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Objective: To characterize the circadian clock in murine cartilage tissue and identify tissue-specific clock target genes, and to investigate whether the circadian clock changes during aging or during cartilage degeneration using an experimental mouse model of osteoarthritis (OA)., Methods: Cartilage explants were obtained from aged and young adult mice after transduction with the circadian clock fusion protein reporter PER2::luc, and real-time bioluminescence recordings were used to characterize the properties of the clock. Time-series microarrays were performed on mouse cartilage tissue to identify genes expressed in a circadian manner. Rhythmic genes were confirmed by quantitative reverse transcription-polymerase chain reaction using mouse tissue, primary chondrocytes, and a human chondrocyte cell line. Experimental OA was induced in mice by destabilization of the medial meniscus (DMM), and articular cartilage samples were microdissected and subjected to microarray analysis., Results: Mouse cartilage tissue and a human chondrocyte cell line were found to contain intrinsic molecular circadian clocks. The cartilage clock could be reset by temperature signals, while the circadian period was temperature compensated. PER2::luc bioluminescence demonstrated that circadian oscillations were significantly lower in amplitude in cartilage from aged mice. Time-series microarray analyses of the mouse tissue identified the first circadian transcriptome in cartilage, revealing that 615 genes (∼3.9% of the expressed genes) displayed a circadian pattern of expression. This included genes involved in cartilage homeostasis and survival, as well as genes with potential importance in the pathogenesis of OA. Several clock genes were disrupted in the early stages of cartilage degeneration in the DMM mouse model of OA., Conclusion: These results reveal an autonomous circadian clock in chondrocytes that can be implicated in key aspects of cartilage biology and pathology. Consequently, circadian disruption (e.g., during aging) may compromise tissue homeostasis and increase susceptibility to joint damage or disease., (© 2013 The Authors. Arthritis & Rheumatism is published by Wiley Periodicals, Inc. on behalf of the American College of Rheumatology.)

Objective: To identify changes in gene expression in mice with osteoarthritis (OA) in order to explore the mechanisms of the disease., Methods: Gene expression profiling was performed in cartilage from mice with surgically induced OA. We used wild-type (WT) mice and Adamts5Δcat mice, in which ADAMTS-5 activity is lacking and aggrecan loss and cartilage erosion are inhibited, to distinguish gene expression changes that are independent of ADAMTS-5 activity and cartilage breakdown. Mechanical instability was introduced into the knee joints of 10-week-old male mice via surgical destabilization of the medial meniscus (DMM). Cartilage from the developing lesion in the destabilized medial meniscus and corresponding regions in sham-operated joints was harvested by microdissection at 1, 2, and 6 weeks postsurgery, and RNA was extracted, amplified, and hybridized to whole-genome microarrays., Results: Several previously identified OA-related genes, including Ptgs2, Crlf1, and Inhba, and novel genes, such as Phdla2 and Il11, were up-regulated in both WT mice and Adamts5Δcat mice, indicating that they are independent of ADAMTS-5 activity. The altered expression of other genes, including Col10a1, the sentinel marker of cartilage hypertrophy, and Wnt/β-catenin pathway genes, required ADAMTS-5 activity. Cell death pathway genes were dysregulated, and Tp53, Foxo4, and Xbp1 endoplasmic reticulum-stress transcriptional networks were activated. Analysis of degradome genes identified up-regulation of many proteases, including Mmp3, Capn2, and the novel cartilage proteases Prss46 and Klk8. Comparison with other studies identified 16 genes also dysregulated in rat and human OA as priorities for study., Conclusion: We have identified, for the first time, several genes that have an ADAMTS-5-independent role in OA, identifying them as possible OA initiation candidates. This work provides new insights into the sequence of gene dysregulation and the molecular basis of cartilage destruction in OA., (Copyright © 2013 by the American College of Rheumatology.)

