RA FLS Research Articles

Human gingival tissue‐derived mesenchymal stem cells inhibit proliferation and invasion of rheumatoid fibroblast‐like synoviocytes via the CD39/CD73 signaling pathway

AbstractBackgroundGingiva‐derived mesenchymal stem cells (GMSCs) suppress immune and inflammatory responses in experimental arthritis models. Here, we determined whether GMSCs suppress the proliferation and invasion of rheumatoid arthritis fibroblast‐like synoviocytes (RA FLSs).MethodsSurface markers of GMSCs were analyzed by flow cytometry. mRNA expression of matrix metalloproteinases and pro‐inflammatory cytokine in RA FLSs was measured by quantitative polymerase chain reaction (PCR). RA FLS proliferation was analyzed by a 5‐ethynyl‐2′‐deoxyuridine assay. To explore the molecular mechanisms, we assessed the effect of GMSCs on RA FLS proliferation by adding indoleamine‐2,3‐dioxygenase (IDO), CD39, or CD73 inhibitors. The invasion was analyzed by a transwell assay. The anti‐inflammatory effects of GMSCs were assessed in a type II collagen‐induced arthritis (CIA) mouse model.ResultsCompared with human dermal fibroblast, GMSCs displayed a higher expression of CD39. Interleukin (IL)‐8, IL‐6, MMP‐1, MMP‐3, MMP‐13, and monocyte chemoattractant protein‐1 mRNA were all decreased in RA FLSs after incubation with GMSCs. GMSCs significantly reduced RA FLS proliferation in a dose‐dependent manner in vitro, which was partly dependent on CD39/CD73 signaling. GMSCs significantly impeded the invasive capacity of RA FLSs in a dose‐dependent manner in vitro. GMSCs infusion delayed arthritis onset and reduced arthritis scores in the CIA model.ConclusionGMSCs are effective to inhibit the aggressive behavior of RA FLSs and treat experimental arthritis, implying their therapeutic potential in RA patients.

Non-Canonical WNT5A Signaling Through RYK Contributes to Aggressive Phenotype of the Rheumatoid Fibroblast-Like Synoviocytes

We hypothesized that WNT5A could contribute to the enhanced migration and invasiveness of rheumatoid arthritis fibroblast-like synoviocytes (RA FLS), which is one of the incompletely understood aspects of the RA FLS aggressive phenotype. This hypothesis is based on the previous evidence of a WNT5A role in both, RA and cell migration. Migration and invasion of RA FLS were assessed after incubation with recombinant Wnt5a (rWnt5a) or silencing of the endogenous WNT5A expression. The expression of WNT5A, WNT receptors, cytokines, chemokines, and metalloproteinases was quantified with RT-PCR. The WNT pathway was explored with gene silencing, antibody and pharmacological inhibition followed by migration assays and phosphoprotein western blots. Here, we reported that rWnt5a promoted migration and invasion of RA FLS, whereas knockdown of the endogenous WNT5A reduced them. These effects were specific to the RA FLS since they were not observed in FLS from osteoarthritis (OA) patients. Also, rWnt5a induced the expression of IL6, IL8, CCL2, CXCL5, MMP1, MMP3, MMP9, and MMP13 from baseline or potentiating the TNF induction, WNT5A signaling required the RYK receptor and was mediated through the WNT/Ca2+ and the ROCK pathway. These pathways involved the RYK and ROCK dependent activation of the p38, ERK, AKT, and GSK3β kinases, but not the activation of JNK. Together these findings indicate that WNT5A contributes to the enhanced migration and invasiveness of RA FLS through RYK and the specific activation of ROCK and downstream kinases.

