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Martina Kaiser Jochen Haag Stephan Sder Brigitte Bau Thomas Aigner 《Arthritis \u0026amp; Rheumatology》2004,50(11):3535-3540
Objective
Bone morphogenetic protein (BMP) and transforming growth factor β (TGFβ) are potent anabolic factors in adult articular chondrocytes. In this study, we investigated whether intracellular inhibitors of BMP and TGFβ signaling, inhibitory Smad6 (I‐Smad6) and I‐Smad7, are expressed in articular chondrocytes in normal and osteoarthritic (OA) cartilage, and whether their expression shows a correlation with the anabolic activity of OA chondrocytes in vivo and after interleukin‐1β (IL‐1β) stimulation in vitro.Methods
RNA isolated directly from normal and OA human knee cartilage as well as from cultured articular chondrocytes was analyzed by (quantitative) polymerase chain reaction technology. Immunolocalization of the I‐Smads was performed on tissue sections and compared with the anabolic cellular activity as documented by in situ hybridization experiments for aggrecan and type II collagen.Results
Both Smad6 and Smad7 were expressed in all samples of normal and OA cartilage. Immunostaining (including confocal microscopy) confirmed the presence of Smad6 and Smad7 in the majority of normal and degenerated articular chondrocytes; localization was mostly cytoplasmic. No correlation between expression of the main anabolic genes and expression of the I‐Smads was found. In cultured articular chondrocytes, stimulation with IL‐1β showed up‐regulation of Smad7, whereas Smad6 was down‐regulated.Conclusion
Both Smad6 and Smad7 are expressed in adult human articular chondrocytes. The primarily cytoplasmic localization suggests permanent activation of the I‐Smads in articular cartilage in vivo. No evidence was found that up‐regulation or down‐regulation of I‐Smads in OA cartilage correlates directly with the anabolic (or catabolic) activity of articular chondrocytes. The regulation in chondrocytes of Smad6 and Smad7 expression by IL‐1β suggests a potentially important role of IL‐1β signaling in chondrocytes, via indirect influencing of the BMP/TGFβ signaling cascade.3.
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Alwin Scharstuhl Elly L. Vitters Peter M. van der Kraan Wim B. van den Berg 《Arthritis \u0026amp; Rheumatology》2003,48(12):3442-3451
Objective
Osteoarthritis (OA) is a joint disease characterized by osteophyte development, fibrosis, and articular cartilage damage. Effects of exogenous transforming growth factor β (TGFβ) isoforms and bone morphogenetic proteins (BMPs) suggest a role for these growth factors in the pathogenesis of OA. The aim of this study was to elucidate the role of endogenous TGFβ and BMP during papain‐induced OA‐like changes in mice.Methods
We used adenoviral overexpression of TGFβ and BMP antagonists to block growth factor signaling. An adenovirus expressing a secreted, pan–specific TGFβ antagonist called murine latency‐associated peptide 1 (mLAP‐1) was used. In addition, we used intracellular inhibitory Smad6 as a BMP antagonist and Smad7 as a TGFβ/BMP inhibitor. Papain was injected into the knee joints of C57BL/6 mice to induce osteophyte development, synovial thickening, and articular cartilage proteoglycan (PG) loss.Results
Intraarticular injection of papain caused increased protein expression of several TGFβ and BMP isoforms in synovium and cartilage. Adenovirus transfection into the joint resulted in a strong expression of the transgenes in the synovial lining. Overexpression of mLAP‐1, Smad6, and Smad7 led to a significant reduction in osteophyte formation compared with that in controls. Smad6 and Smad7 overexpression also significantly decreased synovial thickening. Furthermore, the secreted TGFβ inhibitor mLAP‐1 increased articular cartilage PG loss.Conclusion
Our results indicate a pivotal role of endogenous TGFβ in the development of osteophytes and synovial thickening, implicating endogenous TGFβ in the pathogenesis of OA. In contrast, the prevention of cartilage damage by endogenous TGFβ signifies the protective role of TGFβ in articular cartilage. This is the first study to demonstrate that endogenous BMPs are involved in osteophyte formation and synovial thickening in experimental OA.5.
