Rheumatoid Synovial Cells Research Articles

Potent anti‐inflammatory effects of the narrow spectrum kinase inhibitor RV1088 on rheumatoid arthritis synovial membrane cells

Background and PurposeTo investigate whether a narrow spectrum kinase inhibitor RV1088, which simultaneously targets specific MAPKs, Src and spleen tyrosine kinase (Syk), is more effective at inhibiting inflammatory signalling in rheumatoid arthritis (RA) than single kinase inhibitors (SKIs).Experimental Approachelisas were used to determine the efficacy of RV1088, clinically relevant SKIs and the pharmaceutical Humira on pro-inflammatory cytokine production by activated RA synovial fibroblasts, primary human monocytes and macrophages, as well as spontaneous cytokine synthesis by synovial membrane cells from RA patients. In human macrophages, RNAi knockdown of individual kinases was used to reveal the effect of inhibition of kinase expression on cytokine synthesis.Key ResultsRV1088 reduced TNF-α, IL-6 and IL-8 production in all individual activated cell types with low, nM, IC50s. SKIs, and combinations of SKIs, were significantly less effective than RV1088. RNAi of specific kinases in macrophages also caused only modest inhibition of pro-inflammatory cytokine production. RV1088 was also significantly more effective at inhibiting IL-6 and IL-8 production by monocytes and RA synovial fibroblasts compared with Humira. Finally, RV1088 was the only inhibitor that was effective in reducing TNF-α, IL-6 and IL-8 synthesis in RA synovial membrane cells with low nM IC50s.Conclusions and ImplicationsThis study demonstrates potent anti-inflammatory effect of RV1088, highlighting that distinct signalling pathways drive TNF-α, IL-6 and IL-8 production in the different cell types found in RA joints. As such, targeting numerous signalling pathways simultaneously using RV1088 could offer a more powerful method of reducing inflammation in RA than targeting individual kinases.

Upregulation of Thrombospondin 1 Expression in Synovial Tissues and Plasma of Rheumatoid Arthritis: Role of Transforming Growth Factor-β1 toward Fibroblast-like Synovial Cells

To investigate the role of thrombospondin 1 (TSP-1) in RA. Expression of TSP-1 in synovial tissues was determined by immunohistochemistry. Expression of TSP-1 in rheumatoid fibroblast-like synovial cells (FLS) was investigated by quantitative real-time PCR and ELISA. Correlations among the plasma TSP-1 and other variables in patients with RA were examined. Expression of TSP-1 was increased in rheumatoid synovial tissues. Transforming growth factor-β1 (TGF-β1) clearly increased TSP-1 expression in FLS on both mRNA and protein levels. Changes in plasma TSP-1 were associated with those in 28-joint Disease Activity Score-erythrocyte sedimentation rate and plasma TGF-β1. TSP-1 might be critically involved in the disease process of RA through the TGF-β1/TSP-1 axis.

Quantitative analysis of cadherin-11 and β-catenin signalling during proliferation of rheumatoid arthritis-derived synovial fibroblast cells.

Cadherin-11 (CDH11) is an adhesion molecule that anchors β-catenin and is involved with various functions of synovial fibroblast cells (SFCs) during the development of rheumatoid arthritis (RA). However, the mechanism of CDH11 during RA-SFC proliferation is unclear. The aim of our study was to clarify the involvement of CDH11 and β-catenin signalling during proliferation. IL-1β-induced and tumour necrosis factor-α (TNF-α)-induced cell proliferation, with CDH11 siRNAs, β-catenin-specific siRNAs and a CDH11-neutralizing antibody, were assessed by 5-Bromo-2'-deoxy-uridine ELISA. Using CDH11 siRNAs, there were a 42% reduction in IL-1β-induced proliferation and a 64% reduction in β-catenin protein. When β-catenin siRNAs were applied, there was a 63% reduction in IL-1β-induced proliferation. The median effective concentration (EC50 ) values for IL-1β-induced proliferation via CDH11-mediated β-catenin-dependent, total β-catenin-dependent and β-catenin-independent signalling were 0.0015, 0.016 and 0.18 ng/ml, respectively. Blocking CDH11 ligation with a CDH11-neutralizing antibody did not decrease IL-1β-induced proliferation. CDH11-mediated β-catenin signalling was 42% involved in IL-1β-induced proliferation and had the highest susceptibility to IL-1β among the proliferative signallings analysed in this study. The mode of action for CDH11 during the cell proliferation was likely associated with a pool of β-catenin protein. In contrast, CDH11 and β-catenin were not involved in TNF-α-induced RA-SFC proliferation.