SUMOylation links metabolic and aggressive phenotype of RA FLS

SUMOylation links metabolic and aggressive phenotype of RA FLS

Innovative translational models to study human synovial pathology: Target validation and preclinical imaging

Purpose: Many experiments to study inflammation, hyperplasia, and fibrosis in the synovium have been performed in animal models of RA and OA. However, the predictive value of these models for the screening of potential drugs in RA is variable and for OA, none were sufficiently effective in clinical trials. Translational arthritis research with human cells is often performed in monolayer culture where the absence of extracellular matrix and other cell types results in alterations of cell functions and loss of phenotype. To improve the predictive value of preclinical arthritis research by developing and optimizing innovative translational models to study human synovial pathology in vitro and in vivo. Methods: Synovial biopsies from arthritis patients were obtained during joint replacement surgery and processed for either (1) explant cultures, (2) 3D-synovial micromasses, (3) RA-SCID transplantation studies, and/or (4) a biobank for corresponding mRNA and IHC profiling. For explant culture, 3mm biopsies were cultured for 24 hours w/o various inhibitors, and cytokine production was analyzed by Luminex. 3D-micromasses were generated from primary RA FLS and CD14+ PBMCs, stimulated for 3 weeks with 10 ng/ml TNFα or TGFβ and analyzed by histology, IHC and QPCR. For target validation and preclinical imaging, 6mm biopsies were engrafted into SCID mice. Radio- and fluorescently-labelled anti-FcγRI antibodies were injected intravenously, and targeting was determined by biodistribution, μSPECT/CT and IVIS imaging analysis. Results: Our first assay with human arthritis synovium explants demonstrated to be highly suitable to test the therapeutic efficacy of inhibitors for TNFα, TLR4, p38 and the JAK-pathway, resulting in significantly reduced production of proinflammatory cytokines and chemokines at 24 hours of culture (1) . In contrast to the explant cultures, our 3D synovial micromasses could be followed for weeks. In this second translational model, lining formation was observed at day 7 and the micromasses could be stimulated to mimic RA- or OA-like features of synovial hyperplasia or fibrosis respectively. Long-term exposure to the RA-related cytokine TNFα lead to hyperplasia of the lining and an altered macrophage phenotype characterized by reduced CD163 expression. Conversely, the repair-related growth factor TGFβ induced fibrosis-like changes in the micromass lining, a hallmark of OA. This was accompanied by an increased expression of PLOD2, alpha-SMA, and collagen type 1 (2) Our third preclinical model, the human arthritis synovium SCID mouse, was previously validated using adalimumab, secukinumab, and rituximab (3), and was now used to study FcγRI as a potential marker to image synovitis. Gene expression of FCGRI in synovial explants from RA patients was shown to correlate positively and significantly to gene expression of pro-inflammatory factors IL1B, TNFA, IL8, S100A8 and S100A9 while gene expression of FCGRI in synovial explants from OA patients significantly correlated with S100A9. Interestingly, dual-labelled [111In]In-DTPA-IrDye800CW-anti-FcγRI antibody showed high uptake in the synovial transplants of the engrafted SCID mice, and specifically visualized the subcutaneous synovial grafts by both uSPECT/CT and IVIS imaging. Conclusions: The development of these translational models allows us to bridge the gap between preclinical and clinical drug development research. Whereas our synovial explant assay is ideal for short-term interventions, and the RA-SCID mouse a valuable translational model for in vivo preclinical studies, donor variability and access to sufficient tissue may be challenging. In such cases, the 3D synovial micromass model may be an excellent alternative. Especially in combination, these 3 translational approaches will improve target validation, lead optimization and preclinical development of novel anti-rheumatic drugs. (1) Abdollahi S., J Clin Invest. 2008; 118(1): 205-216. (2) Broeren M. et al., ALTEX. 2019; 36(1):18-28 (3) Koenders et al., Arthritis Rheum. 2012 Jun;64(6):1762-70

Indirubin inhibits the migration, invasion, and activation of fibroblast-like synoviocytes from rheumatoid arthritis patients.

