Stimulation of collagenase 3 expression in synovial fibroblasts of patients with rheumatoid arthritis by contact with a three-dimensional collagen matrix or with normal cartilage when coimplanted in NOD/SCID mice

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 associated 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-1beta or tumor necrosis factor a. 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.     

Membrane type 1 matrix metalloproteinase is a crucial promoter of synovial invasion in human rheumatoid arthritis

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.

     

Oncostatin M in combination with tumor necrosis factor α induces cartilage damage and matrix metalloproteinase expression in vitro and in vivo

Objective

To determine the effects of the proinflammatory cytokine combination of oncostatin M (OSM) and tumor necrosis factor α (TNFα) on cartilage destruction in both in vitro and in vivo model systems.

Methods

The release of collagen and proteoglycan was assessed in bovine cartilage explant cultures, while messenger RNA (mRNA) from bovine chondrocytes was analyzed by Northern blotting. Immunohistochemistry was performed on sections prepared from murine joints following injection of adenovirus vectors encoding murine OSM and/or murine TNFα.

Results

The combination of OSM + TNFα induced significant collagen release from bovine cartilage, accompanied by high levels of active collagenolytic activity. Northern blot analysis indicated that this cytokine combination synergistically induced matrix metalloproteinase 1 (MMP‐1), MMP‐3, and MMP‐13 mRNA. The in vivo data clearly indicated that OSM + TNFα overexpression increased MMP levels and decreased levels of tissue inhibitor of metalloproteinases 1 (TIMP‐1). Specifically, OSM + TNFα induced marked synovial hyperplasia, inflammation, and cartilage and bone destruction with a concomitant increase in MMP expression in both cartilage and synovium and decreased TIMP‐1 expression in the articular cartilage. These effects were markedly greater than those seen with either cytokine alone.

Conclusion

This study demonstrates that OSM + TNFα represents a potent proinflammatory cytokine combination that markedly induces MMP production in both cartilage and synovium, thus promoting joint destruction.

     

Expression,regulation, and signaling of the pattern‐recognition receptor nucleotide‐binding oligomerization domain 2 in rheumatoid arthritis synovial fibroblasts

Objective

Since pattern‐recognition receptors (PRRs), in particular Toll‐like receptors (TLRs), were found to be overexpressed in the synovium of rheumatoid arthritis (RA) patients and to play a role in the production of disease‐relevant molecules, we sought to determine the expression, regulation, and function of the PRR nucleotide‐binding oligomerization domain 2 (NOD‐2) in RA.

Methods

Expression of NOD‐2 in synovial tissues was analyzed by immunohistochemistry. Expression and induction of NOD‐2 in RA synovial fibroblasts (RASFs) were measured by conventional and real‐time polymerase chain reaction (PCR) analyses. Levels of interleukin‐6 (IL‐6) and IL‐8 were measured by enzyme‐linked immunosorbent assay (ELISA) and expression of matrix metalloproteinases (MMPs) by ELISA and/or real‐time PCR. NOD‐2 expression was silenced with small interfering RNA. Western blotting with antibodies against phosphorylated and total p38, JNK, and ERK, as well as inhibitors of p38, JNK, and ERK was performed. Activation of NF‐κB was measured by electrophoretic mobility shift assay.

Results

NOD‐2 was expressed by fibroblasts and macrophages in the synovium of RA patients, predominantly at sites of invasion into articular cartilage. In cultured RASFs, no basal expression of messenger RNA for NOD‐2 was detectable, but was induced by poly(I‐C), lipopolysaccharide, and tumor necrosis factor α. After up‐regulation of NOD‐2 by TLR ligands, its ligand muramyl dipeptide (MDP) increased the expression of IL‐6 and IL‐8 via p38 and NF‐κB. Stimulation with MDP further induced the expression of MMP‐1, MMP‐3, and MMP‐13.

