ADAM‐10 is overexpressed in rheumatoid arthritis synovial tissue and mediates angiogenesis

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.

     

Tie2 receptor tyrosine kinase,a major mediator of tumor necrosis factor α–induced angiogenesis in rheumatoid arthritis

Objective

Rheumatoid arthritis (RA) is an inflammatory disease and an angiogenic disease. However, the molecular mechanisms promoting angiogenesis in RA are not clearly identified. Our objective was to study the role of an endothelium‐specific receptor tyrosine kinase, Tie2, in angiogenesis of inflammatory arthritis.

Methods

Expression of Tie2 and its ligand, angiopoietin 1 (Ang1), in human synovium was examined by immunohistochemistry and Western blot. A novel synovium vascular window model was established to study the role of Tie2 in angiogenesis in vivo. Primary cultured endothelial cells and synoviocytes were used to study tumor necrosis factor α (TNFα)–induced Tie2 and Ang1 expression.

Results

Tie2 was implicated in pathologic angiogenesis. We observed that Tie2 and Ang1 were elevated in human RA synovium. Using a novel collagen‐induced arthritis synovial window model, we demonstrated that Tie2 signaling regulated arthritis angiogenesis in vivo. We also showed that Tie2 mediated TNFα‐induced angiogenesis in a mouse cornea assay. In addition, we observed that TNFα can regulate Tie2 activation in multiple ways that may involve interactions between endothelial cells and synoviocytes. TNFα up‐regulates Tie2 in endothelial cells through nuclear factor κB, and it up‐regulates Ang1 in synoviocytes. These findings suggest paracrine regulation of angiogenesis between endothelial cells and synoviocytes.

Conclusion

This study demonstrates that Tie2 regulates angiogenesis in inflammatory synovium. Tie2 signaling is an important angiogenic mediator that links the proinflammatory cytokine TNFα to pathologic angiogenesis.

     

Control of fibroblast‐like synoviocyte proliferation by macrophage migration inhibitory factor

Objective

The hyperplasia of fibroblast‐like synoviocytes (FLS) is considered essential to the evolution of joint destruction in rheumatoid arthritis (RA), but the mechanisms underlying FLS proliferation remain poorly understood. Macrophage migration inhibitory factor (MIF) is a cytokine that has recently been shown to exert proinflammatory effects on RA FLS. This study sought to identify the mechanisms of activation of FLS by MIF, and to assess the effects of MIF on synovial cell proliferation.

Methods

Human RA FLS were treated with recombinant MIF, interleukin‐1β (IL‐1β), tumor necrosis factor α (TNFα), and/or anti‐MIF monoclonal antibodies (mAb). Proliferation was measured with tritiated thymidine incorporation. Nuclear factor κB (NF‐κB) and mitogen‐activated protein (MAP) kinase activation were measured with immunohistochemistry and Western blotting, respectively.

Results

FLS proliferation was significantly increased by MIF. IL‐1β and TNFα also induced proliferation, but these effects were prevented by neutralization with anti‐MIF mAb. Activation of NF‐κB was induced by IL‐1β, but not by MIF. Anti‐MIF mAb had no effect on IL‐1β–induced NF‐κB nuclear translocation. By contrast, MIF induced phosphorylation of extracellular signal–regulated kinase (ERK) MAP kinase. ERK antagonism, but not NF‐κB antagonism, prevented the effect of MIF on FLS proliferation.

Conclusion

These data suggest that MIF may regulate RA synovial hyperplasia by acting directly and via involvement in the effects of IL‐1β and TNFα. In addition, the effects of MIF on FLS activation are independent of NF‐κB, and dependent on ERK MAP kinase. These data suggest an important therapeutic potential for MIF antagonism in RA.

     

Glucocorticoid‐induced leucine zipper is an endogenous antiinflammatory mediator in arthritis

Objective

Glucocorticoid‐induced leucine zipper (GILZ) is a glucocorticoid‐induced protein, the reported molecular interactions of which suggest that it functions to inhibit inflammation. However, the role of endogenous GILZ in the regulation of inflammation in vivo has not been established. This study was undertaken to examine the expression and function of GILZ in vivo in collagen‐induced arthritis (CIA), a murine model of rheumatoid arthritis (RA), and in RA synoviocytes.

Methods

GILZ expression was detected in mouse and human synovium by immunohistochemistry and in cultured cells by real‐time polymerase chain reaction and permeabilization flow cytometry. GILZ function was assessed in vivo by small interfering RNA (siRNA) silencing using cationic liposome–encapsulated GILZ or control nontargeting siRNA and was assessed in vitro using transient overexpression.

