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1.
Mary‐Clare Miller Hugh B. Manning Abhilash Jain Linda Troeberg Jayesh Dudhia David Essex Ann Sandison Motoharu Seiki Jagdeep Nanchahal Hideaki Nagase Yoshifumi Itoh 《Arthritis \u0026amp; Rheumatology》2009,60(3):686-697
Objective
A hallmark of rheumatoid arthritis (RA) is invasion of the synovial pannus into cartilage, and this process requires degradation of the collagen matrix. The aim of this study was to explore the role of one of the collagen‐degrading matrix metalloproteinases (MMPs), membrane type 1 MMP (MT1‐MMP), in synovial pannus invasiveness.Methods
The expression and localization of MT1‐MMP in human RA pannus were investigated by Western blot analysis of primary synovial cells and immunohistochemical analysis of RA joint specimens. The functional role of MT1‐MMP was analyzed by 3‐dimensional (3‐D) collagen invasion assays and a cartilage invasion assay in the presence or absence of tissue inhibitor of metalloproteinases 1 (TIMP‐1), TIMP‐2, or GM6001. The effect of adenoviral expression of a dominant‐negative MT1‐MMP construct lacking a catalytic domain was also examined.Results
MT1‐MMP was highly expressed at the pannus–cartilage junction in RA joints. Freshly isolated rheumatoid synovial tissue and isolated RA synovial fibroblasts invaded into a 3‐D collagen matrix in an MT1‐MMP–dependent manner. Invasion was blocked by TIMP‐2 and GM6001 but not by TIMP‐1. Invasion was also inhibited by the overexpression of a dominant‐negative MT1‐MMP, which inhibits collagenolytic activity and proMMP‐2 activation by MT1‐MMP on the cell surface. Synovial fibroblasts also invaded into cartilage in an MT1‐MMP–dependent manner. This process was further enhanced by removing aggrecan from the cartilage matrix.Conclusion
MT1‐MMP serves as an essential collagen‐degrading proteinase during pannus invasion in human RA. Specific inhibition of MT1‐MMP–dependent invasion may represent a novel therapeutic strategy for RA.2.
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Laurel Young Amel Katrib Carolyn Cuello Ute Vollmer‐Conna James V. Bertouch Peter J. Roberts‐Thomson Michael J. Ahern Malcolm D. Smith Peter P. Youssef 《Arthritis \u0026amp; Rheumatology》2001,44(2):343-350
Objective
To investigate the effects of intraarticular glucocorticoid treatment on macrophage infiltration, the expression of the chemokines monocyte chemoattractant protein 1 (MCP‐1) and macrophage inflammatory protein 1α (MIP‐1α), and the expression of matrix metalloproteinases 1 and 3 (MMPs 1 and 3) and their inhibitors, the tissue inhibitors of metalloproteinases 1 and 2 (TIMPs 1 and 2), in osteoarthritis (OA) synovial membranes.Methods
Forty patients underwent arthroscopic biopsy before and 1 month after intraarticular injection of glucocorticoids. Twenty‐one patients received 120 mg of methylprednisolone acetate (Depo‐Medrol; Upjohn, Kalamazoo, MI), and 20 patients received placebo (1 patient received placebo in 1 knee and methylprednisolone acetate in the other). Immunoperoxidase staining for the expression of CD68, MCP‐1, MIP‐1α, MMP‐1, MMP‐3, TIMP‐1, and TIMP‐2 was performed, and the immunostaining was quantified by color video image analysis.Results
CD68, MCP‐1, MIP‐1α, MMP‐1, MMP‐3, TIMP‐1, and TIMP‐2 immunostaining was observed in all synovial membranes. Intraarticular glucocorticoid treatment was associated with a small (30%) but statistically significant (P = 0.048) reduction in CD68+ macrophage staining in the synovial lining layer, but there was no change in the CD68 expression in the synovial sublining layer. No significant differences were observed for MCP‐1, MIP‐1α, MMP‐1, MMP‐3, TIMP‐1, and TIMP‐2 immunostaining in the synovial lining or sublining layers.Conclusion
Intraarticular glucocorticoids may reduce CD68+ macrophage infiltration into the synovial lining layer, but not the expression of MCP‐1, MIP‐1α, MMP‐1, MMP‐3, TIMP‐1, and TIMP‐2 in the synovial membrane, in patients with OA.4.
