fibroblast-like synoviocytes

Rheumatoid arthritis (RA), affecting 0.5-1.0% of the adult population worldwide, is a complicated autoimmune disease with systemic sequelae and is characterized by a chronic inflammation of synovial joints and bone erosion [1]. Environmental and genetic factors are two major aspects of RA and account for approximately 60% and 40% of the risk for developing RA, respectively [2]. Although the mechanism underlying RA remains unknown, fibroblast- like synoviocytes (FLS) have been reported to play a critical role in the pathogenesis of RA [3, 4]. In the normal synovium, FLS are a highly differentiated unicellular cell type, governing the provisions of support, nourishment, and lubrication of the joint tissue [5]. However, when an inflammatory response occurs, FLS become hyperplastic, invasive, and highly migratory, contributing to cartilage destruction and thus, RA [6, 7]. Van Hamburg et al. reported that the combination of neutralizing TNF activity and 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2 D 3 ) contribute to controlling human Th17 activity, which induces a pro-inflammatory feedback loop on RA synovial fibroblast interactions and inhibits synovial inflammation [8]. However, the specific mechanism of this function requires a further investigation.

AIA: antigen-induced arthritis; BGLAP: bone gamma-carboxyglutamic acid- containing protein; BSA: bovine serum albumin; CAIA: antibody-induced arthritis; CCL-2: chemokine ligand 2; DMEM: Dulbecco ’ s modified Eagle ’ s medium; ELISA: enzyme-linked immunosorbent assay; FBS: fetal bovine serum; FLS: fibroblast-like synoviocytes; GM-CSF: granulocyte-macrophage colony-stimulating factor; ICAM-1: intercellular adhesion molecule 1; IgG: immunoglobulin G; IL: interleukin; PDGF: platelet-derived growth factor; OA: osteoarthritis; RA: rheumatoid arthritis; RANKL: receptor activator of nuclear factor kappa-B (NF κ -B) ligand; RT-PCR: reverse transcription polymerase chain reaction; Th: T helper; TNF- α : tumour necrosis factor alpha; UDPGD: uridine diphosphoglucose dehydrogenase; VCAM-1: vascular cell adhesion molecule 1; WT: wild-type.

Tendon never restores the complete biological and mechanical properties after healing. Several techniques are available for tissue-engineered biological augmentation for tendon healing like stem cells. Recently, synovium has been investigated as a source of cells for tissue engineering. In the present study, we investigated potentials of fibroblast like synoviocytes (FLSs) in tendon healing. Sixteen rabbits were divided randomly into control and treatment groups. One rabbit was used as a donor of synovial membrane (synovium). The injury model was unilateral complete transection through the middle one third of deep digital flexor tendon (DDFT). Subsequently, the tendon stumps were sutured with 3/0 nylon. In treatment group, 0.1 mL phosphate-buffered saline (PBS) solution containing 1 × 10 6 nucleated cells of FLSs was

In this study, the anti-inflammatory effects of piperine were tested in IL1β-stimulated rheumatoid arthritis fibroblast-like synoviocytes derived from patients with rheumatoid arthritis.[r]

It has been proposed that fibroblast-like synoviocytes (FLS) are actively involved in chronic inflammatory reactions. The molecular mechanisms of their sus- tained activation might be contributed to inflamma- tory cascades. Cadherin-11 is a classical molecule that mediates hemophilic cell-to-cell adhesion in FLS, plays an important role in the development of the normal syno- vium lining layer of the joint [9–11], and has been identi- fied to participate in the cartilage invasion except for its adhesive function [10]. Cadherin-11-deficient mice have a hypoplastic synovial lining, display disorganized synovial reaction to inflammation, and are resistant to inflamma- tory arthritis [12]. Our previous study demonstrated that Cadherin-11 expression in FLS was positively related to the degree of synovitis [13]. As a predominant inflamma- tory factor, TNF-α plays a vital role in the upregulation of Cadherin-11 in FLS. However, the underlying mechanism for the upregulation of Cadherin-11 expression in FLS by the stimulation of TNF-α was rarely investigated.

