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.

Human cartilaginous endplate degeneration is induced by calcium and the extracellular calcium-sensing receptor in the intervertebral disc.

The cartilaginous endplates (CEPs) are thin layers of hyaline cartilage found adjacent to intervertebral discs (IVDs). In addition to providing structural support, CEPs regulate nutrient and metabolic exchange in the disc. In IVD pathogenesis, CEP undergoes degeneration and calcification, compromising nutrient availability and disc cell metabolism. The mechanism(s) underlying the biochemical changes of CEP in disc degeneration are currently unknown. Since calcification is often observed in later stages of IVD degeneration, we hypothesised that elevations in free calcium (Ca2+) impair CEP homeostasis. Indeed, our results demonstrated that the Ca2+ content was consistently higher in human CEP tissue with grade of disc degeneration. Increasing the levels of Ca2+ resulted in decreases in the secretion and accumulation of collagens type I, II and proteoglycan in cultured human CEP cells. Ca2+ exerted its effects on CEP matrix protein synthesis through activation of the extracellular calcium-sensing receptor (CaSR); however, aggrecan content was also affected independent of CaSR activation as increases in Ca2+ directly enhanced the activity of aggrecanases. Finally, supplementing Ca2+ in our IVD organ cultures was sufficient to induce degeneration and increase the mineralisation of CEP, and decrease the diffusion of glucose into the disc. Thus, any attempt to induce anabolic repair of the disc without addressing Ca2+ may be impaired, as the increased metabolic demand of IVD cells would be compromised by decreases in the permeability of the CEP.

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.

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.

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.

A vision on the future of articular cartilage repair.

An AO Foundation (Davos, Switzerland) sponsored workshop "Cell Therapy in Cartilage Repair" from the Symposium "Where Science meets Clinics" (September 5-7, 2013, Davos) gathered leaders from medicine, science, industry, and regulatory organisations to debate the vision of cell therapy in articular cartilage repair and the measures that could be taken to narrow the gap between vision and current practice. Cell-based therapy is already in clinical use to enhance the repair of cartilage lesions, with procedures such as microfracture and articular chondrocyte implantation. However, even though long term follow up is good from a clinical perspective and some of the most rigorous randomised controlled trials in the regenerative medicine/orthopaedics field show beneficial effect, none of these options have proved successful in restoring the original articular cartilage structure and functionality in patients so far. With the remarkable recent advances in experimental research in cell biology (new sources for chondrocytes, stem cells), molecular biology (growth factors, genes), biomaterials, biomechanics, and translational science, a combined effort between scientists and clinicians with broad expertise may allow development of an improved cell therapy for cartilage repair. This position paper describes the current state of the art in the field to help define a procedure adapted to the clinical situation for upcoming translation in the patient.

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.

Spine degeneration in a murine model of chronic human tobacco smokers.

OBJECTIVE: To investigate the mechanisms by which chronic tobacco smoking promotes intervertebral disc degeneration (IDD) and vertebral degeneration in mice. METHODS: Three month old C57BL/6 mice were exposed to tobacco smoke by direct inhalation (4 cigarettes/day, 5 days/week for 6 months) to model long-term smoking in humans. Total disc proteoglycan (PG) content [1,9-dimethylmethylene blue (DMMB) assay], aggrecan proteolysis (immunobloting analysis), and cellular senescence (p16INK4a immunohistochemistry) were analyzed. PG and collagen syntheses ((35)S-sulfate and (3)H-proline incorporation, respectively) were measured using disc organotypic culture. Vertebral osteoporosity was measured by micro-computed tomography. RESULTS: Disc PG content of smoke-exposed mice was 63% of unexposed control, while new PG and collagen syntheses were 59% and 41% of those of untreated mice, respectively. Exposure to tobacco smoke dramatically increased metalloproteinase-mediated proteolysis of disc aggrecan within its interglobular domain (IGD). Cellular senescence was elevated two-fold in discs of smoke-exposed mice. Smoke exposure increased vertebral endplate porosity, which closely correlates with IDD in humans. CONCLUSIONS: These findings further support tobacco smoke as a contributor to spinal degeneration. Furthermore, the data provide a novel mechanistic insight, indicating that smoking-induced IDD is a result of both reduced PG synthesis and increased degradation of a key disc extracellular matrix protein, aggrecan. Cleavage of aggrecan IGD is extremely detrimental as this results in the loss of the entire glycosaminoglycan-attachment region of aggrecan, which is vital for attracting water necessary to counteract compressive forces. Our results suggest identification and inhibition of specific metalloproteinases responsible for smoke-induced aggrecanolysis as a potential therapeutic strategy to treat IDD.

