Seung-Ah Yoo

Serum Amyloid A Binding to Formyl Peptide Receptor-Like 1 Induces Synovial Hyperplasia and Angiogenesis

Serum amyloid A (SAA) is a major acute-phase reactant, and has been demonstrated to mediate proinflammatory cellular responses. Although SAA has been used as an indicator for a variety of inflammatory diseases, the role of SAA in synovial hyperplasia and proliferation of endothelial cells, a pathological hallmark of rheumatoid arthritis (RA), has yet to be elucidated. In this study, we have demonstrated that SAA promotes the proliferation of human fibroblast-like synoviocytes (FLS). In addition, SAA protects RA FLS against the apoptotic death induced by serum starvation, anti-Fas IgM, and sodium nitroprusside. The activity of SAA appears to be mediated by the formyl peptide receptor-like 1 (FPRL1) receptor, as it was mimicked by the WKYMVm peptide, a specific ligand for FPRL1, but completely abrogated by down-regulating the FPRL1 transcripts with short interfering RNA. The effect of SAA on FLS hyperplasia was shown to be caused by an increase in the levels of intracellular calcium, as well as the activation of ERK and Akt, which resulted in an elevation in the expression of cyclin D1 and Bcl-2. Moreover, SAA stimulated the proliferation, migration, and tube formation of endothelial cells in vitro, and enhanced the sprouting activity of endothelial cells ex vivo and neovascularization in vivo. These observations indicate that the binding of SAA to FPRL1 may contribute to the destruction of bone and cartilage via the promotion of synoviocyte hyperplasia and angiogenesis, thus providing a potential target for the control of RA.

Arginine-Rich Anti-Vascular Endothelial Growth Factor (Anti-VEGF) Hexapeptide Inhibits Collagen-Induced Arthritis and VEGF-Stimulated Productions of TNF-α and IL-6 by Human Monocytes

Vascular endothelial growth factor (VEGF) has been suggested to play a critical role in the pathogenesis of rheumatoid arthritis (RA). We previously identified a novel RRKRRR hexapeptide that blocked the interaction between VEGF and its receptor through the screening of peptide libraries. In this study, we investigated whether anti-VEGF peptide RRKRRR (dRK6) could suppress collagen-induced arthritis (CIA) and regulate the activation of mononuclear cells of RA patients. A s.c. injection of dRK6 resulted in a dose-dependent decrease in the severity and incidence of CIA and suppressed synovial infiltration of inflammatory cells in DBA/1 mice. In these mice, the T cell responses to type II collagen (CII) in lymph node cells and circulating IgG Abs to CII were also dose-dependently inhibited by the peptides. In addition, VEGF directly increased the production of TNF-α and IL-6 from human PBMC. Synovial fluid mononuclear cells of RA patients showed a greater response to VEGF stimulation than the PBMC of healthy controls. The major cell types responding to VEGF were monocytes. Moreover, anti-VEGF dRK6 inhibited the VEGF-induced production of TNF-α and IL-6 from synovial fluid mononuclear cells of RA patients and decreased serum IL-6 levels in CIA mice. In summary, we observed first that dRK6 suppressed the ongoing paw inflammation in mice and blocked the VEGF-induced production of proinflammatory cytokines. These data suggest that dRK6 may be an effective strategy in the treatment of RA, and could be applied to modulate various chronic VEGF-dependent inflammatory diseases.

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 alpha (TNFalpha), 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-1beta, FLS from RA patients produced higher amounts of PlGF than did FLS from patients with osteoarthritis. Exogenous PlGF specifically increased the production of TNFalpha 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-1beta and TNFalpha. A novel anti-flt-1 hexapeptide, GNQWFI, abrogated the PlGF-induced increase in TNFalpha 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.