To evaluate the inhibition of indirubin in FLSs migration, invasion, activation, and proliferation in RA FLSs. The levels of IL-6 and IL-8 in cultural supernatants were measured by ELISA. RA FLS migration and invasion in vitro were measured by the Boyden chamber method and the scratch assay. Signal transduction protein expression was measured by western blot. FLS proliferation was detected by Edu incorporation. F-actin was measured by immunofluorescence staining. We found that indirubin reduced migration, invasion, inflammation, and proliferation in RA FLSs. In addition, we demonstrated that indirubin inhibited lamellipodium formation during cell migration. To gain insight into molecular mechanisms, we evaluated the effect of indirubin on PAK1 and MAPK activation. Our results indicated that indirubin inhibited the activity of PAK1 and MAPK. Our observations suggest that indirubin may be protective against joint destruction in RA by regulating synoviocyte migration, invasion, activation, and proliferation.

Grem1 is a key regulator of synovial hyperplasia in rheumatoid arthritis. (CAM1P.162)

Abstract Fibroblast like synoviocyte plays critical roles is the major cell in the inflammatory processes of Rheumatoid arthritis. We attempted to identify a key regulator that can drive aggressive phenotypes of RA FLS. For this, we performed an integrative transcriptome data analysis using three datasets from independent RA studies, a panel of normal tissues, and NCI60 cancer cell line panel. Through the analysis, we identified Gremlin (GREM1), which is robustly up-regulated in RA and predominantly expressed in FLS. GREM1 is identified as bone morphogenetic proteins antagonist and is known to bind BMPs. To investigate potential roles of GREM1 in RA, we first examined the level of GREM1 expression in synovial tissues, synovial fluid (SF) and FLS from RA. Immunohistochemistry and ELISA revealed up-regulation of GREM1 protein in RA compared to OA. Proinflammatory cytokines also induced the expression of GREM1 in RA FLS and their expressions were significantly correlated with GREM1 expression in RA SF. We next investigated cellular functions of GREM1 using RA FLS. GREM1 deficieny by siRNA elicited significant suppression of FLS proliferation, migration and invasion. In contrast, stimulation with rhGREM1 to RA FLS increased cell survival and invasion. Furthermore, we found that GREM1 activates AKT1 and MAPK signaling cascade, as well as antagonizes BMP2. This study demonstrated that GREM1 is a key regulator responsible for synovial hyperplasia and FLS migration.

SAT0058 Slug is induced by benzo(a)pyrene and egf through PI3K/AKT/MTOR pathway and is closely involved in the regulation of the invasive properties of FLS in rheumatoid arthritis

BackgroundSlug, a Snail family of zinc finger transcription factor, plays a critical role in tumor proliferation, invasion and metastasis. We have previously shown that Slug is overexpressed in RA synovial...

SAT0059 Inhibition of CIP2A facilitate C-MYC independent apoptosis of fibroblast-like synoviocytes in rheumatoid arthritis

BackgroundCancerous inhibitor of protein phosphatase 2A (CIP2A) is a recently identified oncoprotein that leads to cellular proliferation in cancer cells by stabilizing c-Myc protein. The effect of CIP2A in stabilizing...

FRI0034 Suppression of hdac5 expression by inflammatory cytokines is required to promote cxcl chemokine production in ra fls

BackgroundHistone deacetylases (HDACs) are important regulators of gene expression and protein function in the immune system. HDAC inhibitors (HDACi) display anti-inflammatory properties in animal and in vitro models of rheumatoid...