Severe osteogenesis imperfecta caused by a small in-frame deletion in CRTAP.

Mutations of proteins involved in posttranslational modification of collagen type I can cause osteogenesis imperfecta (OI) inherited in a recessive pattern. The cartilage-associated protein (CRTAP) is part of a heterotrimeric complex (together with prolyl-3-hydroxylase-1 [P3H1] and cyclophilin B) that 3-hydroxylates the alpha 1 chain of collagen type I at proline residue 986 and plays a collagen chaperon role. CRTAP mutations usually cause severe OI. We report on a patient with OI and a homozygous in-frame deletion in CRTAP and a severe form of OI. The girl was born with markedly deformed long bones. Despite intravenous bisphosphonate treatment, she developed multiple vertebral compression fractures and severe scoliosis and at 4 years of age was able to sit only with support. Although CRTAP transcript levels were normal in the patient's fibroblasts, protein levels of both CRTAP and P3H1 were severely reduced. The degree of 3-hydroxylation at proline residue 986 was also decreased. This report characterizes a patient with a CRTAP small in-frame deletion. We are unaware of prior reports of this finding. We suggest that this deletion affects crucial amino acids that are important for the interaction and/or stabilization of CRTAP and P3H1.

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.

Short- and long-term outcome of patients with pseudo-vitamin D deficiency rickets treated with calcitriol.

BACKGROUND: Pseudo-vitamin D deficiency rickets (PDDR; OMIM 264700) is a rare autosomal recessive disorder caused by mutations in the CYP27B1 gene, leading to an inability to synthesize 1α,25-dihydroxyvitamin D(3) (calcitriol). The long-term (>1 yr) effects of calcitriol replacement treatment have not been reported. MATERIALS AND METHODS: Thirty-nine patients (20 females) with PDDR received calcitriol for periods of 2.0-26 yr. In 21 patients, data were available at diagnosis and during the first 2 yr of treatment with calcitriol. Twenty-five patients had reached their final height at the time of this analysis. RESULTS: The most common presenting features were active rickets, neurological signs, and short stature. Treatment with calcitriol resulted in the normalization of biochemical parameters and mean lumbar spine areal bone mineral density z-scores within 3 months, whereas height z-scores increased more gradually. As to long-term effects, adult patients who had received calcitriol before the pubertal growth spurt (n = 11) had normal height, whereas patients who were treated with calcitriol only after puberty (n = 14) on average were short (height z-score -2.2). Lumbar spine areal bone mineral density z-scores were normal in all patients who had achieved final height. Nine women had 19 pregnancies, which all were without complications. All newborns were eucalcemic at birth. CONCLUSION: Treatment with calcitriol started in infancy results in short- and long-term correction of all clinical, biochemical, and radiological abnormalities related to PDDR.

Development of a whole disc organ culture system to study human intervertebral disc.

