecan degradation products. RESULTS: Complex loading via asymmetric compression induced cell death, an increase in caspase-3 staining (apoptosis), a loss of aggrecan and an increase in aggregate modulus in the concave annulus fibrosis. While an up-regulation of MMP-1, ADAMTS4, IL-1ß, and IL-6 mRNA, and a reduced aggregate modulus were induced in the convex annulus. CONCLUSION: Asymmetric compression had direct deleterious effects on both tissue and cells, suggesting an injurious loading regime that could lead to a degenerative cascade, including cell death, the production of inflammatory mediators, and a shift towards catabolism. This explant model is useful to assess how injurious mechanical loading affects the cellular response which may contribute to the progression of degenerative changes in large animal IVDs, and results suggest that interventions should address inflammation, apoptosis, and lamellar integrity.

Involvement of ADAMTS5 and hyaluronidase in aggrecan degradation and release from OSM-stimulated cartilage.

The relative contribution of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)4 and ADAMTS5 to aggrecan degradation under oncostatin M (OSM) stimulation, the role of the ancillary domains of the aggrecanases on their ability to cleave within the chondroitin sulfate (CS)-2 region, the role of hyaluronidases (HYAL) in stimulating aggrecan release in the absence of proteolysis, and the identity of the hyaluronidase involved in OSM-mediated cartilage breakdown were investigated. Bovine articular cartilage explants were cultured in the presence of interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNFalpha) and/or OSM, or treated with trypsin and/or hyaluronidase. Aggrecan was digested with various domain-truncated isoforms of ADAMTS4 and ADAMTS5. Aggrecan and link protein degradation and release were analyzed by immunoblotting. Aggrecanase and HYAL gene expression were determined. ADAMTS4 was the most inducible aggrecanase upon cytokine stimulation, whereas ADAMTS5 was the most abundant aggrecanase. ADAMTS5 was the most active aggrecanase and was responsible for the generation of an OSM-specific degradation pattern in the CS-2 region. Its ability to cleave at the OSM-specific site adjacent to the aggrecan G3 region was enhanced by truncation of the C-terminal thrombospondin domain, but reduced by further truncation of both the spacer and cysteine-rich domains of the enzyme. OSM has the ability to mediate proteoglycan release through hyaluronan degradation, under conditions where HYAL-2 is the predominant hyaluronidase being expressed. Compared to other catabolic cytokines, OSM exhibits a unique potential at degrading the proteoglycan aggregate, by promoting early robust aggrecanolysis, primarily through the action of ADAMTS5, and hyaluronan degradation.

Glypican and biglycan in the nuclei of neurons and glioma cells: presence of functional nuclear localization signals and dynamic changes in glypican during the cell cycle.

We have investigated the expression patterns and subcellular localization in nervous tissue of glypican, a major glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan that is predominantly synthesized by neurons, and of biglycan, a small, leucine-rich chondroitin sulfate proteoglycan. By laser scanning confocal microscopy of rat central nervous tissue and C6 glioma cells, we found that a significant portion of the glypican and biglycan immunoreactivity colocalized with nuclear staining by propidium iodide and was also seen in isolated nuclei. In certain regions, staining was selective, insofar as glypican and biglycan immunoreactivity in the nucleus was seen predominantly in a subpopulation of large spinal cord neurons. The amino acid sequences of both proteoglycans contain potential nuclear localization signals, and these were demonstrated to be functional based on their ability to target beta-galactosidase fusion proteins to the nuclei of transfected 293 cells. Nuclear localization of glypican beta-galactosidase or Fc fusion proteins in transfected 293 cells and C6 glioma cells was greatly reduced or abolished after mutation of the basic amino acids or deletion of the sequence containing the nuclear localization signal, and no nuclear staining was seen in the case of heparan sulfate and chondroitin sulfate proteoglycans that do not possess a nuclear localization signal, such as syndecan-3 or decorin (which is closely related in structure to biglycan). Transfection of COS-1 cells with an epitope-tagged glypican cDNA demonstrated transport of the full-length proteoglycan to the nucleus, and there are also dynamic changes in the pattern of glypican immunoreactivity in the nucleus of C6 cells both during cell division and correlated with different phases of the cell cycle. Our data therefore suggest that in certain cells and central nervous system regions, glypican and biglycan may be involved in the regulation of cell division and survival by directly participating in nuclear processes.