A1.4 Inflammatory microenvironment of rheumatoid and osteoarthritic joint affects immunomodulatory activity of adipose-derived mesenchymal stem cells

Background and objectives Adipose-derived mesenchymal stem cells (ASCs) have immunomodulatory properties, but their activity is dependent on signals provided by local microenvironment. ASCs administration in rheumatoid arthritis (RA) did not bring consistent results, while in osteoarthritis (OA) it has beneficial effects. It is likely that proinflammatory milieu of rheumatoid joint affects ASCs activity. To test this hypothesis, the function of rheumatoid ASCs (RA-ASCs) and osteoarthritic ASCs (OA-ASCs) isolated from infrapatellar fat pad of the knee joint have been analysed. Materials and methods Infrapatellar fat pads were obtained from 29 RA and 12 OA patients undergoing total knee joint replacement surgery. Then, ASCs were isolated accordingly to routinely applied procedure. ASCs were treated or not with IFNγ or TNF. The expression of indoleamine 2,3-dioxygenase and heme oxygenase 1 mRNA was measured using quantitative PCR, while ASCs secretory potential was assessed by specific ELISAs. Rheumatoid synovial fibroblasts (RA-FLS) or peripheral blood mononuclear cells (PBMCs) from healthy donors were cultured in ASC-conditioned media or co-cultured with ASCs. Next, cytokines secretion and cells proliferation were measured using ELISAs and bromodeoxyuridine/ 3 H-thymidine proliferation assays, respectively. Results RA- and OA-ASCs activity in vitro is comparable, nonetheless some differences in spontaneous secretory activity and heme oxygenase 1 mRNA expression have been observed. Unstimulated RA- and OA-ASCs slightly inhibited PBMCs proliferation and induced IL-10 secretion but increased IL-17A production and failed to suppress release of other proinflammatory factors (TNF, IFNγ, RANTES) by PBMCs. Under TNF stimulation, RA- and OA-ASCs up-regulated IL-6 and MMP-3 secretion in RA-FLS cultures and IL-17A release by PBMCs. ASCs treatment with IFNγ or TNF did not intensify their anti-inflammatory activity. Conclusions Our study demonstrates that immunosuppressive function of RA- and OA-ASCs derived from infrapatellar fat pad is impaired. Possibly, it is due to the localisation of ASCs in the site of inflammation. Moreover, TNF triggers proinflammatory properties of ASCs which indicates that when administered to the rheumatoid joint, ASCs may shift from an anti-inflammatory to proinflammatory phenotype. Acknowledgements This work was supported by the Polish National Science Centre (grant no. 2011/01/N/NZ5/00932) and by the European Union funds (European Social Fund, Human Capital Programme).

Pim-2/mTORC1 Pathway Shapes Inflammatory Capacity in Rheumatoid Arthritis Synovial Cells Exposed to Lipid Peroxidations.

Rheumatoid arthritis is a systemic autoimmune disease characterized by chronic inflammation of multiple joints, with disruption of joint cartilage. The proliferation of synovial fibroblasts in response to multiple inflammation factors is central to the pathogenesis of rheumatoid arthritis. Our previous studies showed that 4-HNE may induce synovial intrinsic inflammations by activating NF-κB pathways and lead to cell apoptosis. However, the molecular mechanisms of how synovial NF-κB activation is modulated are not fully understood. Here, the present findings demonstrated that 4-HNE may induce synovial intrinsic inflammations by mTORC1 inactivation. While ectopic activation of mTORC1 pathway by the overexpression of Pim-2 may disrupt the initiation of inflammatory reactions and maintain synovial homeostasis, our findings will help to uncover novel signaling pathways between inflammations and oxidative stress in rheumatoid arthritis development and imply that Pim-2/mTORC1 pathway may be critical for the initiation of inflammatory reactions in human rheumatoid arthritis synovial cells.