CIP2A facilitates apoptotic resistance of fibroblast-like synoviocytes in rheumatoid arthritis independent of c-Myc expression

The aim of this study is to investigate the effects of CIP2A (Cancerous inhibitor of protein phosphatase 2A) on the apoptosis of RA FLS. Proliferation and apoptotic activity of RA FLS following treatment with CIP2A siRNA or control siRNA were analyzed using MTT assays and Cell Death Detection kit. RA FLS was treated with CIP2A siRNA or control siRNA in 3-, 6-, and 9-day intervals for a Western blot analysis to determine C-Myc expression. To evaluate the signal transduction pathways engaged in apoptosis, caspase-3 activity, caspase-9 activity, PARP, and phosphorylation of the Akt kinase were analyzed by Western blot. Cell viability of RA FLS was significantly lower in the CIP2A siRNA-treated group compared with the control after 7 days (p = 0.022). Apoptosis of RA FLS was significantly higher in the CIP2A siRNA-treated group compared with the control when incubated for 3, 6, and 9 days (p = 0.029, p = 0.021, p = 0.043, respectively). C-Myc expression did not change with the different incubation periods. CIP2A siRNA-treated FLS expressed higher level of activated caspase-3, caspase-9, and PARP (p = 0.014, p = 0.020, p = 0.021, respectively) and lower level of phosphorylated Akt (p = 0.001) compared with those treated with the control siRNA. Our data show that CIP2A expression in RA FLS is an important mediator of dysfunctional apoptosis independent of c-Myc stabilization. Expression of CIP2A may contribute to apoptotic resistance of RA FLS through the activation of Akt and deactivation of caspase-3, caspase-9, and PARP. Inhibition of CIP2A may therefore constitute a novel, promising therapeutic target in RA.

MiR-346 Controls Release of TNF-α Protein and Stability of Its mRNA in Rheumatoid Arthritis via Tristetraprolin Stabilization

TNF-α is a major cytokine implicated in rheumatoid arthritis. Its expression is regulated both at the transcriptional and posttranscriptional levels and recent data demonstrated that miRNAs are implicated in TNF-α response in macrophages. LPS-activated FLS isolated from RA patients express TNF-α mRNA but not the mature protein. This prompted us to look for miRNAs which could be implicated in this anti-inflammatory effect. Using a microarray, we found two miRNAs, miR-125b and miR-939 predicted to target the 3′-UTR of TNF-α mRNA, to be up-regulated in RA FLS in response to LPS, but their repression did not restore mature TNF-α expression in FLS. We showed previously that miR-346, which is upregulated in LPS-activated FLS, inhibited Btk expression that stabilized TNF-α mRNA. Blocking miR-346 reestablished TNF-α expression in activated FLS. Interestingly, transfection of miR-346 in LPS-activated THP-1 cells inhibited TNF-α secretion. We also demonstrated that TTP, a RNA binding protein which inhibited TNF-α synthesis, is overexpressed in activated FLS and that inhibition of miR-346 decreases its expression. Conversely, transfection of miR-346 in LPS-activated THP-1 cells increased TTP mRNA expression and inhibited TNF-α release. These results indicate that miR-346 controls TNF-α synthesis by regulating TTP expression.

Anti-inflammatory and anti-nociceptive effect of Betula platyphylla var. japonica in human interleukin-1β-stimulated fibroblast-like synoviocytes and in experimental animal models