Results

GILZ was readily detectable in the synovium of mice with CIA and was up‐regulated by therapeutic doses of glucocorticoids. Depleting GILZ expression in vivo increased the clinical and histologic severity of CIA and increased synovial expression of tumor necrosis factor and interleukin‐1 (IL‐1), without affecting the levels of circulating cytokines or anticollagen antibodies. GILZ was highly expressed in the synovium of patients with active RA and in cultured RA synovial fibroblasts, and GILZ overexpression in synovial fibroblasts inhibited IL‐6 and IL‐8 release.

Conclusion

Our findings indicate that GILZ functions as an endogenous inhibitor of chronic inflammation via effects on cytokine expression and suggest that local modulation of GILZ expression could be a beneficial therapeutic strategy.

     

Down‐regulation of myeloid cell leukemia 1 by epigallocatechin‐3‐gallate sensitizes rheumatoid arthritis synovial fibroblasts to tumor necrosis factor α–induced apoptosis

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.

     

Heterogeneous requirement of IκB kinase 2 for inflammatory cytokine and matrix metalloproteinase production in rheumatoid arthritis: Implications for therapy

Objective

To investigate the potential role of IκB kinase 1 (IKK‐1) and IKK‐2 in the regulation of nuclear factor κB (NF‐κB) activation and the expression of tumor necrosis factor α (TNFα), as well as interleukin‐1β (IL‐1β), IL‐6, IL‐8, vascular endothelial growth factor (VEGF), and matrix metalloproteinases (MMPs), in rheumatoid arthritis (RA).

Methods

Recombinant adenoviruses expressing β‐galactosidase, dominant‐negative IKK‐1 and IKK‐2, or IκBα were used to infect ex vivo RA synovial membrane cultures and synovial fibroblasts obtained from patients with RA undergoing joint replacement surgery, or human dermal fibroblasts, human umbilical vein endothelial cells (HUVECs), and monocyte‐derived macrophages from healthy volunteers. Then, their effect on the spontaneous or stimulus‐induced release of inflammatory cytokines, VEGF, and MMPs from RA synovial membrane cells was examined.

Results

IKK‐2 was not required for lipopolysaccharide (LPS)–induced NF‐κB activation or TNFα, IL‐6, or IL‐8 production in macrophages, but was essential for this process in response to CD40 ligand, TNFα, and IL‐1. In synovial fibroblasts, dermal fibroblasts, and HUVECs, IKK‐2 was also required for LPS‐induced NF‐κB activation and IL‐6 or IL‐8 production. In RA synovial membrane cells, IKK‐2 inhibition had no effect on spontaneous TNFα production but significantly reduced IL‐1β, IL‐6, IL‐8, VEGF, and MMPs 1, 2, 3, and 13.

Conclusion

Our study demonstrates that IKK‐2 is not essential for TNFα production in RA. However, because IKK‐2 regulates the expression of other inflammatory cytokines (IL‐1β, IL‐6, and IL‐8), VEGF, and MMPs 1, 2, 3, and 13, which are involved in the inflammatory, angiogenic, and destructive processes in the RA joint, it may still be a good therapeutic target.

     

Role of notch‐1 intracellular domain in activation of rheumatoid synoviocytes

Objective

Notch family proteins are transmembrane receptors that control cell fate and proliferation. Rheumatoid arthritis (RA) is characterized by activation and abnormal proliferation/differentiation of synoviocytes. We examined the expression of Notch‐1 and its role in the activation of RA synoviocytes.

Methods

The expression of Notch‐1 protein was detected by a specific antibody raised against the Notch‐1 intracellular domain. Notch‐1 messenger RNA (mRNA) expression in synoviocytes was analyzed by Northern blotting. Notch‐1 protein expression was confirmed by Western blotting with anti–Notch‐1 antibody. To analyze the role of Notch‐1 in synoviocyte proliferation, we examined the effects of antisense Notch‐1 oligonucleotides (ODNs) and MW167, a γ‐secretase inhibitor.

Results

Notch‐1 protein and mRNA were detected in synovium from all study subjects. The nucleus of RA synoviocytes showed strong staining with anti–Notch‐1 antibody, whereas there was predominantly cytoplasmic staining of normal and osteoarthritis (OA) synoviocytes. Western blotting showed a distinct ∼63‐kd protein detected by anti–Notch‐1 antibody in nuclear extracts from RA synoviocytes, indicating that nuclear staining of RA synovium and synoviocytes is likely to be the result of nuclear localization of Notch‐1 intracellular domain (NICD). Furthermore, tumor necrosis factor α (TNFα) increased NICD nuclear translocation in a dose‐dependent manner. Antisense Notch‐1 ODNs partially blocked the proliferation of RA synoviocytes and inhibited TNFα‐induced proliferation in both OA and RA synoviocytes. In addition, γ‐secretase inhibitor, which blocks the production of NICD, also inhibited TNFα‐induced proliferation of RA synoviocytes.