Salahuddin Ahmed Matthew D. Silverman Hubert Marotte Kevin Kwan Natalie Matuszczak Alisa E. Koch 《Arthritis \u0026amp; Rheumatology》2009,60(5):1282-1293
Objective
Overexpression of the antiapoptotic protein myeloid cell leukemia 1 (Mcl‐1) in rheumatoid arthritis (RA) synovial fibroblasts is a major cause of their resistance to tumor necrosis factor α (TNFα)–induced apoptosis. This study was undertaken to evaluate the efficacy of epigallocatechin‐3‐gallate (EGCG) in down‐regulating Mcl‐1 expression and its mechanism of RA synovial fibroblast sensitization to TNFα‐induced apoptosis.Methods
EGCG effects on cultured RA synovial fibroblast cell morphology, proliferation, and viability over 72 hours were determined by microscopy and a fluorescent cell enumeration assay. Caspase 3 activity was determined by a colorimetric assay. Western blotting was used to evaluate the apoptosis mediators poly(ADP‐ribose) polymerase (PARP), Mcl‐1, Bcl‐2, Akt, and nuclear translocation of NF‐κB.Results
In RA synovial fibroblasts, EGCG (5–50 μM) inhibited constitutive and TNFα‐induced Mcl‐1 protein expression in a concentration‐ and time‐dependent manner (P < 0.05). Importantly, EGCG specifically abrogated Mcl‐1 expression in RA synovial fibroblasts and affected Mcl‐1 expression to a lesser extent in osteoarthritis and normal synovial fibroblasts or endothelial cells. Inhibition of Mcl‐1 by EGCG triggered caspase 3 activity in RA synovial fibroblasts, which was mediated via down‐regulation of the TNFα‐induced Akt and NF‐κB pathways. Caspase 3 activation by EGCG also suppressed RA synovial fibroblast growth, and this effect was mimicked by Akt and NF‐κB inhibitors. Interestingly, Mcl‐1 degradation by EGCG sensitized RA synovial fibroblasts to TNFα‐induced PARP cleavage and apoptotic cell death.Conclusion
Our findings indicate that EGCG itself induces apoptosis and further sensitizes RA synovial fibroblasts to TNFα‐induced apoptosis by specifically blocking Mcl‐1 expression and, hence, may be of promising adjunct therapeutic value in regulating the invasive growth of synovial fibroblasts in RA.5.
6.
Dirk Wernicke Claudia Schulze‐Westhoff Peter Petrow Rolf Bruer Josef Zacher Steffen Gay Erika Gromnica‐Ihle 《Arthritis \u0026amp; Rheumatology》2002,46(1):64-74
Objective
To study the expression of collagenase 3 (matrix metalloproteinase 13 [MMP‐13]) and collagenase 1 (MMP‐1) in synovial fibroblasts from patients with rheumatoid arthritis (RA) when cultured within 3‐dimensional collagen gels or coimplanted with normal cartilage in immunodeficient NOD/SCID mice.Methods
Messenger RNA (mRNA) and protein expression of collagenase 3 and collagenase 1 were characterized in synovial and skin fibroblasts by Northern blot and Western blot analysis. The mRNA expression of both collagenases in cell–cartilage implants in NOD/SCID mice was investigated by in situ hybridization in combination with immunohistochemistry of human fibroblasts.Results
Synovial fibroblasts coimplanted with normal cartilage in NOD/SCID mice deeply invaded adjacent cartilage tissue. In this in vivo system of cartilage destruction, collagenase 3 mRNA was induced in synovial fibroblasts at sites of cartilage erosion, while the expression of collagenase 1 mRNA could not be detected. Culture of synovial fibroblasts within 3‐dimensional collagen gels was a ssociated with a marked increase in collagenase 3 mRNA expression and proenzyme production. This stimulatory effect was 1 order of magnitude higher in comparison with a 2–4‐fold increase upon treatment with interleukin‐1 β or tumor necrosis factor α. In contrast, mRNA expression and proenzyme production of collagenase 1 were increased strongly, and to a similar extent, either by contact with 3‐dimensional collagen or by proinflammatory cytokines.Conclusion
The expression of collagenase 3, in contrast to that of collagenase 1, is preferentially stimulated in synovial fibroblasts by 3‐dimensional collagen rather than by proinflammatory cytokines. The induction of collagenase 3 by cell–matrix interactions represents a potential mechanism contributing to the invasive phenotype of synovial fibroblasts at sites of synovial invasion into cartilage in RA.7.
