LXR modulation blocks prostaglandin E2 production and matrix degradation in cartilage and alleviates pain in a rat osteoarthritis model.

Osteoarthritis (OA), the most common arthritic condition in humans, is characterized by the progressive degeneration of articular cartilage accompanied by chronic joint pain. Inflammatory mediators, such as cytokines and prostaglandin E(2) (PGE(2)) that are elevated in OA joints, play important roles in the progression of cartilage degradation and pain-associated nociceptor sensitivity. We have found that the nuclear receptor family transcription factors Liver X Receptors (LXRalpha and -beta) are expressed in cartilage, with LXRbeta being the predominant isoform. Here we show that genetic disruption of Lxrbeta gene expression in mice results in significantly increased proteoglycan (aggrecan) degradation and PGE(2) production in articular cartilage treated with IL-1beta, indicating a protective role of LXRbeta in cartilage. Using human cartilage explants, we found that activation of LXRs by the synthetic ligand GW3965 significantly reduced cytokine-induced degradation and loss of aggrecan from the tissue. Furthermore, LXR activation dramatically inhibited cytokine-induced PGE(2) production by human osteoarthritic cartilage as well as by a synovial sarcoma cell line. These effects were achieved at least partly by repression of the expression of ADAMTS4, a physiological cartilage aggrecanase, and of cyclooxygenase-2 and microsomal prostaglandin E synthase-1, key enzymes in the PGE(2) synthesis pathway. Consistent with our in vitro observations, oral administration of GW3965 potently alleviated joint pain in a rat meniscal tear model of osteoarthritis.

Deletion of active ADAMTS5 prevents cartilage degradation in a murine model of osteoarthritis.

Human osteoarthritis is a progressive disease of the joints characterized by degradation of articular cartilage. Although disease initiation may be multifactorial, the cartilage destruction appears to be a result of uncontrolled proteolytic extracellular matrix destruction. A major component of the cartilage extracellular matrix is aggrecan, a proteoglycan that imparts compressive resistance to the tissue. Aggrecan is cleaved at a specific 'aggrecanase' site in human osteoarthritic cartilage; this cleavage can be performed by several members of ADAMTS family of metalloproteases. The relative contribution of individual ADAMTS proteases to cartilage destruction during osteoarthritis has not been resolved. Here we describe experiments with a genetically modified mouse in which the catalytic domain of ADAMTS5 (aggrecanase-2) was deleted. After surgically induced joint instability, there was significant reduction in the severity of cartilage destruction in the ADAMTS5 knockout mice compared with wild-type mice. This is the first report of a single gene deletion capable of abrogating the course of cartilage destruction in an animal model of osteoarthritis. These results demonstrate that ADAMTS5 is the primary 'aggrecanase' responsible for aggrecan degradation in a murine model of osteoarthritis, and suggest rational strategies for therapeutic intervention in osteoarthritis.

Tenascin-C induces inflammatory mediators and matrix degradation in osteoarthritic cartilage.

BACKGROUND: Tenascin-C (TN-C) is an extracellular matrix glycoprotein that is involved in tissue injury and repair processes. We analyzed TN-C expression in normal and osteoarthritic (OA) human cartilage, and evaluated its capacity to induce inflammatory and catabolic mediators in chondrocytes in vitro. The effect of TN-C on proteoglycan loss from articular cartilage in culture was also assessed. METHODS: TN-C in culture media, cartilage extracts, and synovial fluid of human and animal joints was quantified using a sandwich ELISA and/or analyzed by Western immunoblotting. mRNA expression of TN-C and aggrecanases were analyzed by Taqman assays. Human and bovine primary chondrocytes and/or explant culture systems were utilized to study TN-C induced inflammatory or catabolic mediators and proteoglycan loss. Total proteoglycan and aggrecanase -generated ARG-aggrecan fragments were quantified in human and rat synovial fluids by ELISA. RESULTS: TN-C protein and mRNA expression were significantly upregulated in OA cartilage with a concomitant elevation of TN-C levels in the synovial fluid of OA patients. IL-1 enhanced TN-C expression in articular cartilage. Addition of TN-C induced IL-6, PGE2, and nitrate release and upregulated ADAMTS4 mRNA in cultured primary human and bovine chondrocytes. TN-C treatment resulted in an increased loss of proteoglycan from cartilage explants in culture. A correlation was observed between TN-C and aggrecanase generated ARG-aggrecan fragment levels in the synovial fluid of human OA joints and in the lavage of rat joints that underwent surgical induction of OA. CONCLUSIONS: TN-C expression in the knee cartilage and TN-C levels measured in the synovial fluid are significantly enhanced in OA patients. Our findings suggest that the elevated levels of TN-C could induce inflammatory mediators and promote matrix degradation in OA joints.

Chondral defect repair after the microfracture procedure: a nonhuman primate model.