Conclusion

Not only TLRs, but also the PRR NOD‐2 is expressed in the synovium of RA patients, and activation of NOD‐2 acts synergistically with TLRs in the production of proinflammatory and destructive mediators. Therefore, NOD‐2 might contribute to the initiation and perpetuation of chronic, destructive inflammation in RA.

     

Formation of invadopodia‐like structures by synovial cells promotes cartilage breakdown in collagen‐induced arthritis: Involvement of the protein tyrosine kinase Src

Objective

Invasive synovial fibroblasts are suggested to be the major effectors of cartilage and bone destruction, and this aggressive phenotype can lead to irreversible damage. In cancer cells, invasion across tissue boundaries and metastasis have recently been shown to depend on the capacity of the cells to breach the basement membrane, a process that was linked to the formation of the actin‐rich cell protrusions called invadopodia. This study was undertaken to investigate whether arthritic synovial cells use invadopodia to invade and degrade cartilage components.

Methods

Fibroblast‐like synoviocytes (FLS) from control rats or rats with collagen‐induced arthritis (CIA) were cultured on fluorescent matrix in the presence of Src inhibitors or were transfected with wild‐type or variants of Src kinases. The in vivo effect of Src inhibition on cartilage degradation and invasion was studied in a rat model of CIA.

Results

FLS from rats with CIA produced more invadopodia‐like structures than did FLS from control rats, leading to increased extracellular matrix degradation. Furthermore, c‐Src activation was increased in synovial cells from rats with CIA, and Src activity was found to mediate the formation of invadopodia. Pharmacologic blockade of Src activity by PP2 or intraarticular expression of a c‐Src–specific short hairpin RNA in the CIA model reduced synovial membrane hyperplasia and cartilage degradation, an event linked to decreased invadopodia formation by synovial fibroblasts.

Conclusion

This study demonstrates that inhibition of invadopodia formation in arthritic synovial cells leads to a direct effect on extracellular matrix degradation in vitro and in vivo, making invadopodia a relevant therapeutic target for interfering with this process.

     

Reduced expression and proteolytic susceptibility of lubricin/superficial zone protein may explain early elevation in the coefficient of friction in the joints of rats with antigen‐induced arthritis

Objective

To investigate the effect of arthritis development and progression on the integrity and function of lubricin and the relationship of lubricin to cartilage damage in a rat antigen‐induced arthritis model.

Methods

Arthritis was induced in the right knee joints, using methylated bovine serum albumin and Freund's complete adjuvant. Whole joint friction measurements were performed ex vivo with a modified Stanton pendulum configuration, and coefficients of friction (μ) were determined. Levels of messenger RNA (mRNA) for lubricin, cathepsin B, and interleukin‐1β (IL‐1β) in synovial tissue from control and affected joints were determined by quantitative polymerase chain reaction. Lubricin staining in cartilage was performed using a lubricin‐specific monoclonal antibody.

Results

The μ values in excised right joints following arthritis induction were significantly (P < 0.001) higher than those in excised left joints. Lubricin mRNA expression levels in synovial tissue on days 4 and 7 after arthritis induction were significantly (P < 0.001) lower in the right joints compared with the left joints, whereas levels of cathepsin B and IL‐1β mRNA expression on days 4, 7, and 14 were significantly (P < 0.001) higher in the right joints than the left joints. Lubricin staining was diminished in cartilage from the right joints compared with the left joints.

Conclusion

Elevated coefficients of friction in arthritic joints occur concurrently with enhanced proteolytic degradation by up‐regulated cathepsin B and other proteases, as well as decreased lubricin synthesis by synovial fibroblasts and superficial zone chondrocytes. Loss of joint lubrication is an early event in inflammatory arthropathy. Restoring chondroprotection and preventing potential wear‐induced cartilage degradation may require lubricin supplementation in synovial fluid.