Characterization of an ADAMTS-5-mediated cleavage site in aggrecan in OSM-stimulated bovine cartilage.

OBJECTIVE: In a previous study, we identified a 50-kDa G3-containing aggrecan degradation product in bovine cartilage, released from the tissue after interleukin-1 (IL-1) stimulation in the presence of oncostatin M (OSM). Our objective was to purify, determine the N-terminal sequence of this fragment and verify whether this cleavage could be attributed to a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5 action in vitro. METHODS: Collected media from bovine cartilage explant cultures stimulated with IL-1+OSM were subjected to anion-exchange chromatography. The N-terminal sequence of the fragment of interest in the purified fractions was determined by automated Edman sequencing. Fetal bovine aggrecan was digested with full-length recombinant ADAMTS-4 and ADAMTS-5 and resulting degradation products were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) and immunoblotting using an anti-G3 antiserum and an anti-neoepitope antibody that had been generated to the new N-terminus of the G3 fragment. RESULTS: Characterization of the 50-kDa fragment showed that it possesses chondroitin sulfate (CS) and is the result of a cleavage within the C-terminal portion of the CS-2 domain, adjacent to the G3 region. Sequence analysis identified the cleavage region as TQRPAE(2047)-(2048)ARLEIE, suggesting an aggrecanase-derived product. Using an anti-neoepitope antibody specific for the additional cleavage site, it was shown that the product is generated in vitro upon digestion of aggrecan by ADAMTS-5 and, to a much lesser extent, by ADAMTS-4. CONCLUSIONS: The abundance and rapid rate of release of this degradation product in organ cultures in the presence of OSM suggest that it could result from a unique aggrecan proteolysis mediated by aggrecanases.

The consequence of PRELP overexpression on skin.

PRELP is a member of the small leucine-rich repeat proteoglycan family that is abundantly expressed in many cartilages compared to other connective tissues. To study the consequence of PRELP overexpression in tissues where it is normally expressed at low abundance, transgenic mice were generated in which the human PRELP transgene was placed under control of the CMV promoter. A connective tissue phenotype was observed in the skin, where the organization of collagen fibrils in the dermis was perturbed and the thickness of the hypodermal fat layer was diminished.

Preferential mRNA expression of prostromelysin relative to procollagenase and in situ localization in human articular cartilage.

An imbalance between extracellular proteinases and their inhibitors is thought to underlie cartilage degradation. In cultures of adult cartilage, prostromelysin mRNA levels were much higher than those for procollagenase and this differential was increased in cultures stimulated with IL-1 beta. Analysis of mRNA prepared from freshly isolated chondrocytes showed abundant amounts of prostromelysin mRNA in normal adult cartilage but low levels in the neonate. Not all adult cartilage may possess such high levels of prostromelysin mRNA, as the message levels in the cartilage remaining on late-stage osteoarthritic joints were lower than those in normal adult cartilage. Relative to prostromelysin mRNA, little procollagenase and TIMP mRNA were found in the adult cartilage. In situ hybridization revealed that metalloproteinase mRNAs were localized in chondrocytes of the superficial zone in adult cartilage. However, upon IL-1 beta treatment, chondrocytes in all cartilage zones were observed to express prostromelysin mRNA. Relative to the neonate, the normal adult cartilage appears to have a high degradative potential, if one accepts that steady-state mRNA levels reflect prostromelysin production. As the adult cartilage is not apparently undergoing rapid turnover, it would appear that control of prostromelysin activation may be the major regulatory step in stromelysin-induced cartilage degradation.