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Sohel M. Julovi Tadashi Yasuda Makoto Shimizu Teruko Hiramitsu Takashi Nakamura 《Arthritis \u0026amp; Rheumatology》2004,50(2):516-525
Objective
To investigate the mechanism of the inhibitory action of hyaluronan (HA) on interleukin‐1β (IL‐1β)‐stimulated production of matrix metalloproteinases (MMPs) in human articular cartilage.Methods
IL‐1β was added to normal and osteoarthritic (OA) human articular cartilage in explant culture to stimulate MMP production. Articular cartilage was incubated or preincubated with a clinically used form of 800‐kd HA to assess its effect on IL‐1β‐induced MMPs. Levels of secreted MMPs 1, 3, and 13 in conditioned media were detected by immunoblotting; intracellular MMP synthesis in chondrocytes was evaluated by immunofluorescence microscopy. Penetration of HA into cartilage tissue and its binding to CD44 were analyzed by fluorescence microscopy using fluoresceinated HA. Blocking experiments with anti‐CD44 antibody were performed to investigate the mechanism of action of HA.Results
Treatment and pretreatment with 800‐kd HA at 1 mg/ml resulted in significant suppression of IL‐1β‐stimulated production of MMPs 1, 3, and 13 in normal and OA cartilage explant culture. Fluorescence histocytochemistry revealed that HA penetrated cartilage tissue and localized in the pericellular matrix around chondrocytes. HA‐binding blocking experiments using anti‐CD44 antibody demonstrated that the association of HA with chondrocytes was mediated by CD44. Preincubation with anti‐CD44 antibody, which suppressed IL‐1β‐stimulated MMPs, reversed the inhibitory effect of HA on MMP production that was induced by IL‐1β in normal and OA cartilage.Conclusion
This study demonstrates that HA effectively inhibits IL‐1β‐stimulated production of MMP‐1, MMP‐3, and MMP‐13, which supports the clinical use of HA in the treatment of OA. The action of HA on IL‐1β may involve direct interaction between HA and CD44 on chondrocytes.7.
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Takumi Kakinuma Tadashi Yasuda Takefumi Nakagawa Teruko Hiramitsu Miki Akiyoshi Masao Akagi Tatsuya Sawamura Takashi Nakamura 《Arthritis \u0026amp; Rheumatology》2004,50(11):3495-3503
Objective
To investigate for the presence of oxidized low‐density lipoprotein (ox‐LDL) and lectin‐like oxidized LDL receptor 1 (LOX‐1) in cartilage specimens from rheumatoid arthritis (RA) joints and to determine whether the interaction of ox‐LDL with LOX‐1 can induce matrix metalloproteinase 3 (MMP‐3) in articular cartilage explant culture.Methods
Human articular cartilage specimens obtained from patients with RA, osteoarthritis (OA), and femoral neck fractures were examined for LOX‐1 and ox‐LDL by confocal fluorescence microscopy. The association between ox‐LDL and LOX‐1 was evaluated by immunofluorescence analysis. Articular cartilage specimens from patients with femoral neck fractures were incubated with ox‐LDL, with or without preincubation with neutralizing anti–LOX‐1 antibody. MMP‐3 synthesis by chondrocytes in explant cartilage was evaluated by immunofluorescence, and protein secretion into conditioned medium was monitored by immunoblotting and enzyme‐linked immunosorbent assay.Results
The majority of the RA chondrocytes stained positively with both anti–LOX‐1 and anti–ox‐LDL antibodies; however, no positive cells were found in OA and normal cartilage specimens. Anti–LOX‐1 antibody suppressed the binding of DiI‐labeled ox‐LDL to chondrocytes in explant culture, suggesting that the interaction was mediated by LOX‐1. In contrast to native LDL, ox‐LDL induced MMP‐3 synthesis by articular chondrocytes in association with the induction of LOX‐1, which resulted in enhanced secretion of MMP‐3 into the culture medium. Anti–LOX‐1 antibody reversed ox‐LDL–stimulated MMP‐3 synthesis to control levels.Conclusion
Ox‐LDL, principally mediated by LOX‐1, enhanced MMP‐3 production in articular chondrocytes. Increased accumulation of ox‐LDL with elevated expression of LOX‐1 in RA cartilage indicates a specific role of the receptor–ligand interaction in cartilage pathology in RA.9.