     

Synergistic effects of glycoprotein 130 binding cytokines in combination with interleukin‐1 on cartilage collagen breakdown

Objective

To determine whether other glycoprotein 130 (gp130) binding cytokines can mimic the effects of oncostatin M (OSM) in acting synergistically with interleukin‐1α (IL‐1α) to induce cartilage collagen breakdown and collagenase expression, and to determine which receptors mediate these effects.

Methods

The release of collagen and proteoglycan was assessed in bovine and human cartilage explant cultures. Messenger RNA (mRNA) and protein production from immortalized human chondrocytes (T/C28a4) was analyzed by Northern blotting and specific enzyme‐linked immunosorbent assays. Collagenase activity was measured by bioassay. Cell surface receptors were detected by flow cytometry.

Results

OSM in combination with IL‐1α caused a rapid synergistic induction of matrix metalloproteinase 1 mRNA, which was sustained over a 72‐hour period. Flow cytometric analyses detected both the OSM‐specific receptor and the gp130 receptor at the chondrocyte cell surface, but failed to detect the leukemia inhibitory factor receptor (LIFR). Cartilage degradation assays revealed that, of the gp130 binding cytokines, only OSM and IL‐6, in the presence of its soluble receptor (sIL‐6R), were able to act synergistically with IL‐1α to promote collagen release.

Conclusion

This study demonstrates that IL‐6 can mimic OSM in synergizing with IL‐1α to induce chondrocyte‐mediated cartilage collagen breakdown and collagenase production. In order to have this effect, IL‐6 requires the presence of its soluble receptor. The apparent absence of LIFR explains why other gp130 binding cytokines do not act in synergy with IL‐1α. Since OSM, IL‐6, and sIL‐6R levels have all been shown to be elevated in the rheumatoid joint, our findings suggest that these cytokines may be key mediators of cartilage collagen catabolism in the inflammatory arthritides.

     

Suppression of leukocyte infiltration and cartilage degradation by selective inhibition of pre–B cell colony‐enhancing factor/visfatin/nicotinamide phosphoribosyltransferase: Apo866‐mediated therapy in human fibroblasts and murine collagen‐induced arthritis

Objective

To assess the ability of pre–B cell colony‐enhancing factor (PBEF) to regulate inflammation and degradative processes in inflammatory arthritis, using the small molecule inhibitor APO866 in human fibroblasts in vitro and in murine collagen‐induced arthritis (CIA).

Methods

Enzyme‐linked immunosorbent assays were used to examine regulation of expression of metalloproteinases and chemokines in human fibroblasts. The role of PBEF was further examined using APO866 in mice with CIA, with effects on disease activity assessed using radiography, histology, in vivo imaging, and quantitative polymerase chain reaction (qPCR).

Results

In vitro activation of human fibroblasts with PBEF promoted expression of matrix metalloproteinase 3 (MMP‐3), CCL2, and CXCL8, an effect inhibited by APO866. In mice with CIA, early intervention with APO866 inhibited synovial inflammation, including chemokine‐directed leukocyte infiltration, and reduced a systemic marker of inflammation, serum hyaluronic acid. APO866 blockade led to reduced expression of MMP‐3 and MMP‐13 in joint extracts and to a reduction in a systemic marker of cartilage erosion, serum cartilage oligomeric matrix protein. Radiologic images revealed that APO866 protected against bone erosion, while qPCR demonstrated inhibition of RANKL expression. In mice with established disease, APO866 reduced synovial inflammation and cartilage destruction, and halted bone erosion. In addition, APO866 reduced the activity of MMP‐3, CCL2, and RANKL in vivo, and inhibited production of CCL2 and RANKL in synovial explants from arthritic mice, a result that was reversed with nicotinamide mononucleotide.

Conclusion

These findings confirm PBEF to be an important regulator of inflammation, cartilage catabolism, and bone erosion, and highlight APO866 as a promising therapeutic agent for targeting PBEF activity in inflammatory arthritis.