The structure and function of cartilage proteoglycans.

Cartilage contains a variety of proteoglycans that are essential for its normal function. These include aggrecan, decorin, biglycan, fibromodulin and lumican. Each proteoglycan serves several functions that are determined by both its core protein and its glycosaminoglycan chains. This review discusses the structure/function relationships of the cartilage proteoglycans, and the manner in which perturbations in proteoglycan structure or abundance can adversely affect tissue function.

Expression of lumican in human breast carcinoma.

Lumican mRNA has been identified as being differentially expressed between different regions of the same human breast tumor. In situ hybridization study of 26 independent breast tumors confirmed the presence of lumican mRNA in fibroblast-like cells within stroma and showed a significant increase of its expression in tumor compared to adjacent normal stroma (P < 0.001). Higher lumican expression was associated with higher tumor grade, lower estrogen receptor levels in the tumor, and younger age of the patients (P < 0.05). Reverse transcription-PCR analysis of total RNA extracted from 19 independent breast tissues exhibiting lesions that are thought to parallel tumor progression also suggests that this proteoglycan is differentially expressed during tumor progression.

The effects of vitamin D deficiency on proteoglycan and hyaluronate constituents of chick bone.

The effect of vitamin D deficiency on proteoglycan and hyaluronate constituents of cortical diaphyseal chick bone was studied. Proteoglycans in rachitic bone showed no significant change with respect to their size, composition, or amount relative to other extractable macromolecular components. In contrast, bone hyaluronate levels were raised in chicks fed on diets that were either vitamin D-deficient or depleted in calcium or phosphate, a 7-fold increase being seen in hypocalcaemic vitamin D-deficient chicks. This increase in hyaluronate was not directly related either to the absence of vitamin D or to abnormal levels of blood calcium or phosphate per se; hyaluronate levels are probably regulated by another factor, not yet identified, that is responsive to changes in vitamin D and mineral metabolism.

The use of caesium sulphate density gradient centrifugation to analyse proteoglycans from human articular cartilages of different ages.

Proteoglycan subunits from human articular cartilage were fractionated by caesium sulphate density gradient centrifugation. A single heterogeneous population of molecules was produced whose average density decreased with increasing age of the individual from which they were obtained. At no density did the carbohydrate composition of any adult fraction resemble that of any newborn fraction, although there was considerable overlap in density. However, there was a similarity in amino acid composition between the most dense proteoglycans from the adult and those of least density from the newborn. The carbohydrate content of a 2-year-old proteoglycan was intermediate in composition, with high density fractions resembling the newborn and low density fractions resembling the adult. In addition, the proteoglycans of lowest density in both the newborn and two year preparations showed additional bands on agarose/polyacrylamide gel electrophoresis resembling the adult material. These results indicate that while a core protein of adult composition may occur in the juvenile proteoglycan it need not necessarily be glycosylated in an adult manner, suggesting that glycosylation is to some extent independent of the origin of core protein heterogeneity.

Studies on the interaction of newly secreted proteoglycan subunits with hyaluronate in human articular cartilage.

Newly secreted proteoglycans from adult human cartilage do not interact well with hyaluronate, but attain this ability with time in the extracellular matrix. The conversion process occurs in all types of cartilagenous matrix, as newborn cartilage cultures, chondrosarcoma cultures and adult chondrocyte cultures each secreted proteoglycan subunits which exhibited the delayed aggregation phenomenon. However, the rate of conversion is probably dependent upon the structure of the surrounding matrix and the cell type. In vitro, link protein appears to enhance an initial change in the hyaluronate-binding region of the newly secreted proteoglycan subunits to allows stronger interaction with hyaluronate. In a second step, which is pH- and temperature-dependent, the change becomes irreversible. Thus, in addition to its role in stabilizing the interaction of mature proteoglycan subunits with hyaluronate, link protein may also aid in promoting the conversion of the newly synthesized proteoglycan subunit to a form that is capable of strong interaction with hyaluronate.