Objective: To investigate the role of matrix metalloproteinase 13 (MMP-13; collagenase 3) in osteoarthritis (OA)., Methods: OA was surgically induced in the knees of MMP-13-knockout mice and wild-type mice, and mice were compared. Histologic scoring of femoral and tibial cartilage aggrecan loss (0-3 scale), erosion (0-7 scale), and chondrocyte hypertrophy (0-1 scale), as well as osteophyte size (0-3 scale) and maturity (0-3 scale) was performed. Serial sections were stained for type X collagen and the MMP-generated aggrecan neoepitope DIPEN., Results: Following surgery, aggrecan loss and cartilage erosion were more severe in the tibia than femur (P<0.01) and tibial cartilage erosion increased with time (P<0.05) in wild-type mice. Cartilaginous osteophytes were present at 4 weeks and underwent ossification, with size and maturity increasing by 8 weeks (P<0.01). There was no difference between genotypes in aggrecan loss or cartilage erosion at 4 weeks. There was less tibial cartilage erosion in knockout mice than in wild-type mice at 8 weeks (P<0.02). Cartilaginous osteophytes were larger in knockout mice at 4 weeks (P<0.01), but by 8 weeks osteophyte maturity and size were no different from those in wild-type mice. Articular chondrocyte hypertrophy with positive type X collagen and DIPEN staining occurred in both wild-type and knockout mouse joints., Conclusion: Our findings indicate that structural cartilage damage in a mouse model of OA is dependent on MMP-13 activity. Chondrocyte hypertrophy is not regulated by MMP-13 activity in this model and does not in itself lead to cartilage erosion. MMP-13 deficiency can inhibit cartilage erosion in the presence of aggrecan depletion, supporting the potential for therapeutic intervention in established OA with MMP-13 inhibitors.

Objective: To investigate the role of activated protein C (APC) in cartilage degradation., Methods: Chondrocyte expression of protein C, endothelial protein C receptor (EPCR), and thrombomodulin (TM) were evaluated by reverse transcription-polymerase chain reaction (RT-PCR). APC was immunolocalized in developing joints and in osteoarthritic (OA) cartilage from humans. The effect of APC on aggrecan and collagen degradation was examined in explant cultures of ovine cartilage in control cultures and in cultures stimulated with interleukin-1alpha (IL-1alpha), tumor necrosis factor alpha (TNFalpha), or retinoic acid (RetA), using colorimetric assays and Western blotting. Chondrocyte expression of matrix metalloproteinases (MMPs), ADAMTS, and tissue inhibitor of metalloproteinases (TIMPs) was measured by RT-PCR. MMP-2 and MMP-9 activity was evaluated by gelatin zymography and MMP-13 by fluorogenic assay., Results: Positive cellular immunostaining for APC was found at sites of MMP activity in developing joints and in OA, but not normal, cartilage. Chondrocytes expressed messenger RNA for protein C, EPCR, and TM, with the latter 2 levels increased by IL-1alpha and TNFalpha stimulation. APC augmented aggrecan release and initiated collagen breakdown in IL-1alpha-treated and TNFalpha-treated cartilage, but not in normal or in RetA-treated cartilage. APC-stimulated aggrecan and collagen breakdown were due to MMP activity but were not associated with modulation of MMP, ADAMTS, or TIMP expression. APC resulted in MMP-13 activation in cartilage cultures. APC could not directly activate proMMP-13, but it was associated with increased MMP-2 and MMP-9 activity., Conclusion: APC may be a relevant activator of MMPs in cartilage and may play a role in progressive cartilage degradation in arthritis.

Objective: To develop proteomics to analyze mouse cartilage degradation and correlate transcriptional and translational responses to catabolic stimuli., Methods: Proteomic techniques were used to analyze catabolism in mouse femoral head cartilage. Using specific methods to prepare cartilage extracts and conditioned media for 2-dimensional polyacrylamide gel electrophoresis and subsequent tandem mass spectrometry, we identified novel proteins and fragments released into the media of control, interleukin-1alpha (IL-1alpha)-treated, and all-trans-retinoic acid (RetA)-treated explants. Fluorescence 2-dimensional difference gel electrophoresis was used to quantify protein expression changes. We also measured changes in messenger RNA (mRNA) expression to distinguish transcriptional and posttranslational regulation of released proteins., Results: Differentially abundant proteins in the media of control and treated explants included fragments of thrombospondin 1 and connective tissue growth factor. IL-1alpha stimulated release of the cartilage degeneration marker matrix metalloproteinase 3, as well as proteins with uncharacterized roles in cartilage pathology, such as neutrophil gelatinase-associated lipocalin. RetA stimulated release of the extracellular matrix proteins cartilage oligomeric matrix protein, link protein, and matrilin-3 into the media, which was accompanied by a dramatic reduction in the corresponding mRNA transcript levels. Gelsolin, which has been implicated in cytoskeletal reorganization in arthritis synovial fibroblasts but has not been previously associated with cartilage pathology, was regulated by IL-1alpha and RetA., Conclusion: In this first analysis of mouse cartilage degradation and protein release using proteomics, we identified proteins and fragments, some of which represent novel candidate biomarkers for cartilage degradation. Applying these proteomic techniques to wild-type and genetically modified mouse cartilage will provide insights into the mechanisms of cartilage degeneration.