Proinflammatory Role of Vascular Endothelial Growth Factor in the Pathogenesis of Rheumatoid Arthritis: Prospects for Therapeutic Intervention

Recent experimental and clinical studies have placed new emphasis on the role of angiogenesis in chronic inflammatory disease. Vascular endothelial growth factor (VEGF) and its receptors are the best characterized system in the regulation of rheumatoid arthritis (RA) by angiogenesis. Furthermore, in addition to its angiogenic role, VEGF can act as a direct proinflammatory mediator during the pathogenesis of RA, and protect rheumatoid synoviocytes from apoptosis, which contributes to synovial hyperplasia. Therefore, the developments of synovial inflammation, hyperplasia, and angiogenesis in the joints of RA patients seem to be regulated by a common cue, namely, VEGF. Agents that target VEGF, such as anti-VEGF antibody and aptamer, have yielded promising clinical data in patients with cancer or macular degeneration, and in RA patients, pharmacologic modulations targeting VEGF or its receptor may offer new therapeutic approaches. In this review, the authors integrate current knowledge of VEGF signaling and information on VEGF antagonists gleaned experimentally and place emphasis on the use of synthetic anti-VEGF hexapeptide to prevent VEGF interacting with its receptor.

Hypoxia induces expression of connective tissue growth factor in scleroderma skin fibroblasts

Connective tissue growth factor (CTGF) plays a role in the fibrotic process of systemic sclerosis (SSc). Because hypoxia is associated with fibrosis in several profibrogenic conditions, we investigated whether CTGF expression in SSc fibroblasts is regulated by hypoxia. Dermal fibroblasts from patients with SSc and healthy controls were cultured in the presence of hypoxia or cobalt chloride (CoCl2), a chemical inducer of hypoxia‐inducible factor (HIF)‐1α. Expression of CTGF was evaluated by Northern and Western blot analyses. Dermal fibroblasts exposed to hypoxia (1% O2) or CoCl2 (1–100 µM) enhanced expression of CTGF mRNA. Skin fibroblasts transfected with HIF‐1α showed the increased levels of CTGF protein and mRNA, as well as nuclear staining of HIF‐1α, which was enhanced further by treatment of CoCl2. Simultaneous treatment of CoCl2 and transforming growth factor (TGF)‐β additively increased CTGF mRNA in dermal fibroblasts. Interferon‐γ inhibited the TGF‐β‐induced CTGF mRNA expression dose‐dependently in dermal fibroblasts, but they failed to hamper the CoCl2‐induced CTGF mRNA expression. In addition, CoCl2 treatment increased nuclear factor (NF)‐κB binding activity for CTGF mRNA, while decreasing IκBα expression in dermal fibroblasts. Our data suggest that hypoxia, caused possibly by microvascular alterations, up‐regulates CTGF expression through the activation of HIF‐1α in dermal fibroblasts of SSc patients, and thereby contributes to the progression of skin fibrosis.

Interaction of Vascular Endothelial Growth Factor 165 with Neuropilin-1 Protects Rheumatoid Synoviocytes from Apoptotic Death by Regulating Bcl-2 Expression and Bax Translocation

Rheumatoid arthritis (RA) synoviocytes are resistant to apoptosis and exhibit a transformed phenotype, which might be caused by chronic exposure to genotoxic stimuli including reactive oxygen species and growth factors. In this study, we investigated the role of vascular endothelial growth factor165 (VEGF165), a potent angiogenic factor, and its receptor in the apoptosis of synoviocytes. We demonstrated here that neuropilin-1, rather than fms-like tyrosine kinase-1 and kinase insert domain-containing receptor, is the major VEGF165 receptor in the fibroblast-like synoviocytes. Neuropilin-1 was highly expressed in the lining layer, infiltrating leukocytes, and endothelial cells of rheumatoid synovium. The production of VEGF165, a ligand for neuropilin, was significantly higher in the RA synoviocytes than in the osteoarthritis synoviocytes. The ligation of recombinant VEGF165 to its receptor prevented the apoptosis of synoviocytes induced by serum starvation or sodium nitroprusside (SNP). VEGF165 rapidly triggered phospho-Akt and phospho-ERK activity and then induced Bcl-2 expression in the rheumatoid synoviocytes. The Akt or ERK inhibitor cancelled the protective effect of VEGF165 on SNP-induced synoviocyte apoptosis. Moreover, VEGF165 blocks SNP-induced Bcl-2 down-regulation as well as SNP-induced Bax translocation from the cytosol to the mitochondria. The down-regulation of the neuropilin-1 transcripts by short interfering RNA caused spontaneous synoviocyte apoptosis, which was associated with both the decrease in Bcl-2 expression and the increase in Bax translocation to mitochondria. Collectively, our data suggest that the interaction of VEGF165 with neuropilin-1 is crucial to the survival of rheumatoid synoviocytes and provide important implications for the abnormal growth of synoviocytes and therapeutic intervention in RA.