Traditional medicine has widely been used Betula platyphylla var. japonica to treat various inflammatory diseases including arthritis. To determine the anti-inflammatory, anti-nociceptive, and anti-arthritic effects of Betula platyphylla in interleukin-1β (IL-1β)-stimulated fibroblast-like synoviocytes from human rheumatoid arthritis and in nociceptive and inflammatory animal model. The inflammatory mediators such as IL-6, tumor necrosis factor (TNF)-α matrix metalloproteinase (MMP)-1, MMP-13, inducible nitric oxide synthesis (iNOS), nitrites, prostaglandin E(2) (PGE(2)) and cyclo-oxygenase 2 (COX-2) activity of Betula platyphylla were tested in IL-1β-stimulated fibroblast-like synoviocytes. Tail withdrawal in response to thermal stimulation in tail flick test or paw flinching and shaking in response to sc hind paw formalin injection was measured 1h after oral administration of Betula platyphylla. The former was evaluated with a paw pressure test, and the latter was measured using the squeaking score, and paw volume in inflammatory arthritis tests. Betula platyphylla significantly inhibited proliferation of IL-1β-induced synoviocytes. Betula platyphylla reduced the levels of inflammatory mediators, such as IL-6, TNF-α, MMP-1, MMP13, and PGE(2). In particular, Betula platyphylla significantly inhibited the releases of nitrites and iNOS, as well as release of NFκB, into the nucleus of IL-1β-treated synoviocytes, even at concentrations as low as 1μg/ml. Oral administrant of Betula platyphylla at 400mg/kg significantly decreased about 27.8% of tail flick withdrawal and inhibited about the number of paw flinches in both phases 1 and 2 of the formalin test. In the carrageenan-induced acute pain and arthritis model, Betula platyphylla dose dependently reduced the nociceptive threshold and the arthritic symptoms at day 8, respectively, and Betula platyphylla at 400mg/kg markedly reduced the inflammatory area about 48% in the ankle joints. This capacity of Betula platyphylla at 400mg/kg was similar to that of the celecoxib-2 inhibitor in carrageenan-induced nociceptive and inflammatory arthritis model. These results suggest that Betula platyphylla has anti-nociceptive and anti-inflammatory effects in IL-1β-stimulated RA FLS and in an animal model of arthritis. Thus, the use of Betula platyphylla as a pharmaceutical candidate for the treatment of arthritis should be further studied.

Effects of resveratrol on proliferation and apoptosis of TNF-α induced rheumatoid arthritis fibroblast-like synoviocytes

To observe the effect of resveratrol (Res) on in vitro proliferation and apoptosis of TNF-alpha induced rheumatoid arthritis fibroblast-like synoviocytes (RA FLS), and further to investigate the PI3 K/Akt/BAD signal mechanism. The inhibition rate of RA FLS was examined by MTT assay. Cell cycle and the amount of apoptotic cells was measured by flow cytometry. PI3K/Akt/BAD signal transduction proteins expression was measured by western blot. The living cells measured by MTT dose and time-dependently reduced in Res groups. In Res groups, the fraction of living cells in the S-phase and G2/M-phase decreased respectively, while that in G1-phase increased, the difference was statistically significant compared with the TNF-alpha group (P < 0.05). Flow cytometry demonstrated that the apoptosis rate increased with increased Res concentration. Res inhibited TNF-alpha induced phosphorylation of Akt and BAD in RA FLS. Res can inhibit RA FLS proliferation and induce apoptosis through inhibition of PI3K/Akt/BAD signalling pathway. Res may provide a new therapeutic approach in treatment of RA.

Programmed Cell Death 5 Factor Enhances Triptolide-Induced Fibroblast-Like Synoviocyte Apoptosis of Rheumatoid Arthritis

To study the effect of programmed cell death 5 (PDCD5) on apoptosis of rheumatoid arthritis fibroblast-like synoviocyte (RAFLS) induced by triptolide. Cultured synovial cells in vitro from RA patients were transfected with Ad-PDCD5. In protein level, expression of PDCD5 protein in Ad-PDCD5 transfected RAFLS was detected by Western blot. RAFLS transfected with Ad-PDCD5 were cultured in presence or absence of triptolide and RAFLs apoptosis was determined by flow cytometry. Transfection of RAFLS with increasing concentration of Ad-PDCD5 (50-300 MOI) resulted in dose-dependent increase of PDCD5 production. Apoptotic cells percentage of no transfection group, Ad-null group and Ad-PDCD5 group were, respectively, (22.41 +/- 3.87)%, (28.77 +/- 12.97)%, and (48.87 +/- 12.69)%. Alternatively, transfection without triptolide stimuli had no effect. The data showed that gene transfection of PDCD5 alone without triptolide was not sufficient to activate RAFLS apoptosis; PDCD5 acted as an enhancer rather than inductor of apoptosis. Overexpression of PDCD5 could enhance apoptosis of RA FLS induced by triptolide; PDCD5 may be a potential therapeutic target to RA.