Objective: To evaluate histologic, immunohistochemical, and molecular changes in tendon induced by altered strain in a large-animal model., Methods: A full-thickness partial-width laceration of the infraspinatus tendon was created in 5 sheep, while 5 sham-operated sheep were used as controls. Sheep were killed after 4 weeks, and 4 differentially stressed tendon regions (tensile or near bone attachment from overstressed or stress-deprived halves) were evaluated for histopathology, proteoglycan (PG) accumulation, and characterization of glycosaminoglycans and aggrecan catabolites. Gene expression of matrix components, enzymes, and inhibitors was analyzed by reverse transcriptase-polymerase chain reaction., Results: Histopathologic changes were detected in both overstressed and stress-deprived tensile tendon, but only in stress-deprived tendon near bone. In overstressed and stress-deprived tensile tendon, levels of keratan sulfate, chondroitin 4-sulfate, and chondroitin 6-sulfate were increased. In overstressed tensile tendon, levels of ADAMTS-generated aggrecan catabolites were increased. There was increased matrix metalloproteinase 13 (MMP-13) and decreased fibromodulin and decorin expression in all regions. Increased MMP-1, MMP-9, MMP-14, and ADAMTS-1 expression, and decreased type II collagen expression were restricted to stress-deprived tendon. In stress-deprived bone-attachment regions, messenger RNA (mRNA) for aggrecan was decreased, and ADAMTS was increased. In overstressed tensile tendon, aggrecan mRNA was increased, and ADAMTS was decreased., Conclusion: The distinct molecular changes in adjacent tissue implicate altered strain rather than humoral factors in controlling abnormal tenocyte metabolism, and highlight the importance of regional sampling. Tendon abnormalities induced by increased strain are accompanied by increased aggrecan, decreased ADAMTS, and low PG expression, which may negatively impact the structural integrity of the tissue and predispose to rupture.

Objective: The major proteases responsible for aggrecan turnover in articular cartilage are the aggrecanases (ADAMTS-4 and ADAMTS-5). Although several studies have demonstrated C-terminal truncation of these aggrecanases, the mechanism and importance of this processing are poorly understood. The objective of this study was to further investigate ADAMTS-4 and ADAMTS-5 C-terminal truncation in a porcine model in vitro culture system., Methods: Chondrocyte-agarose cultures with well-established extracellular matrices were treated with or without interleukin-1 (IL-1), for a variety of different culture time periods. Cultures were analyzed for release of sulfated glycosaminoglycan, aggrecanase-generated interglobular domain (IGD)-aggrecan cleavage, and the presence of ADAMTS-4 and ADAMTS-5 isoforms. Inhibition of aggrecanase activity with monoclonal antibodies, tissue inhibitor of metalloproteinases 3 (TIMP-3), and cycloheximide pretreatment were used to identify ADAMTS isoforms involved in IGD-aggrecan catabolism., Results: Multiple isoforms, including possible zymogens, of ADAMTS-4 and ADAMTS-5 were sequestered within the extracellular matrix formed by 3-week chondrocyte-agarose cultures. IL-1 exposure induced production of a low molecular weight (37 kd) isoform of ADAMTS-4. This isoform was capable of degrading exogenous aggrecan at the IGD-aggrecanase site, was inhibited by TIMP-3, was blocked after preincubation with an antibody to a sequence in the catalytic domain of ADAMTS-4, and required de novo synthesis in the presence of IL-1 for its generation., Conclusion: In porcine chondrocyte-agarose cultures, a 37-kd ADAMTS-4 isoform appears to be the major matrix protease responsible for the IGD-aggrecanase activity detected in response to exposure to IL-1. This conclusion contradicts that of recent studies of transgenic knockout mice and highlights the need to determine the roles of the different aggrecanase(s) in human disease.