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9.
Astrid Jüngel Oliver Distler Ursula Schulze‐Horsel Lars C. Huber Huy Riem Ha Beat Simmen Joachim R. Kalden David S. Pisetsky Steffen Gay Jrg H. W. Distler 《Arthritis \u0026amp; Rheumatology》2007,56(11):3564-3574
Objective
Microparticles are small vesicles that are released from activated or dying cells and that occur abundantly in the synovial fluid of patients with rheumatoid arthritis (RA). The goal of these studies was to elucidate the mechanisms by which microparticles activate synovial fibroblasts to express a proinflammatory phenotype.Methods
Microparticles from monocytes and T cells were isolated by differential centrifugation. Synovial fibroblasts were cocultured with increasing numbers of microparticles. Gene expression was analyzed by real‐time polymerase chain reaction and confirmed by Western blotting and enzyme immunoassay. Arachidonic acid labeled with tritium was used to study the transport of biologically active lipids by microparticles. The roles of NF‐κB and activator protein 1 (AP‐1) signaling were analyzed with electrophoretic mobility shift assay and transfection with small interfering RNA and IκB expression vectors.Results
Microparticles strongly induced the synthesis of cyclooxygenase 2 (COX‐2), microsomal prostaglandin E synthase 1 (mPGES‐1), and prostaglandin E2 (PGE2). In contrast, no up‐regulation of COX‐1, mPGES‐2, cytosolic PGES, or phospholipase A2 was observed. The induction of PGE2 was blocked by selective inhibition of COX‐2. Microparticles activated NF‐κB, AP‐1, p38, and JNK signaling in synovial fibroblasts. Inhibition of NF‐κB, AP‐1, and JNK signaling reduced the stimulatory effects. Arachidonic acid was transported from leukocytes to fibroblasts by microparticles. Arachidonic acid derived from microparticles was converted to PGE2 by synovial fibroblasts.Conclusion
These results demonstrate that microparticles up‐regulate the production of PGE2 in synovial fibroblasts by inducing COX‐2 and mPGES‐1. These data provide evidence for a novel mechanism by which microparticles may contribute to inflammation and pain in RA.10.
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Sophie Flood Rheinallt Parri Anwen Williams Victor Duance Deborah Mason 《Arthritis \u0026amp; Rheumatology》2007,56(8):2523-2534
Objective
Patients with rheumatoid arthritis (RA) have increased concentrations of the amino acid glutamate in synovial fluid. This study was undertaken to determine whether glutamate receptors are expressed in the synovial joint, and to determine whether activation of glutamate receptors on human synoviocytes contributes to RA disease pathology.Methods
Glutamate receptor expression was examined in tissue samples from rat knee joints and in human fibroblast‐like synoviocytes (FLS). FLS from 5 RA patients and 1 normal control were used to determine whether a range of glutamate receptor antagonists influenced expression of the proinflammatory cytokine interleukin‐6 (IL‐6), enzymes involved in matrix degradation and cytokine processing (matrix metalloproteinase 2 [MMP‐2] and MMP‐9), and the inhibitors of these enzymes (tissue inhibitor of metalloproteinases 1 [TIMP‐1] and TIMP‐2). IL‐6 concentrations were determined by enzyme‐linked immunosorbent assay, MMP activity was measured by gelatin zymography, and TIMP activity was determined by reverse zymography. Fluorescence imaging of intracellular calcium concentrations in live RA FLS stimulated with specific antagonists was used to reveal functional activation of glutamate receptors that modulated IL‐6 or MMP‐2.Results
Ionotropic and metabotropic glutamate receptor subunit mRNA were expressed in the patella, fat pad, and meniscus of the rat knee and in human articular cartilage. Inhibition of N‐methyl‐D ‐aspartate (NMDA) receptors in RA FLS increased proMMP‐2 release, whereas non‐NMDA ionotropic glutamate receptor antagonists reduced IL‐6 production by these cells. Stimulation with glutamate, NMDA, or kainate (KA) increased intracellular calcium concentrations in RA FLS, demonstrating functional activation of specific ionotropic glutamate receptors.Conclusion
Our findings indicate that activation of NMDA and KA glutamate receptors on human synoviocytes may contribute to joint destruction by increasing IL‐6 expression.14.