Lipid peroxidation-mediated inflammation promotes cell apoptosis through activation of NF-κB pathway in rheumatoid arthritis synovial cells.

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic inflammation of multiple joints. The central pathogenesis of RA is the proliferation of synovial fibroblasts in response to inflammatory cytokines. However, some of the targeted therapies for inflammation reactions do not display significant clinical improvement after initiation of therapy. Thus, the relationship between inflammatory responses and RA therapy is still incompletely understood. In the present study, we proposed to determine whether enhanced inflammations may lead to cell apoptosis in rheumatoid arthritis synoviocytes. Our results indicated that products of lipid peroxidations, 4-HNE, may induce synovial intrinsic inflammations by activating NF-κB pathways and it may lead to cell apoptosis. Pharmacological inhibition of NF-κB activation may reduce the 4-HNE mediated inflammation responses and subsequent cell apoptosis. Our results may help to clarify the role of inflammations on RA development and imply that blocking NF-κB activation may be partly beneficial for human RA therapy. These findings might provide a mechanism-based rationale for developing new strategy to RA clinical therapy.

Anti-inflammatory effects of a novel ricinoleic acid poloxamer gel system for transdermal delivery

Our previous study showed that the use of ricinoleic acid as an oil phase resulted in the formation of a stable pluronic lecithin organogel (PLO gel) with better thixotropic properties and higher permeation of ketoprofen than the isopropyl palmitate PLO gel. This study aims to evaluate and compare the in vitro and in vivo anti-inflammatory effects of the ricinoleic acid PLO gel system with isopropyl palmitate PLO gel. Ketoprofen was used as a model drug. In vitro anti-inflammatory activity and cell viability tests were performed in human rheumatoid arthritis synovial fibroblast cell line using a blank ricinoleic acid PLO gel and compared to that of the isopropyl palmitate PLO gel. In vivo anti-inflammatory activity of ricinoleic acid PLO gel containing 10% ketoprofen was evaluated and compared with the isopropyl palmitate PLO gel in a carrageenan-induced rat paw edema model. The results from the in vitro study showed that the blank ricinoleic acid PLO gel possessed significantly higher anti-inflammatory activity than isopropyl palmitate PLO gel at 1mM concentration (p<0.05), while both the gel formulations had no significant cytotoxic activity. Further in vivo testing of the formulation showed that the ricinoleic acid PLO gel formulation was significantly more effective in reducing pain and edema when compared to the isopropyl palmitate PLO gel. In addition, the ricinoleic acid PLO gel formulation markedly inhibited the synthesis of prostaglandin E2. In conclusion, the efficacy of PLO gels used in pain management may be enhanced by using ricinoleic acid instead of isopropyl palmitate as an oil phase.

Hempseed oil induces reactive oxygen species- and C/EBP homologous protein-mediated apoptosis in MH7A human rheumatoid arthritis fibroblast-like synovial cells

Ethnopharmacological relevanceThe medicinal efficacy of hempseed (Cannabis sativa L.), which is rich in polyunsaturated fatty acids, in atopic dermatitis, inflammation, and rheumatoid arthritis (RA) has been suggested for centuries. Hempseed has been used as a treatment for these diseases in Korean and Chinese folk medicine. The aim of the study is to investigate the effects of hempseed oil (HO) on MH7A human RA fibroblast-like synovial cells. Materials and methodsMH7A cells were used to study the anti-rheumatoid effects of hempseed (Cannabis sativa L., cv. Cheungsam/Cannabaceae) oil by investigating cell viability, apoptosis, lipid accumulation, oxidative stress, and endoplasmic reticulum (ER) stress-induced apoptosis. ResultsHO treatment reduced the survival rate of MH7A cells and promoted apoptotic cell death in a time- and dose-dependent manner. Both lipid accumulation and the level of intracellular reactive oxygen species (ROS) increased in HO-treated MH7A cells. Co-treatment with the antioxidant Tiron effectively abrogated the cytotoxic effects of HO; the ROS level was reduced, cell viability was recovered, and apoptotic cell death was significantly diminished. Moreover, HO-treated cells exhibited increased expression of the major ER stress markers, glucose-regulated protein 78 and C/EBP homologous protein (CHOP). The siRNA-mediated knockdown of CHOP prevented HO-induced apoptosis. ConclusionsOur results suggest that HO treatment induced lipid accumulation, ROS production, CHOP expression, and apoptosis in MH7A cells, and that CHOP functions as an anti-rheumatoid factor downstream of HO in MH7A cells.