Objective: To determine the role of the proteinase ADAMTS-1 in normal and accelerated catabolism of aggrecan in articular and growth plate cartilage of mice., Methods: Expression of ADAMTS-1 was determined using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of RNA isolated from microdissected chondrocytes from different zones of mouse growth plate and articular cartilage. Real-time RT-PCR for ADAMTS-4, ADAMTS-5, and ADAMTS-9 was performed on femoral head cartilage of wild-type (WT) and ADAMTS-1-knockout (KO) mice. Histologic and immunohistologic evaluation of growth plate and articular cartilage was performed in WT and KO mice from birth to 12 weeks of age. The effect of ADAMTS-1 ablation on cartilage proteoglycan loss was studied in antigen-induced arthritis (AIA). Aggrecan catabolism in WT and KO mice was studied in an in vitro model of cartilage degradation, by quantitation of glycosaminoglycan loss and histologic, immunohistologic, and Western immunoblot analyses., Results: ADAMTS-1 messenger RNA (mRNA) was expressed in normal mouse articular and growth plate cartilage and was up-regulated in terminal hypertrophic differentiation of growth plate chondrocytes. There was no difference in mRNA levels in the cartilage of WT compared with KO mice for the other potential aggrecanases ADAMTS-4, ADAMTS-5, or ADAMTS-9. ADAMTS-1-KO mice were significantly smaller than their WT littermates; however, no morphologic differences between the genotypes were evident in growth plate or articular cartilage from birth to skeletal maturity (12-16 weeks). Similarly, no difference in cartilage aggrecan content or presence of aggrecan degradation products was detected between WT and KO mice. There was no difference between WT and KO mice in the degree of synovial inflammation or depletion of cartilage aggrecan in AIA. There was no difference between WT and KO cartilage in either basal or stimulated aggrecan loss in vitro; however, subtle changes in the aggrecanase-generated aggrecan catabolites were observed in interleukin-1-treated cartilage., Conclusion: Although ADAMTS-1 is expressed in articular and growth plate cartilage and is able to cleave aggrecan at physiologically relevant sites, our results indicate that it does not play a significant nonredundant role in normal cartilage and bone development and growth. Similarly, ablation of ADAMTS-1 offered no protection from accelerated aggrecanolysis in an inflammatory model of arthritis or in an in vitro model of early cartilage degradation. ADAMTS-1 does not appear to be a viable target for treatment of cartilage destruction in arthritis.

Objective: Protease-activated receptor 1 (PAR-1) is the cell surface receptor for thrombin. It is unclear whether thrombin contributes to inflammation other than by effects on coagulation. We investigated the proinflammatory participation of PAR-1 in antigen-induced arthritis (AIA)., Methods: Arthritis was induced by intraarticular injection of methylated bovine serum albumin (mBSA) in preimmunized PAR-1-deficient (PAR-1(-/-)) and wild-type (WT) mice. The disease was assessed after 7 days by histologic examination of knee joints after decalcification and Safranin O/toluidine blue staining. Serum levels of anti-mBSA IgG, interferon-gamma, and interleukin-4 (IL-4) were determined by enzyme-linked immunosorbent assay. T cell proliferation response was determined by measuring the incorporation of (3)H-thymidine. Cytokine messenger RNA (mRNA) expression was detected in synovial tissues and peritoneal cells by real-time polymerase chain reaction., Results: Arthritis severity was significantly reduced in PAR-1(-/-) mice compared with WT mice (P = 0.017). Analysis of individual aspects of joint histology revealed significant reductions in synovial exudates (P < 0.001), cartilage degradation (P < 0.01), and bone damage (P = 0.05) in PAR-1(-/-) mice. Synovial IL-1, IL-6, and matrix metalloproteinase 13 (MMP-13) mRNA was significantly reduced in PAR-1(-/-) mice. The titers of antigen-specific serum anti-mBSA total IgG, IgG1, and IgG2a were significantly reduced, and serum IL-4 was significantly increased in arthritic PAR-1(-/-) mice. In contrast, no difference was observed in antigen-induced T cell proliferation between PAR-1(-/-) and WT mice. In vitro, thrombin-induced (but not lipopolysaccharide-induced) IL-1, IL-6, and MMP-13 mRNA expression was significantly impaired in PAR-1(-/-) mice compared with WT controls., Conclusion: These data demonstrate the requirement of PAR-1 for the expression of AIA, the development of an antigen-specific Ig response, thrombin-induced macrophage cytokine and MMP expression, and the inhibitory effect of PAR-1 on serum IL-4. We conclude that PAR-1 plays a significant role in this model of arthritis.