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P. J. T. Koshy C. J. Lundy A. D. Rowan S. Porter D. R. Edwards A. Hogan I. M. Clark T. E. Cawston 《Arthritis \u0026amp; Rheumatology》2002,46(4):961-967
Objective
Previous studies have reported elevated levels of interleukin‐1 (IL‐1) and oncostatin M (OSM) in rheumatoid joints, as well as the synergistic degradation of human articular cartilage by this cytokine combination. The present study was undertaken to investigate the ability of IL‐1 and OSM to modulate gene expression of matrix metalloproteinase (MMP), ADAM, and ADAM‐TS (ADAM with thrombospondin motifs) family members in human chondrocytes.Methods
T/C28a4 human chondrocytes were stimulated for 2–48 hours with IL‐1 and/or OSM. Total RNA was harvested, reverse transcribed, and assessed by real‐time polymerase chain reaction for the expression of various MMP, ADAM, and ADAM‐TS messenger RNAs (mRNA). Results were normalized to 18S ribosomal RNA.Results
IL‐1 and OSM synergized to markedly induce the expression of the collagenases MMP‐1, MMP‐8, and MMP‐13 as well as MMP‐3, an activator of proMMPs. Expression of mRNA for MMPs 1, 3, and 13 was induced early, whereas that of MMP‐8 mRNA occurred late. Gene expression of MMP‐14, an MMP that degrades collagen and activates proMMP‐13, was elevated by this combination. IL‐1 and OSM also synergized to induce gene expression of the aggrecanase ADAM‐TS4, but not ADAM‐TS5.Conclusion
These data indicate that the potent cartilage‐degrading properties of the combination of IL‐1 and OSM are potentially mediated by a synergistic induction of the aggrecan‐degrading enzyme ADAM‐TS4 and the collagen‐degrading enzymes MMP‐1, MMP‐8, MMP‐13, and MMP‐14, although differences in the magnitude of response and in the time course of induction were observed. A role for MMPs 3 and 14 in the activation of proMMPs may also be implicated.12.
Tadashi Yasuda A. Robin Poole Makoto Shimizu Takefumi Nakagawa Sohel M. Julovi Hirokazu Tamamura Nobutaka Fujii Takashi Nakamura 《Arthritis \u0026amp; Rheumatology》2003,48(5):1271-1280
Objective
To investigate the mechanism of induction of matrix metalloproteinases (MMPs) by a 40‐kd COOH‐terminal heparin‐binding fibronectin fragment (HBFN‐f) containing III12–14 and IIICS domains in human articular cartilage in culture.Methods
Human articular cartilage was removed from macroscopically normal femoral heads and cultured with HBFN‐f. MMP secretion into conditioned media was analyzed by immunoblotting (MMPs 1 and 13) and by gelatin zymography (MMPs 2 and 9). Type II collagen cleavage by collagenase was monitored in culture by immunoassay. Involvement of specific peptide‐binding domains in HBFN‐f and the involvement of CD44 were assessed with synthetic peptides and an anti‐CD44 antibody. Immunofluorescence histochemistry was performed using fluorescein isothiocyanate–conjugated anti‐CD44 antibody.Results
HBFN‐f stimulated production of MMPs 1, 2, 9, and 13 in association with type II collagen cleavage by collagenase in human articular cartilage. Peptide V (WQPPRARI) of HBFN‐f, which can bind cell surface heparan sulfate proteoglycan (HSPG), blocked MMP induction by HBFN‐f, while the scrambled peptide V (RPQIPWAR) had no effect. Peptide CS‐1 of 25 amino acids in IIICS of HBFN‐f caused no significant effect. Treatment of cartilage with anti‐CD44 antibody or HSPG resulted in significant inhibition of HBFN‐f–stimulated MMP production. Preincubation with peptide V blocked binding of the anti‐CD44 antibody to chondrocytes in cartilage.Conclusion
Interaction of the peptide V sequence in HBFN‐f with glycosaminoglycans, such as those in CD44, plays an important role in HBFN‐f–stimulated MMP production in articular cartilage. Because CD44 is up‐regulated in osteoarthritic and rheumatoid arthritic cartilage, the role of the interaction between CD44 and HBFN‐f in these pathologies should be of relevance and should be studied further.13.