Harris Perlman Nadine Nguyen Hongtao Liu Joy Eslick Sybille Esser Kenneth Walsh Terry L. Moore Richard M. Pope 《Arthritis \u0026amp; Rheumatology》2003,48(11):3096-3101
Objective
To characterize the expression pattern of tumor necrosis factor–related apoptosis‐inducing ligand (TRAIL) and its cognate receptors (TRAIL R1, R2, R3, and R4) on rheumatoid arthritis (RA) synovial fluid (SF) lymphocytes and monocyte/macrophages and on cultured RA synovial fibroblasts.Methods
The expression of TRAIL and TRAIL receptors on RA SF lymphocytes and monocyte/macrophages, normal macrophages, and RA synovial fibroblasts was examined by flow cytometry with previously characterized monoclonal antibodies. The ability of adenoviral‐mediated delivery of TRAIL to induce macrophage or RA synovial fibroblast apoptosis was examined by flow cytometry.Results
By flow cytometry, neither TRAIL nor its cognate receptors was detectable on RA SF lymphocytes or RA synovial fibroblasts. In contrast, RA SF macrophages expressed TRAIL R3, a decoy receptor (P < 0.01 versus isotype control), but not TRAIL, or TRAIL R1, R2, or R4. Normal peripheral blood–derived monocyte‐differentiated macrophages expressed TRAIL R2 (P < 0.01), but not TRAIL or the other TRAIL receptors. Adenoviral‐mediated delivery of TRAIL had no effect on the survival of normal macrophages or RA synovial fibroblasts but readily induced apoptosis in the prostate cancer cell line (PC‐3) that expressed TRAIL R1 and R2.Conclusion
TRAIL R1 and R2, which are required for signal transmission by TRAIL, were not detected on RA SF lymphocytes, macrophages, or synovial fibroblasts. These observations do not support a potential therapeutic role for TRAIL in RA.15.
Anthony P. Hollander Kevin P. Corke Anthony J. Freemont Claire E. Lewis 《Arthritis \u0026amp; Rheumatology》2001,44(7):1540-1544
Objective
To determine if the rheumatoid synovium is a suitable target for hypoxia‐regulated gene therapy.Methods
Sequential sections of wax‐embedded synovial membrane samples were obtained from 10 patients with rheumatoid arthritis (RA), 10 with primary osteoarthritis (OA), and from 6 healthy controls. Membrane sections from each patient were immunostained for hypoxia‐inducible factor 1α (HIF‐1α) and CD68 (a pan–macrophage marker).Results
HIF‐1α was expressed abundantly by macrophages in most rheumatoid synovia, predominantly close to the intimal layer but also in the subintimal zone. There was markedly lower expression of HIF‐1α in OA synovia, and it was absent from all of the healthy synovia.Conclusion
These observations indicate that macrophages transduced with a therapeutic gene under the control of a hypoxia‐inducible promoter could be administered to RA patients systemically. Migration of these cells to synovial tissue would result in the transgene being switched on in diseased joints but not in healthy tissues.16.