Effects of Thapsigargin on the Proliferation and Survival of Human Rheumatoid Arthritis Synovial Cells

A series of experiments have been carried out to investigate the effects of different concentrations of thapsigargin (0, 0.001, 0.1, and 1 μM) on the proliferation and survival of human rheumatoid arthritis synovial cells (MH7A). The results showed that thapsigargin can block the cell proliferation in human rheumatoid arthritis synovial cells in a time- and dose-dependent manner. Results of Hoechst staining suggested that thapsigargin may induce cell apoptosis in MH7A cells in a time- and dose-dependent manner, and the percentages of cell death reached 44.6% at thapsigargin concentration of 1 μM treated for 4 days compared to the control. The protein and mRNA levels of cyclin D1 decreased gradually with the increasing of thapsigargin concentration and treatment times. Moreover, the protein levels of mTORC1 downstream indicators pS6K and p4EBP-1 were reduced by thapsigargin treatment at different concentrations and times, which should be responsible for the reduced cyclin D1 expressions. Our results revealed that thapsigargin may effectively impair the cell proliferation and survival of MH7A cells. The present findings will help to understand the molecular mechanism of fibroblast-like synoviocytes proliferations and suggest that thapsigargin is of potential for the clinical treatment of rheumatoid arthritis.

A preliminary study on the characterization of follicular helper T (Tfh) cells in rheumatoid arthritis synovium

Rheumatoid arthritis (RA) is a chronic and systematic autoimmune inflammatory disease. Recently, a novel T cell subset, follicular helper CD4 T cell (Tfh cells) was found in relation to the pathogenesis and progression of RA, and increased numbers of circulating Tfh cells were found in RA patients. However, there is little evidence regarding the localization of Tfh cells in synovium tissues from RA patients, owing to the lack of an available method to characterize their localization in tissue. The aim of our present study was to characterize the Tfh cells in rheumatoid synovium tissues from RA patients by using immunohistochemistry and triple-fluorescence immunostaining methods. Our results showed that specific staining of CD4, CXCR5 and ICOS could be found on infiltrating immune cells in rheumatoid synovium tissues. The use of triple-fluorescence immunostaining and confocal laser scanning showed immunolocalization of CD4+CXCR5+ICOS+T cells (Tfh cells) in the rheumatoid synovium tissues, whereas these signals were absent in osteoarthritis (OA) synovium and in normal synovium tissues. Thus the data from our present preliminary study support the notion that CD4+CXCR5+ICOS+Tfh cells could be found in rheumatoid synovium tissues from RA patients, indicating the possibility that this T cell subset in synovium tissues may have important roles in the pathogenesis and progression of RA.

The Histone Deacetylase Inhibitors MS-275 and SAHA Suppress the p38 Mitogen-Activated Protein Kinase Signaling Pathway and Chemotaxis in Rheumatoid Arthritic Synovial Fibroblastic E11 Cells

MS-275 (entinostat) and SAHA (vorinostat), two histone deacetylase (HDAC) inhibitors currently in oncological trials, have displayed potent anti-rheumatic activities in rodent models of rheumatoid arthritis (RA). To further elucidate their anti-inflammatory mechanisms, the impact of MS-275 and SAHA on the p38 mitogen-activated protein kinase (MAPK) signaling pathway and chemotaxis was assessed in human rheumatoid arthritic synovial fibroblastic E11 cells. MS-275 and SAHA significantly suppressed the expression of p38α MAPK, but induced the expression of MAPK phosphatase-1 (MKP-1), an endogenous suppressor of p38α in E11 cells. At the same time, the association between p38α and MKP-1 was up-regulated and consequently, the activation (phosphorylation) of p38α was inhibited. Moreover, MS-275 and SAHA suppressed granulocyte chemotactic protein-2 (GCP-2), monocyte chemotactic protein-2 (MCP-2) and macrophage migration inhibitory factor (MIF) in E11 cells in a concentration-dependent manner. Subsequently, E11-driven migration of THP-1 and U937 monocytes was inhibited. In summary, suppression of the p38 MAPK signaling pathway and chemotaxis appear to be important anti-rheumatic mechanisms of action of these HDAC inhibitors.