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Christopher B. Forsyth Judit Pulai Richard F. Loeser 《Arthritis \u0026amp; Rheumatology》2002,46(9):2368-2376
Objective
To determine if integrin‐mediated signaling results in activation of chondrocyte mitogen‐activated protein (MAP) kinases that lead to increased expression of matrix metalloproteinase 13 (MMP‐13; collagenase 3), a potent mediator of cartilage matrix degradation.Methods
Human articular chondrocytes isolated from normal ankle and knee cartilage obtained from tissue donors were cultured in monolayers. The cells were treated with a 120‐kd fibronectin fragment (FN‐f) that binds the α5β1 integrin or with antibodies to specific integrin receptors. Activation of MAP kinases was determined by immunoblotting with phosphospecific antibodies. MMP production was measured by gelatin zymography, and MMP‐13 production and activation were determined by immunoblotting and by a fluorogenic peptide assay.Results
Human articular chondrocytes were found to respond to the 120‐kd FN‐f and to adhesion‐blocking antibodies to the α2β1 and α5β1 integrins with increased phosphorylation of the extracellular signal–regulated kinase 1 (ERK1)/ERK2, c‐Jun N‐terminal kinase (JNK), and p38 MAP kinases. Intact FN and integrin‐blocking antibodies to α1, α3, and αVβ3 and a nonblocking α5 antibody had no effect. After MAP kinase activation, increased phosphorylation of c‐Jun and the nuclear factor κB inhibitor was noted, followed by increased pro‐ and activated MMP‐13 in the conditioned media. Inhibitors of mitogen‐activated protein kinase kinase, p38, and JNK were each able to inhibit increased MMP‐13 production, while the interleukin‐1 receptor antagonist (IL‐1Ra) protein did not. However, the IL‐1Ra partially inhibited FN‐f–induced activation of MMP‐13.Conclusion
Integrin‐mediated MAP kinase signaling stimulated by FN‐f is associated with increased production and release of pro‐ and active MMP‐13. Autocrine production of IL‐1 appears to result in additional MMP‐13 activation. These processes may play a key role in feedback loops responsible for progressive cartilage degradation in arthritis.15.
L. Xu C. M. Flahiff B. A. Waldman D. Wu B. R. Olsen L. A. Setton Y. Li 《Arthritis \u0026amp; Rheumatology》2003,48(9):2509-2518
Objective
To investigate whether heterozygosity for a loss‐of‐function mutation in the gene encoding the α1 chain of type XI collagen (Col11a1) in mice (chondrodysplasia, cho) causes osteoarthritis (OA), and to understand the biochemical and biomechanical effects of this mutation on articular cartilage in knee and temporomandibular (TM) joints.Methods
Articular cartilage from the knee and TM joints of mice heterozygous for cho (cho/+) and their wild‐type littermates (+/+) was examined. The morphologic properties of cartilage were evaluated, and collagen fibrils were examined by transmission electron microscopy. Immunohistochemical staining was performed to examine the protein expression levels of matrix metalloproteinase 3 (MMP‐3) and MMP‐13 in knee joints. In 6‐month‐old animals, fixed‐charge density was determined using a semiquantitative histochemical method, and tensile stiffness was determined using an osmotic loading technique.Results
The diameter of collagen fibrils in articular cartilage of knee joints from heterozygous cho/+ mice was increased relative to that in control cartilage, and histologic analysis showed OA‐like degenerative changes in knee and TM joints, starting at age 3 months. The changes became more severe with aging. At 3 months, protein expression for MMP‐3 was increased in knee joints from cho/+ mice. At 6 months, protein expression for MMP‐13 was higher in knee joints from cho/+ mice than in joints from their wild‐type littermates, and negative fixed‐charge density was significantly decreased. Moreover, tensile stiffness in articular cartilage of knee joints from cho/+ mice was moderately reduced and was inversely correlated with the increase in articular cartilage degeneration.Conclusion
Heterozygosity for a loss‐of‐function mutation in Col11a1 results in the development of OA in the knee and TM joints of cho/+ mice. Morphologic and biochemical evidence of OA appears to precede significant mechanical changes, suggesting that the cho mutation leads to OA through a mechanism that does not initially involve mechanical factors.16.