     

Inhibition of fibroblast activation protein and dipeptidylpeptidase 4 increases cartilage invasion by rheumatoid arthritis synovial fibroblasts

Objective

Since fibroblasts in the synovium of patients with rheumatoid arthritis (RA) express the serine proteases fibroblast activation protein (FAP) and dipeptidylpeptidase 4 (DPP‐4)/CD26, we undertook the current study to determine the functional role of both enzymes in the invasion of RA synovial fibroblasts (RASFs) into articular cartilage.

Methods

Expression of FAP and DPP‐4/CD26 by RASFs was analyzed using fluorescence‐activated cell sorting and immunocytochemistry. Serine protease activity was measured by cleavage of fluorogenic substrates and inhibited upon treatment with L‐glutamyl L‐boroproline. The induction and expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in RASFs were detected using real‐time polymerase chain reaction. Densitometric measurements of MMPs using immunoblotting confirmed our findings on the messenger RNA level. Stromal cell–derived factor 1 (SDF‐1 [CXCL12]), MMP‐1, and MMP‐3 protein levels were measured using enzyme‐linked immunosorbent assay. The impact of FAP and DPP‐4/CD26 inhibition on the invasiveness of RASFs was analyzed in the SCID mouse coimplantation model of RA using immunohistochemistry.

Results

Inhibition of serine protease activity of FAP and DPP‐4/CD26 in vitro led to increased levels of SDF‐1 in concert with MMP‐1 and MMP‐3, which are downstream effectors of SDF‐1 signaling. Using the SCID mouse coimplantation model, inhibition of enzymatic activity in vivo significantly promoted invasion of xenotransplanted RASFs into cotransplanted human cartilage. Zones of cartilage resorption were infiltrated by FAP‐expressing RASFs and marked by a significantly higher accumulation of MMP‐1 and MMP‐3, when compared with controls.

Conclusion

Our results indicate a central role for the serine protease activity of FAP and DPP‐4/CD26 in protecting articular cartilage against invasion by synovial fibroblasts in RA.

     

Crucial role of macrophages in matrix metalloproteinase–mediated cartilage destruction during experimental osteoarthritis : Involvement of matrix metalloproteinase 3

Objective

To explore the involvement of synovial macrophages in early cartilage damage in osteoarthritis (OA), and to identify the role of matrix metalloproteinase 3 (MMP‐3) in the pathology of early and late OA.

Methods

The role of synovial macrophages in MMP‐mediated damage in OA was studied by depleting synovial macrophages prior to elicitation of a collagenase‐induced instability model of OA. The expression of MMP in synovium and cartilage was monitored using TaqMan analysis. In spontaneous and induced OA, cartilage pathology was scored in MMP‐3–knockout mice and control mice, by histologic assessment and VDIPEN staining.

Results

On day 14 following induction of OA, MMP‐mediated neoepitopes were detected in cartilage from mice with mild experimental OA (mean ± SD positively stained surface area 20 ± 3.2%). Remarkably, by depleting synovial macrophages prior to induction of OA, the generation of MMP‐induced neoepitopes was largely prevented (mean ± SD positively stained surface area 5 ± 1%; P< 0.001), indicating an important role for synovial macrophages in the occurrence of MMP‐mediated cartilage damage. We observed a strong decrease in MMP‐3 and MMP‐9 expression in synovial but not cartilage tissue in macrophage‐depleted joints. Among 2‐year‐old mice, spontaneous OA–like changes in the lining layer were significantly decreased in MMP‐3–knockout mice compared with control mice. Even more striking was the 67% reduction in the occurrence of severe cartilage damage in MMP‐3–knockout mice. In addition, MMP‐mediated VDIPEN expression was significantly decreased, indicating reduced MMP‐mediated cartilage breakdown.

Conclusion

The results of this study prove that MMP‐3 is involved in the generation of severe cartilage damage in murine OA. Synovial macrophages are crucial in early MMP activity and appear to mediate MMP production in synovium rather than cartilage.