The anti-malaria agent artesunate inhibits expression of vascular endothelial growth factor and hypoxia-inducible factor-1α in human rheumatoid arthritis fibroblast-like synoviocyte

Increasing evidence indicates that the anti-malarial agent artemisinin and its derivatives may exert anti-angiogenic effect. In the present study, we explored the effect of artesunate, a artemisinin derivative, on TNFα- and hypoxia-induced expression of hypoxia inducible factor-1α (HIF-1α) and secretion of vascular endothelial growth factor (VEGF) and inteleukin-8 (IL-8) in human rheumatoid arthritis fibroblast-like synoviocytes (RA FLS), and further investigated the signal mechanism by which this compound modulates HIF-1α, VEGF and IL-8 expression. RA FLS obtained from patients with active rheumatoid arthritis were pretreated with artesunate, and then stimulated with TNFα and hypoxia. Production of VEGF and IL-8 was measured by ELISA. Nuclear location of HIF-1α was measured by confocal fluorescence microscopy. HIF-1α and other signal transduction proteins expression was measured by Western blot. Artesunate decreased the secretion of VEGF and IL-8 from TNFα- or hypoxia-stimulated RA FLS in a dose-dependent manner. Artesunate also inhibited TNFα- or hypoxia-induced nuclear expression and translocation of HIF-1α. We also showed that artesunate prevented Akt phosphorylation, but did not find evidence that phosphorylation of p38 and ERK was affected. TNFα- or hypoxia-induced secretion of VEGF and IL-8 and expression of HIF-1α were hampered by treatment with the PI3 kinase inhibitor LY294002, suggesting that inhibition of PI3 kinase/Akt activation might inhibit VEGF and IL-8 secretion and HIF-1α expression induced by TNFα or hypoxia. Our results suggest that artesunate inhibits angiogenic factor expression in RA FLS, and provide novel evidence that, as a low-cost agent, artesunate may have therapeutic potential for RA.

A Critical Role for Adrenomedullin-Calcitonin Receptor-Like Receptor in Regulating Rheumatoid Fibroblast-Like Synoviocyte Apoptosis

Rheumatoid arthritis (RA) is characterized by fibroblast-like synoviocyte (FLS) hyperplasia, which is partly ascribable to decreased apoptosis. In this study, we show that adrenomedullin (ADM), an antiapoptotic peptide, is constitutively secreted in larger amounts by FLS from joints with RA (RA-FLS) than with osteoarthritis (OA-FLS). ADM secretion was regulated by TNF-alpha. Peptidylglycine alpha-amidating monooxygenase, the ADM-processing enzyme, was expressed at the mRNA level by both RA-FLS and OA-FLS. Constituents of the ADM heterodimeric receptor calcitonin receptor-like receptor (CRLR)/receptor activity-modifying protein (RAMP)-2 were up-regulated at the mRNA and protein levels in cultured RA-FLS compared with OA-FLS. ADM induced rapid intracellular cAMP production in FLS and reduced caspase-3 activity, DNA fragmentation, and chromatin condensation in RA-FLS exposed to apoptotic conditions, indicating that CRLR/RAMP-2 was fully functional. ADM-induced cAMP production was less marked in OA-FLS than in RA-FLS, suggesting differences in receptor regulation and expression. ADM dose-dependently inhibited RA-FLS apoptosis, and this effect was reversed by the 22-52 ADM antagonist peptide. ADM inhibited RA-FLS apoptosis triggered by extrinsic and intrinsic pathways. Our data suggest that ADM may prevent or reduce RA-FLS apoptosis, via up-regulation of its functional receptor CRLR/RAMP-2. Regulation of ADM secretion and/or CRLR/RAMP-2 activation may constitute new treatment strategies for RA.