BACKGROUND: The extent and time course of chondral defect healing after microfracture in humans are not well described. Although most physicians recommend a period of activity and weightbearing restriction to protect the healing cartilage, there are limited data on which to base decisions regarding the duration of such restrictions. HYPOTHESIS: Evaluation of the status of chondral defect repair at different time points after microfracture in a primate model may provide a rationale for postoperative activity recommendations. STUDY DESIGN: Descriptive laboratory study. METHODS: Full-thickness chondral defects created on the femoral condyles and trochlea of 12 cynomolgus macaques were treated with microfracture and evaluated by gross and histologic examination at 6 and 12 weeks. RESULTS: At 6 weeks, there was limited chondral repair and ongoing resorption of subchondral bone. By 12 weeks, the defects were completely filled and showed more mature cartilage and bone repair. CONCLUSION: In the primate animal model, significant improvements in the extent and quality of cartilage repair were observed from the 6- to 12-week time points after microfracture. CLINICAL RELEVANCE: The poor status of the defect repair at 6 weeks and the ongoing healing observed from the 6- to 12-week time points may indicate that the repair is vulnerable during this initial postoperative period. Assuming the goal of postoperative weightbearing and activity restriction in patients after microfracture is to protect immature repair tissue, this study lends support to extending such recommendations longer than 6 weeks.

Prevention of cartilage degeneration in a rat model of osteoarthritis by intraarticular treatment with recombinant lubricin.

OBJECTIVE: Lubricin, also referred to as superficial zone protein and PRG4, is a synovial glycoprotein that supplies a friction-resistant, antiadhesive coating to the surfaces of articular cartilage, thereby protecting against arthritis-associated tissue wear and degradation. This study was undertaken to generate and characterize a novel recombinant lubricin protein construct, LUB:1, and to evaluate its therapeutic efficacy following intraarticular delivery in a rat model of osteoarthritis (OA). METHODS: Binding and localization of LUB:1 to cartilage surfaces was assessed by immunohistochemistry. The cartilage-lubricating properties of LUB:1 were determined using a custom friction testing apparatus. A cell-binding assay was performed to quantify the ability of LUB:1 to prevent cell adhesion. Efficacy studies were conducted in a rat meniscal tear model of OA. One week after the surgical induction of OA, LUB:1 or phosphate buffered saline vehicle was administered by intraarticular injection for 4 weeks, with dosing intervals of either once per week or 3 times per week. OA pathology scores were determined by histologic analysis. RESULTS: LUB:1 was shown to bind effectively to cartilage surfaces, and facilitated both cartilage boundary lubrication and inhibition of synovial cell adhesion. Treatment of rat knee joints with LUB:1 resulted in significant disease-modifying, chondroprotective effects during the progression of OA, by markedly reducing cartilage degeneration and structural damage. CONCLUSION: Our findings demonstrate the potential use of recombinant lubricin molecules in novel biotherapeutic approaches to the treatment of OA and associated cartilage abnormalities.

The influence of enrichment devices on development of osteoarthritis in a surgically induced murine model.

This study measured the influence of three different environmental enrichment devices (EEDs) on the severity of osteoarthritis (OA) in a surgically induced murine model. The development of OA requires joint movement after surgical instability induced by destabilization of the medial meniscus at 10 weeks of age. We evaluated the hypothesis that animals behavioral activity levels may influence the severity of the disease by investigating the effect of different EEDs on mouse activity and correlating this to OA severity. Thirty male 129S6/SvEvTac mice were housed in groups of five and provided with nesting material and one of three different EEDs: a heavy plastic tube (CPVC), Shepherd Shack (SS), or Tecniplast Mouse House (TMH). We videorecorded the cages throughout the study and constructed an ethogram. Eight weeks after surgery we euthanized the mice and performed a histologic examination of the knees to score the severity of OA based on the different housing systems, correlating the scores with behavioral activity levels for each cage. OA was higher in the mice with CPVC and TMH devices in their cages, whereas the mice with SS devices exhibited less cartilage damage; however, although we observed increased behavioral activity in mice with the CPVC tube and TMH and less in mice with the SS, the statistical results were not significant. The histological results of OA and the ethogram correlated to support our hypothesis that the type of EED plays an indirect role in the severity of the disease by modifying the activity levels of mice. In activity-dependent studies, the impact of an EED needs to be evaluated before change the environment.

In vivo osteoarthritis target validation utilizing genetically-modified mice.