Selective Blockade of Tumor Necrosis Factor Receptor I Inhibits Proinflammatory Cytokine and Chemokine Production in Human Rheumatoid Arthritis Synovial Membrane Cell Cultures

To determine whether selective blockade of tumor necrosis factor receptor I (TNFRI) affects spontaneous proinflammatory cytokine and chemokine production in ex vivo-cultured human rheumatoid arthritis synovial membrane mononuclear cells (MNCs) and to compare this response to that of TNF ligand blockade using etanercept. A bispecific, single variable-domain antibody (anti-TNFRI moiety plus an albumin binding moiety [TNFRI-AlbudAb]) was used to selectively block TNFRI. Inhibition of TNFα-mediated responses in cell lines expressing TNFRI/II confirmed TNFRI-AlbudAb potency, human rhabdomyosarcoma cell line KYM-1D4 cytotoxicity, and human umbilical vein endothelial cell (HUVEC) vascular cell adhesion molecule 1 (VCAM-1) upregulation. Eighteen RA synovial membrane MNC suspensions were cultured for 2 days or 5 days, either alone or in the presence of TNFRI-AlbudAb, control-AlbudAb, or etanercept. Proinflammatory cytokines and chemokines in culture supernatants were measured by enzyme-linked immunosorbent assays. A mixed-effects statistical analysis model was used to assess the extent of TNFRI selective blockade, where the results were expressed as the percentage change with 95% confidence intervals (95% CIs). TNFRI-AlbudAb inhibited TNFα-induced KYM-1D4 cell cytotoxicity (50% inhibition concentration [IC50 ] 4 nM) and HUVEC VCAM-1 up-regulation (IC50 12 nM) in a dose-dependent manner. In ex vivo-cultured RA synovial membrane MNCs, selective blockade of TNFRI inhibited the production of proinflammatory cytokines and chemokines to levels similar to those obtained with TNF ligand blockade, without inducing cellular toxicity. Changes in cytokine levels were as follows: -23.5% (95% CI -12.4, -33.2 [P = 0.004]) for granulocyte-macrophage colony-stimulating factor, -33.4% (95% CI -20.6, -44.2 [P ≤ 0.0001]) for interleukin-10 (IL-10), -17.6% (95% CI 3.2, -34.2 [P = 0.0880]) for IL-1β, and -19.0% (95% CI -3.4, -32.1 [P = 0.0207]) for IL-6. Changes in chemokine levels were as follows: -34.2% (-14.4, -49.4 [P = 0.0030]) for IL-8, -56.6% (-30.7, -72.9 [P = 0.0011]) for RANTES, and -24.9% (2, -44.8 [P = 0.0656]) for monocyte chemotactic protein 1. In ex vivo-cultured RA synovial membrane MNCs, although a limited role of TNFRII cannot be ruled out, TNFRI signaling was found to be the dominant pathway leading to proinflammatory cytokine and chemokine production. Thus, selective blockade of TNFRI could potentially be therapeutically beneficial over TNF ligand blockade by retaining the beneficial TNFRII signaling.

AB0084 Non-canonical nf-kappab signaling induces a pro-angiogenic response in rheumatoid arthritis synovial fibroblasts and endothelial cells

BackgroundIn rheumatoid arthritis (RA) leukocytes migrate to the joints and gain entry into the synovial tissue through increased permeability of local endothelial cells[1]. These immune cells activate synovial fibroblasts (SF)...