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Seiji Kubo Gregory M. Cooper Tomoyuki Matsumoto Julie A. Phillippi Karin A. Corsi Arvydas Usas Guangheng Li Freddie H. Fu Johnny Huard 《Arthritis \u0026amp; Rheumatology》2009,60(1):155-165
Objective
To investigate the effect of vascular endothelial growth factor (VEGF) stimulation and the effect of blocking VEGF with its antagonist, soluble Flt‐1 (sFlt‐1), on chondrogenesis, using muscle‐derived stem cells (MDSCs) isolated from mouse skeletal muscle.Methods
The direct effect of VEGF on the in vitro chondrogenic ability of mouse MDSCs was tested using a pellet culture system, followed by real‐time quantitative polymerase chain reaction (PCR) and histologic analyses. Next, the effect of VEGF on chondrogenesis within the synovial joint was tested, using genetically engineered MDSCs implanted into rat osteochondral defects. In this model, MDSCs transduced with a retroviral vector to express bone morphogenetic protein 4 (BMP‐4) were coimplanted with MDSCs transduced to express either VEGF or sFlt‐1 (a VEGF antagonist) to provide a gain‐ and loss‐of‐function experimental design. Histologic scoring was used to compare cartilage formation among the treatment groups.Results
Hyaline‐like cartilage matrix production was observed in both VEGF‐treated and VEGF‐blocked (sFlt‐1–treated) pellet cultures, but quantitative PCR revealed that sFlt‐1 treatment improved the expression of chondrogenic genes in MDSCs that were stimulated to undergo chondrogenic differentiation with BMP‐4 and transforming growth factor β3 (TGFβ3). In vivo testing of articular cartilage repair showed that VEGF‐transduced MDSCs caused an arthritic change in the knee joint, and sFlt‐1 improved the MDSC‐mediated repair of articular cartilage, compared with BMP‐4 alone.Conclusion
Soluble Flt‐1 gene therapy improved the BMP‐4– and TGFβ3‐induced chondrogenic gene expression of MDSCs in vitro and improved the persistence of articular cartilage repair by preventing vascularization and bone invasion into the repaired articular cartilage.18.