     

Matriptase is a novel initiator of cartilage matrix degradation in osteoarthritis

Objective

Increasing evidence implicates serine proteinases in pathologic tissue turnover. The aim of this study was to assess the role of the transmembrane serine proteinase matriptase in cartilage destruction in osteoarthritis (OA).

Methods

Serine proteinase gene expression in femoral head cartilage obtained from either patients with hip OA or patients with fracture to the neck of the femur (NOF) was assessed using a low‐density array. The effect of matriptase on collagen breakdown was determined in cartilage degradation models, while the effect on matrix metalloproteinase (MMP) expression was analyzed by real‐time polymerase chain reaction. ProMMP processing was determined using sodium dodecyl sulfate–polyacrylamide gel electrophoresis/N‐terminal sequencing, while its ability to activate proteinase‐activated receptor 2 (PAR‐2) was determined using a synovial perfusion assay in mice.

Results

Matriptase gene expression was significantly elevated in OA cartilage compared with NOF cartilage, and matriptase was immunolocalized to OA chondrocytes. We showed that matriptase activated proMMP‐1 and processed proMMP‐3 to its fully active form. Exogenous matriptase significantly enhanced cytokine‐stimulated cartilage collagenolysis, while matriptase alone caused significant collagenolysis from OA cartilage, which was metalloproteinase‐dependent. Matriptase also induced MMP‐1, MMP‐3, and MMP‐13 gene expression. Synovial perfusion data confirmed that matriptase activates PAR‐2, and we demonstrated that matriptase‐dependent enhancement of collagenolysis from OA cartilage is blocked by PAR‐2 inhibition.

Conclusion

Elevated matriptase expression in OA and the ability of matriptase to activate selective proMMPs as well as induce collagenase expression make this serine proteinase a key initiator and inducer of cartilage destruction in OA. We propose that the indirect effects of matriptase are mediated by PAR‐2, and a more detailed understanding of these mechanisms may highlight important new therapeutic targets for OA treatment.

     

Blocking vascular endothelial growth factor with soluble Flt‐1 improves the chondrogenic potential of mouse skeletal muscle–derived stem cells

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.

     

Tumor necrosis factor α activation of the apoptotic cascade in murine articular chondrocytes is associated with the induction of metalloproteinases and specific pro‐resorptive factors

Objective

Tumor necrosis factor α (TNFα) blockade provides substantive reduction of the symptoms of rheumatoid arthritis (RA). While the biologic actions of TNFα have been well characterized in immune and synovial cells, which are known to be major contributors to the progression of cartilage destruction in RA, the current studies were designed to assess the direct effects of TNFα on chondrocytes.

Methods

We examined the expression of several groupings of messenger RNA (mRNA) that define key biologic pathways that have previously been associated with either the general actions of TNFα or cartilage destruction, in murine articular chondrocytes isolated from wild‐type mice and TNFα receptor–null (p55/p75^−/−) mice.

Results

TNFα induced the expression of multiple mRNA that facilitate apoptosis and lead to apoptosis‐induced cell death. The induction of apoptosis was accompanied by the increased expression of several factors involved in the regulation of skeletal tissue proteolysis and resorption. Quantitative increases from 2‐fold to >10‐fold were seen for inducible nitric oxide synthase, matrix metalloproteinase 3, macrophage colony‐stimulating factor, and osteoprotegerin mRNA expression. The dependence of the induction of these mRNA on TNFα was confirmed by comparison with the effects of TNFα on chondrocytes isolated from receptor‐null mice.

Conclusion

These findings demonstrate that TNFα alters the expression of a complex array of genes within murine chondrocytes that contribute to the destruction of joint surfaces, independent of its actions on synovial and immune cells. Further studies are needed to clarify the biologic actions of TNFα in human cartilage cells.