SAT0064 The expression of HDAC6 is reduced in rheumatoid arthritis synovial fibroblasts

BackgroundSince rheumatoid synovial cells produce large amounts of various cytokines and proteases, up to 30% of all newly synthesized proteins are unfolded, leading to a cellular stress condition. Histone-deacetylase (HDAC)...

TNF-α modulates expression of the circadian clock gene Per2 in rheumatoid synovial cells

Objectives: To study the effect of tumour necrosis factor (TNF)-α, responsible for the inflammation and circadian rhythm of rheumatoid arthritis (RA), on the expression of circadian clock genes in primary cultured human rheumatoid synovial cells.Method: The expression of circadian clock genes, including circadian locomotor output cycles kaput (Clock), brain and muscle Arnt-like protein-1 (Bmal1), period (Per)1/2, and cryptochrome (Cry)1/2, and the proline and acidic amino acid-rich basic leucine zipper (PAR bZip) genes, a transcriptional activator of Per2, including D site of albumin promoter binding protein (Dbp), hepatic leukaemia factor (Hlf), and thyrotroph embryonic factor (Tef), and a transcriptional repressor of Per2, E4-binding protein 4 (E4bp4), in TNF-α-stimulated synovial cells was determined by real-time polymerase chain reaction (PCR). The D-box motifs in the Per2 promoter were mutated by site-directed mutagenesis, and the promoter activity of the Per2 gene was examined using the luciferase assay.Results: TNF-α enhanced the mRNA expression of Bmal1 and Cry1 but did not affect that of Clock, Per1, or Cry2. However, TNF-α inhibited the mRNA expression of the Per2 gene, as well as Dbp, Hlf, and Tef, but enhanced the mRNA expression of E4bp4. Furthermore, TNF-α inhibited the transcriptional activity of the wild-type Per2 gene in a manner dependent on the D-box 1 and D-box 2 motifs in the Per2 promoter.Conclusions: TNF-α modulates the expression of the Per2 gene through the D-box binding proteins DBP, HLF, TEF, and E4BP4, in rheumatoid synovial cells, and thereby may contribute to the pathogenesis of RA.

Celastrol Induced DNA Damage, Cell Cycle Arrest, and Apoptosis in Human Rheumatoid Fibroblast-Like Synovial Cells

Celastrol is one of the principal active ingredients of Tripterygium wilfordii Hook.f., a toxic Chinese medical herb traditionally prescribed for controlling pain and inhibiting inflammation in various chronic inflammatory diseases, including rheumatoid arthritis (RA). Resistance to apoptosis of fibroblast-like synoviocytes is considered a major characteristic of RA. In this study, we test celastrol's cytotoxic effect and potential mechanisms in human rheumatoid synovial fibroblasts (RA-FLS). In the cytotoxic assay, we found that celastrol dose-dependently decreased RA-FLS viability and increased LDH release. The apoptotic nuclear morphology was observed after celastrol treatment as determined by DAPI fluorescence staining. Flow cytometry analysis with PI and Annexin V revealed that celastrol induced RA-FLS cell cycle arrest in the G2/M phase and apoptosis. Furthermore, celastrol dramatically increased expression of Bax/Bcl-2, proteolytic cleavage of Caspase-3, -9, PARP, and decreased expression of FasR. In addition, celastrol treatment resulted in DNA damage. Collectively, we concluded that celastrol inhibits RA-FLS proliferation by inducing DNA damage, cell cycle arrest, and apoptosis in vitro, which might provide data for its application in RA treatment.

RING-finger type E3 ubiquitin ligase inhibitors as novel candidates for the treatment of rheumatoid arthritis

Rheumatoid arthritis (RA) significantly affects quality of life. We recently cloned synoviolin, a RING-type E3 ubiquitin ligase implicated in the endoplasmic reticulum-associated degradation (ERAD) pathway. Synoviolin is highly expressed in rheumatoid synovial cells and may be involved in the pathogenesis of RA. Inhibition of synoviolin activity is a potentially useful therapeutic approach for the treatment of RA. We conducted a high-throughput screen of small molecules to find inhibitors of synoviolin autoubiquitination activity. We identified two classes of small molecules, named LS-101 and LS-102, which inhibited synoviolin activity. LS-102 selectively inhibited synoviolin enzymatic activity, while LS-101 inhibited a broad array of RING-type E3 ligases. Moreover, these inhibitors suppressed the proliferation of rheumatoid synovial cells, and significantly reduced the severity of disease in a mouse model of RA. Our results suggest that inhibition of synoviolin is a potentially useful approach in the treatment of RA.