A matrix protein of Mr 55,000 that accumulates in human articular cartilage with age.

Adult human articular cartilage contains a protein of Mr 55,000 which is deficient in newborn cartilage. In the adult the molecule represents one of the most abundant non-collagenous, non-proteoglycan molecules in 4 M guanidinium chloride extracts of the tissue. The molecular size of the protein on SDS-PAGE remains constant under reducing and non-reducing conditions, suggesting that it does not exist as a disulphide-bonded multimer, nor do intramolecular disulphide bonds greatly influence its conformation. The protein has the ability to interact with some immunoglobulin preparations making its detection possible by Western blotting with some non-specific antisera. Labeling with [3H]leucine in organ culture indicates that protein of this size is being made by the chondrocytes. However, during purification the newly synthesized molecules do not behave as the resident protein on ion-exchange chromatography, suggesting that the protein may accumulate with age rather than being a major synthetic product of the adult chondrocytes. Amino terminal protein sequence analysis indicates that the N-terminus of the protein is blocked. Sequences derived from peptides generated with cyanogen bromide do not show homology with previously characterized proteins. Molecules of a similar size and composition have been described in bovine cartilage.

Physicochemical properties of cartilage proteoglycans extracted by lanthanum chloride.

Bovine nasal cartilage was extracted with 0.5 M LaCl3 and the extract then diluted with nine volumes of water. The resulting precipitated (PLaCl3) contained the proteoglycan subunits, together with minor protein components, but was essentially free from hyaluronic acid. The properties of PLaCl3 were investigated by chemical analysis, electrophoresis, viscometry and analytical ultracentrifugation, and the results compared with those for proteoglycan obtained by caesium chloride density gradient centrifugation of 2 M CaCl2 cartilage extracts. Proteoglycan subunits (A1D1) prepared from PLaCl3 showed identical properties to those obtained from other high ionic strength cartilage extracts.

O-Linked oligosaccharides of human articular cartilage proteoglycan.

O-linked oligosaccharides and keratan sulphate chains have been isolated from the proteoglycan subunits of human articular cartilage. The oligosaccharides possessed a size and chemical composition similar to the equivalent moieties present in the proteoglycan subunits of the Swarm rat chondrosarcoma. Furthermore, the size and chemical composition of the oligosaccharides showed little change with the age of the individual from whom the proteoglycan was obtained. In contrast, the keratan sulphate chains appeared to increase in chain length with increased age of the individual. The total number of keratan sulphate and oligosaccharide chains per core protein decreased with age, but it was not clear whether there was any change in the ratio of the two components with respect to one another.

Thirty-three novel COL1A1 and COL1A2 mutations in patients with osteogenesis imperfecta types I-IV.

Osteogenesis imperfecta (OI) is a heritable disease of bone characterized by low bone mass and bone fragility. Six different types of OI have been described to date, based on clinical phenotype and histological findings. The genetic defect in many patients with OI types I-IV is due to mutations in the genes encoding type I collagen, while patients with OI types V and VI show no evidence of mutations in the COL1A1/COL1A2 genes. Here we report thirty-three novel mutations in patients with types I-IV OI. Sixteen mutations were in COL1A1 and seventeen were in COL1A2. Most mutations resulted in substitutions for glycine: one of these, a doublet GG>CC transversion, created a unique Gly-->Pro missense mutation in the triple helical domain of COL1A2. Two rare triple helical Gly-->Glu substitutions in COL1A2 are also described. In addition, there were six single-base deletion mutations resulting in frameshifts, seven splice junction mutations, and a 9-bp triple helix insertion associated with a severe (OI II) phenotype. The variety of mutations described in the COL1A1/COL1A2 genes giving rise to an OI phenotype is in accordance with the clinical heterogeneity of the disease. Hum Mutat 17:434, 2001.