APC/C: Anaphase promoting complex/cyclosome; CE-MS: Capillary electrophoresis-mass spectrometry; DMEM: Dulbecco ’ s modified Eagle ’ s medium; DMSO: Dimethyl sulfoxide; ELISA: Enzyme-linked immunosorbent assay; FAO: Fatty acid oxidation; FBS: Fetal bovine serum; FLS: Fibroblast-like synoviocytes; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; GC/MS: Gas chromatography-mass spectrometry; GLS: Glutaminase; HK: Hexokinase; IL: Interleukin; LPS: Lipopolysaccharide; MCT4: Monocarboxylate transporter; MMP: Matrix metalloproteinase; OA: Osteoarthritis; PCR: Polymerase chain reaction; PDGF: Platelet-derived growth factor; PDK: Pyruvate dehydrogenase kinase; RA: Rheumatoid arthritis; sIL-6R: Soluble interleukin-6 receptor; siRNA: Small interfering RNA; TCA: Tricarboxylic acid; TNF: Tumor necrosis factor; Treg: Regulatory T; ZyA: Zymosan A

Effect of adiponectin on the production of vascular endothelial growth factor and matrix metalloproteinases in rheumatoid arthritis fibroblast-like synoviocytes Next, to evaluate whether adiponectin stimulates the produc- tion of VEGF and MMPs for angiogenesis of pannus and joint destruction in RA FLSs, VEGF and MMP production was eval- uated in the supernatants of cell cultures treated with adi- ponectin or IL-1β (Figure 2). Both adiponectin and IL-1β strongly stimulated the production of VEGF, MMP-1, and MMP-13 in RA FLSs. However, the expressions of MMP-2 and MMP-9 were not increased by either adiponectin or IL-1β at the mRNA or protein level. Consistent with the mRNA levels (data not shown), the protein levels of VEGF, MMP-1, and MMP-13 elevated by treatment with 10 μg/mL adiponectin were similar to those after treatment with 1 ng/mL IL-1β. The difference between adiponectin and IL-1β was not statistically significant, suggesting that adiponetin may have a role in the production of VEGF and MMPs like IL-1β does.

Genetic studies have shed light on our understanding of the causes of autoimmune diseases by identifying shared and unique risk loci among these diseases. However, in rheumatoid arthritis (RA), only a fraction of disease sus- ceptibility can be explained by genetic variation [1], and the temporal link between the break of self-tolerance and development of clinical disease remains elusive because circulating autoantibodies are detectable long before the onset of arthritis [2]. In RA, stromal cells in the joint, fibroblast-like synoviocytes (FLS), exhibit an imprinted and epigenetically maintained aggressive phenotype, pre- disposing them to participate in an inflammatory positive feedback loop in response to the cues from the synovial environment [3]. Identifying local tissue conditions able to initiate and perpetuate the ensuing inflammatory cycle is therefore of critical importance to understanding and intervening in the disease process.

In the present study, the in vitro effect of various concentrations of doxycycline were evaluated in cultured equine fibroblast-like synoviocytes viability, using the 3-(4,4-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) reduction assay and trypan blue exclusion. To the best knowledge of the authors, no study to date has investigated the cytotoxicity of doxycycline in equine FLSs.

AIA: antigen-induced arthritis; BGLAP: bone gamma-carboxyglutamic acid- containing protein; BSA: bovine serum albumin; CAIA: antibody-induced arthritis; CCL-2: chemokine ligand 2; DMEM: Dulbecco’s modified Eagle’s medium; ELISA: enzyme-linked immunosorbent assay; FBS: fetal bovine serum; FLS: fibroblast-like synoviocytes; GM-CSF: granulocyte-macrophage colony-stimulating factor; ICAM-1: intercellular adhesion molecule 1; IgG: immunoglobulin G; IL: interleukin; PDGF: platelet-derived growth factor; OA: osteoarthritis; RA: rheumatoid arthritis; RANKL: receptor activator of nuclear factor kappa-B (NFκ-B) ligand; RT-PCR: reverse transcription polymerase chain reaction; Th: T helper; TNF-α: tumour necrosis factor alpha; UDPGD: uridine diphosphoglucose dehydrogenase; VCAM-1: vascular cell adhesion molecule 1; WT: wild-type.