Epirubicin potentiates recombinant adeno‐associated virus type 2/5–mediated TRAIL expression in fibroblast‐like synoviocytes and augments the antiarthritic effects of rAAV2/5‐TRAIL

Synovial cells in rheumatoid synovium display abnormal proliferation, which leads to joint destruction. TRAIL has been described as a proapoptotic factor in fibroblast-like synoviocytes (FLS). This study was undertaken to investigate the functions of rAAV2/5-TRAIL in human FLS and in arthritic mice. Primary human FLS were infected with rAAV2/5-TRAIL in the presence or absence of epirubicin. Transgene expression was monitored by both enzyme-linked immunosorbent assay and flow cytometry. The disease-modulating activity of epirubicin plus rAAV2/5-TRAIL was investigated in mice with collagen-induced arthritis (CIA). Subtoxic doses of epirubicin potentiated rAAV2/5-mediated TRAIL expression in FLS and simultaneously enhanced the sensitivity of FLS to TRAIL. Epirubicin treatment up-regulated death receptor 4 (DR-4) and DR-5 expression and down-regulated FLIP expression, thereby enhancing the activation of procaspase 3, procaspase 8, and procaspase 9. An in vivo study showed that the combination of rAAV2/5-TRAIL gene therapy and epirubicin chemotherapy provided augmented antiarthritic effects in a mouse model of CIA. The intraarticular injection of rAAV2/5-TRAIL combined with epirubicin treatment significantly reduced the severity and incidence of CIA and joint swelling in the animals. Histologic evaluations revealed that inflammatory cell infiltration, cartilage destruction, and bone erosion were significantly reduced in the joints of the mice receiving the synthetic treatment. Results of a viral genome copy number assay indicated that epirubicin dramatically augmented the expression of rAAV2/5-TRAIL without altering its tissue distribution. These results suggest that epirubicin enhances the antiarthritic effect of rAAV2/5-TRAIL and that combination treatment might be an important therapeutic alternative, with practical significance for rheumatoid arthritis.

A novel pathogenic role of the ER chaperone GRP78/BiP in rheumatoid arthritis

An accumulation of misfolded proteins can trigger a cellular survival response in the endoplasmic reticulum (ER). In this study, we found that ER stress-associated gene signatures were highly expressed in rheumatoid arthritis (RA) synoviums and synovial cells. Proinflammatory cytokines, such as TNF and IL-1β, increased the expression of GRP78/BiP, a representative ER chaperone, in RA synoviocytes. RA synoviocytes expressed higher levels of GRP78 than osteoarthritis (OA) synoviocytes when stimulated by thapsigargin or proinflammatory cytokines. Down-regulation of Grp78 transcripts increased the apoptosis of RA synoviocytes while abolishing TNF- or TGF-β-induced synoviocyte proliferation and cyclin D1 up-regulation. Conversely, overexpression of the Grp78 gene prevented synoviocyte apoptosis. Moreover, Grp78 small interfering RNA inhibited VEGF(165)-induced angiogenesis in vitro and also significantly impeded synoviocyte proliferation and angiogenesis in Matrigel implants engrafted into immunodeficient mice. Additionally, repeated intraarticular injections of BiP-inducible factor X, a selective GRP78 inducer, increased synoviocyte proliferation and angiogenesis in the joints of mice with experimental OA. In contrast, mice with Grp78 haploinsufficiency exhibited the suppression of experimentally induced arthritis and developed a limited degree of synovial proliferation and angiogenesis. In summary, this study shows that the ER chaperone GRP78 is crucial for synoviocyte proliferation and angiogenesis, the pathological hallmark of RA.

GCIP, Id like HLH protein, negatively regulates cell proliferation of rheumatoid synovial cells via interaction with CBP

GCIP, Id like HLH protein, negatively regulates cell proliferation of rheumatoid synovial cells via interaction with CBP