Conclusion

Our results demonstrate the expression of Notch‐1 in synoviocytes and the presence of Notch‐1 fragment in the nuclei of RA synoviocytes and suggest the involvement of Notch‐1 signaling in the TNFα‐induced proliferation of RA synoviocytes.

     

The influence of synovial fluid on adenovirus‐mediated gene transfer to the synovial tissue

Objective

To determine the effect of synovial fluid (SF) from rheumatoid arthritis (RA) patients on adenovirus type 5 (Ad5)–mediated gene transfer to synoviocytes, and to explore new strategies for vector development based on the neutralization data obtained.

Methods

SF was derived from 63 randomly selected RA patients. Ten samples were used to study the effect of SF on Ad5‐mediated gene transfer in synoviocytes. IgG and <100‐kd fractions were purified from these 10 SF, and their effect on gene transfer was determined. Neutralizing activity against wild‐type Ad5 (wt‐Ad5), wt‐Ad26, wt‐Ad34, wt‐Ad35, and wt‐Ad48 was tested in the SF from the remaining 53 patients.

Results

Seven of 10 SF samples inhibited Ad5‐mediated gene transfer. Purified antibodies exhibited inhibition patterns similar to those seen with unfractionated SF. In 5 of 10 SF samples, low molecular weight fractions inhibited gene transfer at low dilutions. Neutralization of wt‐Ad35 by SF from RA patients was less frequent than neutralization of other wt‐Ad tested (4% versus 42–72%; n = 53).

Conclusion

SF from 70% of the RA patients contained neutralizing antibodies that hamper Ad5‐mediated gene transfer to synoviocytes. The activity of neutralizing antibodies may be circumvented in the majority of RA patients when vectors based on an Ad35 backbone are used.

     

Fractalkine is a novel chemoattractant for rheumatoid arthritis fibroblast‐like synoviocyte signaling through MAP kinases and Akt

Objective

Fibroblast‐like synoviocytes (FLS) are a major constituent of the hyperplastic synovial pannus that aggressively invades cartilage and bone during the course of rheumatoid arthritis (RA). Fractalkine (FKN/CX₃CL1) expression is up‐regulated in RA synovium and RA synovial fluid. While RA FLS express the FKN receptor, CX₃CR1, the pathophysiologic relevance of FKN stimulation of RA FLS is not understood. This study was undertaken to better characterize the relationship between FKN and the RA FLS that both produce it and express its receptor.

Methods

RA FLS were subjected to chemotaxis and proliferation assays, Western blotting, enzyme‐linked immunosorbent assays, and filamentous actin staining to characterize the relationship between FKN and RA FLS.

Results

FKN secretion by RA FLS was regulated mainly by tumor necrosis factor α. Stimulation of RA FLS with FKN led to significant cytoskeletal rearrangement but no proliferation. Chemotaxis assays revealed that FKN was a novel chemoattractant for RA FLS. Stimulation of RA FLS with FKN resulted in activation of MAP kinases and Akt. JNK, ERK‐1/2, and Akt (at both Ser‐473 and Thr‐308) were each up‐regulated in a time‐dependent manner. Inhibition of ERK‐1/2–mediated signaling, but not JNK or Akt, significantly repressed FKN‐induced RA FLS migration.

Conclusion

These findings indicate a novel role of FKN in regulating RA FLS cytoskeletal structure and migration. FKN specifically induces RA FLS phosphorylation of the MAP kinases JNK and ERK‐1/2, as well as full activation of Akt.

     

Cilostazol enhances apoptosis of synovial cells from rheumatoid arthritis patients with inhibition of cytokine formation via Nrf2‐linked heme oxygenase 1 induction

Objective

To assess the effects of cilostazol in inhibiting proliferation and enhancing apoptosis in synovial cells from patients with rheumatoid arthritis (RA).

Methods

Synovial cell proliferation was measured by MTT assay. The expression of NF‐κB, IκBα, Bcl‐2, Bax, heme oxygenase 1 (HO‐1), and Nrf2 was determined by Western blotting.

Results

Cilostazol suppressed synovial cell proliferation by arresting the G₂/M phases of the cell cycle, and this was reversed by KT5720, an inhibitor of protein kinase A. Cilostazol increased the number of TUNEL‐positive cells, with increased cytochrome c release and apoptosis‐inducing factor translocation as well as increased caspase 3 activation. Cilostazol (10 μM) and cobalt protoporphyrin IX (CoPP) increased HO‐1 messenger RNA and protein expression. These effects were suppressed by zinc protoporphyrin IX (ZnPP), an HO‐1 inhibitor. Cilostazol and CoPP significantly increased IκBα in the cytosol and decreased NF‐κB p65 expression in the nucleus. Increased expression of tumor necrosis factor α (TNFα), interleukin‐1β (IL‐1β), and IL‐6 induced by lipopolysaccharide was attenuated by cilostazol and CoPP, and this was reversed by ZnPP. In mice with collagen‐induced arthritis treated with cilostazol (10 and 30 mg/kg/day), paw thickness was decreased with increased apoptotic cells in the joints. In synovial cells transfected with small interfering RNA (siRNA) targeting HO‐1, cilostazol did not suppress expression of TNFα, IL‐1β, and IL‐6, in contrast to findings with negative control cells. Cilostazol‐ and CoPP‐induced HO‐1 expression was diminished in cells transfected with Nrf2 siRNA.