Objective: To determine if n-3 polyunsaturated fatty acid (PUFA) supplementation (versus treatment with n-6 polyunsaturated or other fatty acid supplements) affects the metabolism of osteoarthritic (OA) cartilage., Methods: The metabolic profile of human OA cartilage was determined at the time of harvest and after 24-hour exposure to n-3 PUFAs or other classes of fatty acids, followed by explant culture for 4 days in the presence or absence of interleukin-1 (IL-1). Parameters measured were glycosaminoglycan release, aggrecanase and matrix metalloproteinase (MMP) activity, and the levels of expression of messenger RNA (mRNA) for mediators of inflammation, aggrecanases, MMPs, and their natural tissue inhibitors (tissue inhibitors of metalloproteinases [TIMPs])., Results: Supplementation with n-3 PUFA (but not other fatty acids) reduced, in a dose-dependent manner, the endogenous and IL-1-induced release of proteoglycan metabolites from articular cartilage explants and specifically abolished endogenous aggrecanase and collagenase proteolytic activity. Similarly, expression of mRNA for ADAMTS-4, MMP-13, and MMP-3 (but not TIMP-1, -2, or -3) was also specifically abolished with n-3 PUFA supplementation. In addition, n-3 PUFA supplementation abolished the expression of mRNA for mediators of inflammation (cyclooxygenase 2, 5-lipoxygenase, 5-lipoxygenase-activating protein, tumor necrosis factor alpha, IL-1alpha, and IL-1beta) without affecting the expression of message for several other proteins involved in normal tissue homeostasis., Conclusion: These studies show that the pathologic indicators manifested in human OA cartilage can be significantly altered by exposure of the cartilage to n-3 PUFA, but not to other classes of fatty acids.

Objective: To determine the effect of cyclosporin A (CSA) on aggrecanase- and matrix metalloproteinase (MMP)-mediated catabolism of proteoglycan (aggrecan) in articular cartilage explants stimulated with interleukin-1 (IL-1) in a culture system that mimics early pathologic processes associated with arthritic disease., Methods: Proteoglycan (glycosaminoglycan) and lactate quantification, Western immunoblot analyses of aggrecan degradation products, reverse transcription-polymerase chain reaction analyses of aggrecanase-1, aggrecanase-2 (ADAM-TS4, ADAM-TS5, respectively), MMP-1, MMP-3, MMP-13, tissue inhibitor of metalloproteinases 1 (TIMP-1), TIMP-2, and TIMP-3 messenger RNA (mRNA) expression in articular cartilage explant cultures, and electrophoretic mobility shift assay analysis of nuclear factor of activated T cells (NF-AT) transcription factor activation were used., Results: CSA inhibited, in a dose-dependent and noncytotoxic manner, aggrecanase-mediated proteoglycan catabolism and loss from IL-1-stimulated cartilage explants. There was no evidence of MMP-mediated aggrecan catabolism in this in vitro model. Treatment of articular cartilage explant cultures with 10 ng/ml of IL-1alpha up-regulated the expression of mRNA for ADAM-TS4, ADAM-TS5, MMP-1, MMP-3, and MMP-13. The expression of ADAM-TS4, ADAM-TS5, and MMP-13 was abrogated by the inclusion of 10 microM CSA in the culture medium. NF-AT activation was observed in chondrocytes but could not be inhibited by preincubation with 10 microM CSA., Conclusion: CSA can inhibit IL-1-induced aggrecanase-mediated proteoglycan catabolism in articular cartilage explants maintained in culture for 4 days, thus demonstrating molecular mechanisms whereby CSA may be an effective therapy for degenerative joint disease.