STUDY TYPE: Basic science Objective: Low back pain is one of the most common health problems1 and is strongly associated with intervertebral disc degeneration, (IVD). Current treatments remove the symptoms without reversing or even retarding the underlying problem. Development of new therapy for the regeneration of the degenerative IVD is complicated by the lack of a validated long-term organ culture model in which therapeutic candidates can be studied. The object of this study was to develop, optimize, and validate an organ culture model for human IVD, allowing for the study of degeneration and the potential for regeneration of the human IVD. METHODS: From eleven donors, an average of 5-6 IVDs were obtained. Inclusion criteria were; age between 50 and 70 years old, no history of cancer, chemotherapy, diabetes, or liver cirrhosis. An x-ray of the harvested spine was done to assess the grade of degeneration. Three different methods for isolating the discs were studied: with bony endplate (BEP), without endplate (NEP), and with cartilage endplate (CEP). Discs were cultured for 4 weeks without external load, in Dulbecco's modified eagle media with glucose and fetal bovine serum (FBS). Four different combinations of concentrations of glucose and FBS were compared: low glucose-low FBS, low glucose-high FBS, high glucose-low FBS, and high glucose-high FBS.2 Short-term cultures (1 week) were performed to compare the cell viability of the three methods of isolating the discs. Swelling potential on NEP and CEP discs from the same donor were evaluated. After four weeks of culture, a 4 mm punch was taken from CEP discs and cell viability was evaluated using a live/dead assay with confocal microscopy. RESULTS: Analyzing the potential of swelling in CEP discs, there was an increase in volume to a maximum of 25% and retention of shape and morphology. Whereas in NEP discs, there was an excessive deformation and a two-fold time increase in volume than CEP discs. The cell viability in short-term cultures is around 40%-50% in the BEP model, 50%-60% in the NEP model and > 96% in the CEP model. BEP isolated discs show endplate necrosis that begins after 4 days of culture. Cell viability in CEP discs was evaluated at 4 weeks in three different areas of the disc: nucleus pulposus, inner annulus fibrosus, and outer annulus fibrosus. We found no difference in live cells (> 96%) between the four different concentrations of FBS and glucose (Table 1). [Table: see text] CONCLUSIONS: We have developed a novel method to isolate human IVDs and optimized the culture conditions. The CEP method has been proven to be superior to the previous models (NEP and BEP) in cell viability and maintaining physiologic swelling.3 In the long-term cultures, the CEP system maintained sufficient nutrient supply and high cell survival in all regions of the discs even with low concentrations of FBS and glucose. The availability of an intact disc organ culture system has a considerable advantage over the culture of isolated disc cells, as it maintains the cells in their unique microenvironment, making any response to catabolic or anabolic agents more physiologically relevant.

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.

Identification of opticin, a member of the small leucine-rich repeat proteoglycan family, in human articular tissues: a novel target for MMP-13 in osteoarthritis.

OBJECTIVE: One of the proteoglycan families is the small leucine-rich proteoglycans (SLRPs) that are characterized by their association with collagen fibrils and/or some glycosaminoglycans. Opticin is a glycoprotein and class III member of the SLRP family, which was initially identified in the vitreous humour of the eye. In this study, we first investigated whether opticin is expressed and produced in normal and OA human articular tissues/cells. Further, we investigated the ability of the key metalloprotease involved in cartilage pathology, MMP-13, to cleave human cartilage opticin. METHODS: Opticin gene expression was investigated in normal and OA human chondrocytes, synovial fibroblasts, and subchondral bone osteoblasts by reverse transcriptase-polymerase chain reaction (RT-PCR). Opticin protein production was determined in normal and OA synovial membrane and cartilage by immunohistochemistry. Opticin was isolated from human cartilage using guanidinium chloride extraction, and human MMP-13-induced opticin degradation analyzed by Western blotting. Finally, the opticin MMP-13 cleavage site was determined. RESULTS: Opticin was expressed in human chondrocytes, synovial fibroblasts and subchondral osteoblasts, and the protein identified in synovial membrane and cartilage. At the protein level, OA cartilage showed a slightly higher level of opticin positive stained chondrocytes than normal cartilage; this did not reach statistical significance. However, in contrast with OA, normal cartilage demonstrated a high level of matrix staining in the superficial zone of the tissue, suggesting that in the OA cartilage matrix, opticin is degraded. Data also showed that cartilage opticin could be cleaved by MMP-13 after only 2h of incubation, indicating a preferential substrate compared to other SLRPs for this enzyme. Microsequencing revealed a major cleavage site at the G(104)/L(105)LAAP and a minor at P(109)/A(110)NHPG upon MMP-13 exposure. CONCLUSION: We demonstrated, for the first time, that opticin is expressed and produced in human articular tissues. Our data also showed that opticin in OA cartilage is degraded in a process that could be mediated by MMP-13. As opticin may contribute towards the structural stability of cartilage, its cleavage by MMP-13 may predispose cartilage to degeneration, particularly at the surface.

Mechanism of proteoglycan aggregate degradation in cartilage stimulated with oncostatin M.