PUMA (p53-upregulated modulator of apoptosis) is a pro- apoptotic gene that can induce rapid cell death through a p53- dependent mechanism. However, the efficacy of PUMA gene therapy to induce synovial apoptosis in rheumatoid arthritis might have limited efficacy if p53 expression or function is deficient. To evaluate this issue, studies were performed to determine whether p53 is required for PUMA-mediated apoptosis in fibroblast-like synoviocytes (FLS). p53 protein was depleted or inhibited in human FLS by using p53 siRNA or a dominant-negative p53 protein. Wild-type and p53 -/- murine FLS

Rheumatoid arthritis (RA) is an immune-mediated disorder induced by chronic and refractory autoimmunity in the synovial joints, which leads to the damage of the cartilage and bone. RA affects approximately 1% of the population globally, although its complex pathogenesis is not fully understood.[1, 2] The fibroblast-like synoviocytes (FLS) play a critical role in the progression of RA. These cells normally line the synovium, but in RA they proliferate in an uncon- trolled manner and form the pannus tissue, a tumor-like structure that causes significant damage to the joints [3, 4]. In the synovial tissue, FLS secrete cytokines that induce persistent inflammation and degradation of the cartilage and bone[5 – 7]. Some pro-inflammatory cytokines like IL-6

Fibroblast-like synoviocytes (FLS) isolated from joints of rheumatoid arthritis (RA) patients display proliferative and invasive prop- erties reminiscent of those of malignant tumor cells. Rac small GTPases play an important role in tumor cell proliferation and in- vasion. We therefore investigated the potential role of Rac proteins in the proliferative and invasive behavior of RA-FLS. We showed that inhibiting Rac activity with the Rac-specific small molecule inhibitor NSC23766 causes a strong inhibition of RA-FLS proliferation, without affecting cell survival. Rac inhibition also results in a strong reduction in RA-FLS invasion through reconstituted extracellular matrix and a less marked inhibition of two-dimensional migration as measured by monolayer wound healing. We also showed that small interfering RNA-mediated depletion of Rac1 inhibits RA-FLS proliferation and invasion to a similar extent as NSC23766. These results demonstrate for the first time that Rac proteins play an important role in the aggressive behavior of FLS isolated from RA patients. In addition, we observed that inhibiting Rac proteins prevents JNK activation and that the JNK inhibitor SP600125 strongly inhibits RA-FLS invasion, suggesting that Rac-mediated JNK activation contributes to the role of Rac proteins in the invasive behavior of RA-FLS. In conclusion, Rac-controlled signaling pathways may present a new source of drug targets for therapeutic intervention in RA.

Figure 1 Effects of mithramycin on gliostatin/thymidine phosphorylase (GLS/TP) promoter activity based on deletion constructs. Closed ovals indicate Sp1-binding sites. Open ovals indicate interferon-stimulated response element (ISRE) and gamma-activated sequence (GAS) sequences. Fibroblast-like synoviocytes were transiently transfected with deletion constructs and then incubated with mithramycin (100 nM, shaded column, or 300 nM, closed column) for 24 hours. Control cells were incubated without mithramycin (open column). Data were normalized by measuring the luminescent reaction of the internal control. Results are presented as mean ± standard error of the mean of five determinations. Statistical significance was calculated by using the Mann-Whitney U test: compared with samples without mithramycin *P < 0.05, **P < 0.01; compared with samples with pTP-Luc1 without mithramycin † P < 0.01.