Inhibition of synovial hyperplasia, rheumatoid T cell activation, and experimental arthritis in mice by sulforaphane, a naturally occurring isothiocyanate

OBJECTIVE To investigate whether sulforaphane (SFN), an isothiocyanate derived from cruciferous vegetables such as broccoli, regulates synoviocyte hyperplasia and T cell activation in rheumatoid arthritis (RA). METHODS Synoviocyte survival was assessed by MTT assay. The levels of Bcl-2, Bax, p53, and pAkt were determined by Western blot analysis. Cytokine concentrations in culture supernatants from mononuclear cells were analyzed by enzyme-linked immunosorbent assay. The in vivo effects of SFN were examined in mice with experimentally induced arthritis. RESULTS SFN induced synoviocyte apoptosis by modulating the expression of Bcl-2/Bax, p53, and pAkt. In addition, nonapoptotic doses of SFN inhibited T cell proliferation and the production of interleukin-17 (IL-17) and tumor necrosis factor alpha (TNFalpha) by RA CD4+ T cells stimulated with anti-CD3 antibody. Anti-CD3 antibody-induced increases in the expression of retinoic acid-related orphan receptor gammat and T-bet were also repressed by SFN. Moreover, the intraperitoneal administration of SFN to mice suppressed the clinical severity of arthritis induced by injection of type II collagen (CII), the anti-CII antibody levels, and the T cell responses to CII. The production of IL-17, TNFalpha, IL-6, and interferon-gamma by lymph node cells and spleen cells from these mice was markedly reduced by treatment with SFN. Anti-CII antibody-induced arthritis in mice was also alleviated by SFN injection. CONCLUSION SFN was found to inhibit synovial hyperplasia, activated T cell proliferation, and the production of IL-17 and TNFalpha by rheumatoid T cells in vitro. The antiarthritic and immune regulatory effects of SFN, which were confirmed in vivo, suggest that SFN may offer a possible treatment option for RA.

NF‐AT5 is a critical regulator of inflammatory arthritis

OBJECTIVE To investigate the role of NF-AT5, an osmoprotective transcription factor, in synovial hyperplasia and angiogenesis in patients with rheumatoid arthritis (RA). METHODS The expression of NF-AT5 in synovial tissue and synoviocytes from RA patients was examined by immunohistochemistry and Western blot analysis, respectively. Messenger RNA (mRNA) in RA synoviocytes and human umbilical vein endothelial cells (HUVECs) transfected with dummy small interfering RNA (siRNA) or NF-AT5 siRNA were profiled using microarray technology. Assays to determine synoviocyte apoptosis and proliferation were performed in the presence of NF-AT5 siRNA. VEGF₁₆₅-induced angiogenesis was assessed by measuring the proliferation, tube formation, and wound migration of HUVECs. Experimental arthritis was induced in mice by injection of anti-type II collagen antibody. RESULTS NF-AT5 was highly expressed in rheumatoid synovium, and its activity was increased by proinflammatory cytokines, such as interleukin-1β and tumor necrosis factor α. The mRNA profiling of synoviocytes and HUVECs transfected with NF-AT5-targeted siRNA revealed 3 major changes in cellular processes associated with the pathogenesis of RA: cell cycle and survival, angiogenesis, and cell migration. Consistent with these results, NF-AT5 knockdown in RA synoviocytes and HUVECs inhibited their proliferation/survival and impeded angiogenic processes in HUVECs. Mice with NF-AT5 haploinsufficiency (NF-AT5(+/-)) developed a very limited degree of synovial proliferation, as seen on histologic analysis, and decreased angiogenesis, and they exhibited a nearly complete suppression of experimentally induced arthritis. CONCLUSION NF-AT5 regulates synovial proliferation and angiogenesis in chronic arthritis.