Conclusion

Cilostazol suppressed proliferation of synovial cells from RA patients by enhancing apoptosis, and also inhibited cytokine production via mediation of cAMP‐dependent protein kinase activation–coupled Nrf2‐linked HO‐1 expression.

     

Gadd45β deficiency in rheumatoid arthritis: Enhanced synovitis through JNK signaling

Objective

JNK‐mediated cell signaling plays a critical role in matrix metalloproteinase (MMP) expression and joint destruction in rheumatoid arthritis (RA). Gadd45β, which is an NF‐κB–regulated gene, was recently identified as an endogenous negative regulator of the JNK pathway, since it could block the upstream kinase MKK‐7. This study was carried out to evaluate whether low Gadd45β expression in RA enhances JNK activation and overproduction of MMPs in RA, and whether Gadd45β deficiency increases arthritis severity in passive K/BxN murine arthritis.

Methods

Activation of the NF‐κB and JNK pathways and Gadd45β expression were analyzed in human synovium and fibroblast‐like synoviocytes (FLS) using quantitative polymerase chain reaction, immunoblotting, immunohistochemistry, electrophoretic mobility shift assay, and luciferase reporter constructs. Gadd45β^−/− and wild‐type mice were evaluated in the K/BxN serum transfer model of inflammatory arthritis, and clinical signs of arthritis, osteoclast formation, and bone erosion were assessed.

Results

Expression levels of the Gadd45β gene and protein were unexpectedly low in human RA synovium despite abundant NF‐κB activity. Forced Gadd45β expression in human FLS attenuated tumor necrosis factor–induced signaling through the JNK pathway, reduced the activation of activator protein 1, and decreased the expression of MMP genes. Furthermore, Gadd45β deficiency exacerbated K/BxN serum–induced arthritis in mice, dramatically increased signaling through the JNK pathway, elevated MMP3 and MMP13 gene expression in the mouse joints, and increased the synovial inflammation and number of osteoclasts.

Conclusion

Deficient Gadd45β expression in RA can contribute to activation of JNK, exacerbate clinical arthritis, and augment joint destruction. This process can be mitigated by enhancing Gadd45β expression or by inhibiting the activity of JNK or its upstream regulator, MKK‐7.

     

Role of placenta growth factor and its receptor flt‐1 in rheumatoid inflammation: A link between angiogenesis and inflammation

Objective

To investigate the direct effects of placenta growth factor (PlGF) and its specific receptor, flt‐1, which are known to mediate angiogenesis, on the inflammatory process of rheumatoid arthritis (RA).

Methods

Expression of PlGF and flt‐1 in the synovial tissue of RA patients was examined using immunohistochemistry. Enzyme‐linked immunosorbent assay was used to determine the concentrations of PlGF, tumor necrosis factor α (TNFα), and interleukin‐6 (IL‐6) in culture supernatants of either mononuclear cells or synoviocytes. The flt‐1 expression level in mononuclear cells was analyzed by flow cytometry. Experimental arthritis was induced in mice either by immunization with type II collagen (CII) or by injection of anti‐CII antibody.

Results

PlGF was highly expressed in the synovium of RA patients, and its primary source was fibroblast‐like synoviocytes (FLS). When stimulated with IL‐1β, FLS from RA patients produced higher amounts of PlGF than did FLS from patients with osteoarthritis. Exogenous PlGF specifically increased the production of TNFα and IL‐6 in mononuclear cells from RA patients (but not those from healthy controls) via a calcineurin‐dependent pathway. The response to PlGF was associated with increased expression of flt‐1 on RA monocytes, which could be induced by IL‐1β and TNFα. A novel anti–flt‐1 hexapeptide, GNQWFI, abrogated the PlGF‐induced increase in TNFα and IL‐6 production, and also suppressed CII‐induced arthritis and serum IL‐6 concentrations in mice. Moreover, genetic ablation of PlGF prevented the development of anti‐CII antibody–induced arthritis in mice.

Conclusion

Our data suggest that enhanced expression of PlGF and flt‐1 may contribute to rheumatoid inflammation by triggering production of proinflammatory cytokines. The use of the novel anti–flt‐1 peptide, GNQWFI, may be an effective strategy for the treatment of RA.