OBJECTIVE: To investigate the potential synergistic and differential effects of cytokine combinations on proteoglycan aggregate catabolism in cartilage. METHODS: Bovine articular cartilage explants were maintained in organ culture and subjected to stimulation with cytokine combinations including interleukin-1alpha (IL-1alpha), IL-1beta, IL-6, IL-17, tumor necrosis factor-alpha (TNFalpha) and oncostatin M (OSM). Aggrecan, link protein and hyaluronan (HA) release and degradation were analyzed, and the effect of the hyaluronidase inhibitor apigenin was investigated. RESULTS: For all cytokine mixtures studied cleavage of aggrecan only by aggrecanase action was apparent. However, OSM acting synergistically with IL-1 or TNFalpha produced a rapid release of all proteoglycan aggregate components due to both aggrecan and HA degradation. This was abolished by the hyaluronidase inhibitor, apigenin. In addition, in the presence of OSM a low molecular weight aggrecan G3 product was observed, suggesting altered aggrecanase cleavage activity is induced by this cytokine. CONCLUSIONS: Under cytokine stimulation, aggrecan release from cartilage may take place via proteolysis of the aggrecan core protein or via depolymerization of HA, with the latter mechanism being induced by OSM. OSM is associated with joint inflammation and its participation may account for the more rapid loss of aggrecan from articular cartilage in the inflammatory arthritides, compared to osteoarthritis.

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.

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.

The cleavage of biglycan by aggrecanases.

OBJECTIVE: Aggrecanase-1 [a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4] and aggrecanase-2 (ADAMTS-5) have been named for their ability to degrade the proteoglycan aggrecan. While this may be the preferred substrate for these enzymes, they are also able to degrade other proteins. The aim of this work was to determine whether the aggrecanases could degrade biglycan and decorin. METHODS: Biglycan, decorin and aggrecan were purified from human and bovine cartilage and subjected to degradation by recombinant aggrecanase-1 or aggrecanase-2. In vitro degradation was assessed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) and immunoblotting, and the cleavage site in biglycan was determined by N-terminal amino acid sequencing. SDS/PAGE and immunoblotting were also used to assess in situ degradation in both normal and arthritic human articular cartilage. RESULTS: Both aggrecanase-1 and aggrecanase-2 are able to cleave bovine and human biglycan at a site within their central leucine-rich repeat regions. Cleavage occurs at an asparagine-cysteine bond within the fifth leucine-rich repeat. In contrast, the closely related proteoglycan decorin is not a substrate for the aggrecanases. Analysis of human articular cartilage from osteoarthritic (OA) and rheumatoid arthritic (RA) joints showed that a biglycan degradation product of equivalent size is present in the extracellular matrix. No equivalent degradation product was, however, detectable in normal adult human articular cartilage. CONCLUSION: Biglycan, which is structurally unrelated to aggrecan, can act as a substrate for aggrecanase-1 and aggrecanase-2, and these proteinases may account for at least part of the biglycan degradation that is present in arthritic cartilage.

Link protein can retard the degradation of hyaluronan in proteoglycan aggregates.

OBJECTIVE: Loss of articular cartilage and intervertebral disc function in arthritis or disc degeneration is associated with degradation of the proteoglycan (PG) aggregates by either proteolysis of aggrecan or hyaluronan (HA) degradation. The aim of this work was to determine whether degradation of HA in PG aggregate degradation is influenced by link protein (LP) stabilization of the PG aggregates. METHODS: Aggrecan and LP were prepared from fetal bovine epiphyseal cartilage, and PG aggregates were formed in the presence or absence of LP. The PG aggregates were exposed to hyaluronidase or free radicals to promote HA degradation. Degradation of HA, aggrecan and LP were assessed by gel filtration chromatography and polyacrylamide gel electrophoresis. RESULTS: High concentrations of hyaluronidase cleaved both PG aggregates between each aggrecan molecule, whereas low concentrations gave much less cleavage of the LP-stabilized aggregate. High free radical concentrations gave extensive cleavage of all components of both PG aggregates, whereas low concentrations are more selective for HA damage and to a much lesser extent in the LP-stabilized aggregates. Thus the presence of LP caused a diminution in the capacity of both catabolic agents to degrade HA as long as levels of the degradative agents were not excessive. CONCLUSION: In addition to stabilizing the PG aggregates towards dissociation, LP may also help protect the PG aggregates from degradation under conditions where tissue catabolism is promoted.