Objective
To examine the effects of a fibronectin (FN) fragment containing the COOH‐terminal heparin‐binding domain (HBFN‐f) on chondrocyte‐mediated type II collagen (CII) cleavage by collagenase and proteoglycan (PG) degradation in articular cartilage in explant culture.Methods
Intact FN or HBFN‐f was added to explant cultures of mature bovine articular cartilage. We investigated collagenase‐mediated cleavage of CII caused by HBFN‐f in explant cultures using a new immunoassay for detection and measurement of the primary collagenase cleavage site of CII. CII denaturation in cartilage was also measured using a specific enzyme‐linked immunosorbent assay. Degradation of PG (principally aggrecan) was analyzed by a dye‐binding assay. APMA and/or a matrix metalloproteinase 13 (MMP‐13) preferential inhibitor or interleukin‐1 receptor antagonist (IL‐1Ra) were added to some cultures to examine the presence of latent procollagenase or the involvement of MMP‐13 or IL‐1, respectively, in cartilage breakdown induced by HBFN‐f. Secretion of MMP‐3 and MMP‐13 into media was detected by immunoblotting.Results
In contrast to intact FN, HBFN‐f was shown to stimulate CII cleavage by collagenase in a dose‐dependent manner following PG degradation, similar to cartilage breakdown induced by IL‐1. Treatment with HBFN‐f also resulted in elevated denaturation of CII. Immunoblotting demonstrated that HBFN‐f enhanced pro–matrix metalloproteinase 13 (proMMP‐13) production as well as that of proMMP‐3. APMA, which activates latent proMMPs, enhanced the HBFN‐f–mediated cleavage of CII by collagenase. An MMP‐13 preferential inhibitor or IL‐1Ra suppressed HBFN‐f–induced collagen cleavage to control levels.Conclusion
Our data demonstrate that HBFN‐f can induce early PG degradation and subsequent CII cleavage. The latter is probably mediated by early proMMP‐13 induction involving an IL‐1–dependent pathway. Activation of latent collagenase is delayed. This new information, together with existing data on other FN fragments, reveals that increased levels of these fragments, found in diseased joints such as in osteoarthritis and rheumatoid arthritis, may stimulate cartilage breakdown by mechanisms of the kind demonstrated in the present study.19.
Robert Terkeltaub Bing Yang Martin Lotz Ru Liu‐Bryan 《Arthritis \u0026amp; Rheumatology》2011,63(7):1928-1937
Objective
Interleukin‐1β (IL‐1β) and tumor necrosis factor α (TNFα) stimulate chondrocyte matrix catabolic responses, thereby compromising cartilage homeostasis in osteoarthritis (OA). AMP‐activated protein kinase (AMPK), which regulates energy homeostasis and cellular metabolism, also exerts antiinflammatory effects in multiple tissues. This study was undertaken to test the hypothesis that AMPK activity limits chondrocyte matrix catabolic responses to IL‐1β and TNFα.Methods
Expression of AMPK subunits was examined, and AMPKα activity was ascertained by the phosphorylation status of AMPKα Thr172 in human knee articular chondrocytes and cartilage by Western blotting and immunohistochemistry, respectively. Procatabolic responses to IL‐1β and TNFα, such as release of glycosaminoglycan, nitric oxide, and matrix metalloproteinases 3 and 13 were determined by dimethylmethylene blue assay, Griess reaction, and Western blotting, respectively, in cartilage explants and chondrocytes with and without knockdown of AMPKα by small interfering RNA.Results
Normal human knee articular chondrocytes expressed AMPKα1, α2, β1, β2, and γ1 subunits. AMPK activity was constitutively present in normal articular chondrocytes and cartilage, but decreased in OA articular chondrocytes and cartilage and in normal chondrocytes treated with IL‐1β and TNFα. Knockdown of AMPKα resulted in enhanced catabolic responses to IL‐1β and TNFα in chondrocytes. Moreover, AMPK activators suppressed cartilage/chondrocyte procatabolic responses to IL‐1β and TNFα and the capacity of TNFα and CXCL8 (IL‐8) to induce type X collagen expression.Conclusion
Our findings indicate that AMPK activity is reduced in OA cartilage and in chondrocytes following treatment with IL‐1β or TNFα. AMPK activators attenuate dephosphorylation of AMPKα and procatabolic responses in chondrocytes induced by these cytokines. These observations suggest that maintenance of AMPK activity supports cartilage homeostasis by protecting cartilage matrix from inflammation‐induced degradation.20.
Arjen B. Blom Sarah M. Brockbank Peter L. van Lent Henk M. van Beuningen Jeroen Geurts Nozomi Takahashi Peter M. van der Kraan Fons A. van de Loo B. Wim Schreurs Kristen Clements Peter Newham Wim B. van den Berg 《Arthritis \u0026amp; Rheumatology》2009,60(2):501-512