Calcineurin Is Expressed and Plays a Critical Role in Inflammatory Arthritis

Calcineurin is a calcium-activated phosphatase to mediate lymphocyte activation and neuron signaling, but its role in inflammatory arthritis remains largely unknown. In this study, we demonstrate that calcineurin was highly expressed in the lining layer, infiltrating leukocytes, and endothelial cells of rheumatoid synovium. The basal expression levels of calcineurin were higher in the cultured synoviocytes of rheumatoid arthritis patients than those of osteoarthritis patients. The calcineurin activity in the synoviocytes was increased by the stimulation with proinflammatory cytokines such as IL-1β and TNF-α. Moreover, rheumatoid arthritis synoviocytes had an enlarged intracellular Ca2+ store and showed a higher degree of [Ca2+]i release for calcineurin activity than osteoarthritis synoviocytes when stimulated with either TNF-α or phorbol myristate acetate. IL-10, an anti-inflammatory cytokine, failed to increase the Ca2+ and calcineurin activity. The targeted inhibition of calcineurin by the overexpression of calcineurin-binding protein 1, a natural calcineurin antagonist, inhibited the production of IL-6 and matrix metalloproteinase-2 by rheumatoid synoviocytes in a similar manner to the calcineurin inhibitor, cyclosporin A. Moreover, the abundant calcineurin expression was found in the invading pannus in the joints of mice with collagen-induced arthritis. In these mice, calcineurin activity in the cultured synovial and lymph node cells correlated well with the severity of arthritis, but which was suppressed by cyclosporin A treatment. Taken together, our data suggest that the abnormal activation of Ca2+ and calcineurin in the synoviocytes may contribute to the pathogenesis of chronic arthritis and thus provide a potential target for controlling inflammatory arthritis.

Hydroxychloroquine potentiates Fas‐mediated apoptosis of rheumatoid synoviocytes

Inadequate apoptosis may contribute to the synovial hyperplasia associated with rheumatoid arthritis (RA). The Fas‐associated death domain protein (FADD)‐like interleukin (IL)‐1β‐converting enzyme (FLICE)‐inhibitory protein (FLIP), which is an apoptotic inhibitor, has been implicated in the resistance to Fas‐mediated apoptosis of synoviocytes. This study investigated whether hydroxychloroquine (HCQ), an anti‐rheumatic drug, induces the apoptosis of rheumatoid synoviocytes, and modulates the expression of FLIP. Fibroblast‐like synoviocytes (FLS) were prepared from the synovial tissues of RA patients, and were cultured with various concentrations of HCQ in the presence or absence of the IgM anti‐Fas monoclonal antibodies (mAb) (CH11). Treatment with HCQ, ranging from 1 to 100 µM, induced the apoptosis of FLS in a dose‐ and time‐dependent manner. The increase in synoviocytes apoptosis by HCQ was associated with caspase‐3 activation. A combined treatment of HCQ and anti‐Fas mAb increased FLS apoptosis and caspase‐3 activity synergistically, compared with either anti‐Fas mAb or HCQ alone. The Fas expression level in the FLS was not increased by the HCQ treatment, while the FLIP mRNA and protein levels were decreased rapidly by the HCQ treatment. Moreover, time kinetics analysis revealed that the decreased expression of FLIP by HCQ preceded the apoptotic event that was triggered by HCQ plus anti‐Fas mAb. Taken together, HCQ increases the apoptosis of rheumatoid synoviocytes by activating caspase‐3, and also sensitizes rheumatoid synoviocytes to Fas‐mediated apoptosis. Our data suggest that HCQ may exert its anti‐rheumatic effect in rheumatoid joints through these mechanisms.