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Takeo Isozaki Bradley J. Rabquer Jeffrey H. Ruth G. Kenneth Haines Alisa E. Koch 《Arthritis \u0026amp; Rheumatology》2013,65(1):98-108
Objective
To examine the expression of ADAM‐10 in rheumatoid arthritis (RA) synovial tissue (ST) and the role it plays in angiogenesis.Methods
ADAM‐10 expression was determined using immunohistology, Western blotting, and quantitative polymerase chain reaction. In order to examine the role of ADAM‐10 in angiogenesis, we performed in vitro Matrigel tube formation and chemotaxis assays using human microvascular endothelial cells (HMVECs) transfected with control or ADAM‐10 small interfering RNA (siRNA). To determine whether ADAM‐10 plays a role in angiogenesis in the context of RA, we performed Matrigel assays using a coculture system of HMVECs and RA synovial fibroblasts.Results
Endothelial cells and lining cells within RA ST expressed high levels of ADAM‐10 compared with cells within osteoarthritis ST and normal ST. ADAM‐10 expression was significantly elevated at the protein and messenger RNA levels in HMVECs and RA synovial fibroblasts stimulated with proinflammatory mediators compared with unstimulated cells. ADAM‐10 siRNA–treated HMVECs had decreased endothelial cell tube formation and migration compared with control siRNA–treated HMVECs. In addition, ADAM‐10 siRNA–treated HMVECs from the RA synovial fibroblast coculture system had decreased endothelial cell tube formation compared with control siRNA–treated HMVECs.Conclusion
These data show that ADAM‐10 is overexpressed in RA and suggest that ADAM‐10 may play a role in RA angiogenesis. ADAM‐10 may be a potential therapeutic target in inflammatory angiogenic diseases such as RA.19.
Fabia Brentano Olivier Schorr Renate E. Gay Steffen Gay Diego Kyburz 《Arthritis \u0026amp; Rheumatology》2005,52(9):2656-2665
Objective
To assess the expression of Toll‐like receptor 3 (TLR‐3) protein in synovial tissues and cultured synovial fibroblasts obtained from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and to investigate the consequences of stimulation of cultured synovial fibroblasts with TLR‐3 ligands.Methods
TLR‐3 expression in synovial tissues was determined by immunohistochemistry and immunofluorescence, and expression in cultured RA synovial fibroblasts (RASFs) was determined by fluorescence‐activated cell sorting and real‐time polymerase chain reaction techniques. TLR‐3 signaling was assessed by incubating RASFs with poly(I‐C), lipopolysaccharide, palmitoyl‐3‐cysteine‐serine‐lysine‐4, or necrotic synovial fluid cells from RA patients in the presence or absence of hydroxychloroquine or Benzonase. Subsequent determination of interferon‐β (IFNβ), CXCL10, CCL5, and interleukin‐6 (IL‐6) protein production in the culture supernatants was performed by enzyme‐linked immunosorbent assays.Results
TLR‐3 protein expression was found to be higher in RA synovial tissues than in OA synovial tissues. TLR‐3 expression was localized predominantly in the synovial lining, with a majority of the TLR‐3–expressing cells coexpressing fibroblast markers. Stimulation of cultured RASFs with the TLR‐3 ligand poly(I‐C) resulted in the production of high levels of IFNβ, CXCL10, CCL5, and IL‐6 protein. Similarly, coincubation of RASFs with necrotic synovial fluid cells from patients with RA resulted in up‐regulation of these cytokines and chemokines in a TLR‐3–dependent manner.Conclusion
Our findings demonstrate the expression of TLR‐3 in RA synovial tissue and the activation of RASFs in vitro by the TLR‐3 ligand poly(I‐C) as well as by necrotic RA synovial fluid cells, and indicate that RNA released from necrotic cells might act as an endogenous TLR‐3 ligand for the stimulation of proinflammatory gene expression in RASFs.20.
Elaine Beaulieu Devi Ngo Leilani Santos Yuan Hang Yang Malcolm Smith Christian Jorgensen Virginie Escriou Daniel Scherman Gabriel Courties Florence Apparailly Eric F. Morand 《Arthritis \u0026amp; Rheumatology》2010,62(9):2651-2661