Osteoarthritis (OA) is a progressive disease of cartilage degradation that significantly impacts quality of life. There are currently no effective treatments and, while a large number of potential therapeutic targets exist, most have not been validated in vivo. The range of OA models in the mouse has dramatically expanded in the last decade, beyond spontaneous models, to include genetically modified transgenic, knockout (KO) and knock-in (KI) mice that can develop premature cartilage degeneration reminiscent of OA. In addition, instability models of OA, either induced by intra-articular (IA) collagenase or surgery, are providing a set of tools to assist in the identification of disease-modifying OA drug (DMOAD) targets. These models are now vital tools to dissect the pathways essential to the pathogenesis of OA. Two targets, ADAMTS (a disintegrin and metalloproteinase with thrombospondin-like motifs)-5 and IL-1beta (interleukin-1 beta), have been validated in the surgical destabilization of the medial meniscus model (DMM) in KO mice. Other potential targets evaluated in instability models, either showed no disease modification or a worsening of disease, suggesting that those targets have no role, a protective role or that other, more destructive enzymes etc., can overcompensate. Development of small molecule or protein antagonist inhibitors of therapeutic targets require many years to bring to clinical trials and often confront potency and safety issues which impede successful progress. Validation, or confirmation of therapeutic targets in vivo is most clearly and efficiently obtained by using KO studies, than by creating potent and selective DMOADs to multiple potential targets. While the results in the mouse will not always transpose to the human condition, the track record of mouse knockouts corresponding to the human phenotype have been excellent. These results indicate that the evaluation of genetically modified mice will become increasingly important as we unravel the genes contributing to OA.

Double-knockout of ADAMTS-4 and ADAMTS-5 in mice results in physiologically normal animals and prevents the progression of osteoarthritis.

OBJECTIVE: To phenotypically characterize ADAMTS-4- and ADAMTS-5-double-knockout mice, and to determine the effect of deletion of ADAMTS-4 and ADAMTS-5 on the progression of osteoarthritis (OA) in mice. METHODS: Mice lacking the catalytic domain of ADAMTS-4 and ADAMTS-5 were crossed to generate ADAMTS-4/5-double-knockout animals. Twelve-week-old and 1-year-old male and female ADAMTS-4/5-double-knockout mice were compared with age- and sex-matched wild-type (WT) mice by evaluating terminal body weights, organ weights, clinical pathology parameters, PIXImus mouse densitometry findings, and macroscopic and microscopic observations. ADAMTS-4/5-double-knockout mice were challenged by surgical induction of joint instability to determine the importance of these genes in the progression of OA. Articular and nonarticular cartilage explants from WT and ADAMTS-4/5-double-knockout mice were treated with interleukin-1 (IL-1) plus retinoic acid ex vivo, to examine proteoglycan degradation. RESULTS: There were no genotype-related phenotype differences between ADAMTS-4/5-double-knockout and WT mice through 1 year of age, with the exception that female ADAMTS-4/5-double-knockout mice had a lower mean terminal body weight at the 12-week time point. Eight weeks after surgical induction of joint instability, OA was significantly less severe in ADAMTS-4/5-double-knockout mice compared with WT mice. Following stimulation of cartilage explants with IL-1 plus retinoic acid, aggrecanase-mediated degradation in ADAMTS-4/5-double-knockout mice was ablated, to a level comparable with that in ADAMTS-5-knockout mice. CONCLUSION: Dual deletion of ADAMTS-4 and ADAMTS-5 generated mice that were phenotypically indistinguishable from WT mice. Deletion of ADAMTS-4/5 provided significant protection against proteoglycan degradation ex vivo and decreased the severity of murine OA. These effects in the ADAMTS-4/5-double-knockout mice were comparable with those observed with deletion of ADAMTS-5 alone.

Characterization of and osteoarthritis susceptibility in ADAMTS-4-knockout mice.

OBJECTIVE: To determine the importance of the enzymatic activity of ADAMTS-4 in normal growth and development and to evaluate the role of ADAMTS-4 in the progression of osteoarthritis (OA). METHODS: We generated catalytic domain-deleted ADAMTS-4-transgenic mice and performed extensive gross and histologic analyses of various organs. The mice were challenged by surgical induction of joint instability leading to OA, to determine the importance of the enzymatic activity of ADAMTS-4 in the progression of the disease. The response of wild-type (WT) and ADAMTS-4-knockout (ADAMTS-4-KO) articular cartilage to interleukin-1 and retinoic acid challenge in vitro was also evaluated. RESULTS: ADAMTS-4-KO mice up to 1 year of age exhibited no gross or histologic abnormalities in 36 tissue sites examined. Despite evidence of ADAMTS-4 expression and activity in growth plates of WT mice, catalytic silencing of this proteinase caused no abnormalities in skeletal development, growth, or remodeling. There was no effect of ADAMTS-4 knockout on the progression or severity of OA 4 weeks or 8 weeks after surgical induction of joint instability. Enzymatic cleavage of aggrecan at the TEGE(373-374)ARGS site was clearly evident after exposure of articular cartilage from ADAMTS-4-KO mice to inflammatory cytokines. CONCLUSION: Although expression of the ADAMTS-4 gene has been found in many tissues throughout the body, deletion of enzymatic activity did not appear to have any effect on normal growth and physiology. Our study provides evidence that ADAMTS-4 is the primary aggrecanase in murine growth plates; however, deletion of its enzymatic activity did not affect normal long bone remodeling. Our results also lead to the hypothesis that, in the mouse, ADAMTS-4 is not the primary enzyme responsible for aggrecan degradation at the TEGE(373-374)ARGS site. The elucidation of the relative importance of ADAMTS-4 in the pathologic process of human OA will require examination of human OA tissues and evidence of disease modification in patients following therapeutic intervention.