CIS: cytokine-inducible SH2; (D)MEM: (Dulbecco ’ s) modified Eagle ’ s medium; DMSO: dimethyl sulfoxide; ELISA: enzyme-linked immunosorbent assay; FLS: fibroblast-like synoviocytes; H & E: hematoxylin and eosin; IL: interleukin; IFN: interferon; JAK2: Janus activated kinase; M-CSF: macrophage colony- stimulating factor; MNCs: mononuclear cells; MTX: methotrexate; MTT: 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetra zolium bromide; NFAT: nuclear factor of activated T cells; NF- κ B: nuclear factor- κ B; OPG: osteoprotegerin; PBMC: peripheral blood mononuclear cells; PBS: phosphate-buffered saline; RA: rheumatoid arthritis; RANKL: receptor activator of NF- κ B ligand; RNasin: ribonuclease inhibitor; RT-PCR: real time-polymerase chain reaction; sIL-6R: soluble IL-6 receptor; siRNA: small interfering RNA; SOCS3: suppressor of cytokine signaling 3; STAT3: signal transducer and activator of transcription 3; TNF: tumor necrosis factor; TRAP: tartrate-resistant acid phosphatase.

Methods: The expression of resistin and adenylate cyclase-associated protein 1 (CAP1), a receptor for resistin, was examined immunohistochemically in synovial tissue. CAP1 expression in in vitro cultured fibroblast-like synoviocytes (FLSs) was assessed with a reverse transcription-polymerase chain reaction (PCR) and western blotting. The gene expression of resistin-stimulated FLSs was evaluated by RNA sequencing (RNA-Seq) and quantitative real-time PCR. Concentrations of chemokine (C-X-C motif) ligand (CXCL) 8, chemokine (C-C motif) ligand (CCL) 2, interleukin (IL)- 1 β , IL-6 and IL-32 in culture supernatants were measured by enzyme-linked immunosorbent assay. Small interfering RNA (siRNA) for CAP1 was transfected into FLSs in order to examine inhibitory effects.

Figure 1 Effect of IFN-γ 0.1 ng/ml on IL-32 mRNA expression and IL-32 release by rheumatoid arthritis RA fibroblast-like synoviocytes FLSs.. e IL32 release in culture supernatants was de[r]

Introduction: Fibroblast-like synoviocytes (FLS) play an important role in the pathogenesis of rheumatoid arthritis (RA). This study aimed to investigate the role of glucose 6-phosphate isomerase (GPI) in the proliferation of RA-FLS. Methods: The distribution of GPI in synovial tissues from RA and osteoarthritis (OA) patients was examined by immunohistochemical analysis. FLS were isolated and cultured, cellular GPI level was detected by real-time polymerase chain reaction (PCR) and Western blot analysis, and secreted GPI was detected by Western blot and enzyme-linked immunosorbent assay (ELISA). Doxorubicin (Adriamycin, ADR) was used to induce apoptosis. Cell proliferation was determined by MTS assay. Flow cytometry was used to detect cell cycle and apoptosis. Secreted pro-inflammatory cytokines were measured by ELISA.

Expression of intercellular adhesion molecule (ICAM)-1 in fibroblast- like synoviocytes (FLS) (a) and U937 cells (b) upon stimulation with IL-18 (x-axis: staining intensity; y-axis: number of events). Broken and continuous lines show ICAM-1 expression in control conditions and IL- 18-stimulated cells, respectively. The strongest induction of ICAM-1 by IL-18 (a) was 95.1, versus 73.9 in controls ( ∆ MFI = +28.6%), and (b) 82.1, versus 31.7 in U937 cells ( ∆ MFI = +159%). The result of each one of three similar independent experiments is represented. CD54PE = fluorescence intensity of cells, stained with phycoerythrin-labeled CD54 monoclonal antibodies; MFI = mean fluorescence intensity.

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease of unclear etiology. This study was conducted to identify critical factors involved in the synovial hyperplasia in RA pathology. We applied cDNA microarray analysis to profile the gene expressions of RA fibroblast-like synoviocytes (FLSs) from patients with RA. We found that the MLN51 (metastatic lymph node 51) gene, identified in breast cancer, is remarkably upregulated in the hyperactive RA FLSs. However, growth- retarded RA FLSs passaged in vitro expressed small quantities of MLN51. MLN51 expression was significantly enhanced in the FLSs when the growth-retarded FLSs were treated with granulocyte – macrophage colony-stimulating factor (GM-CSF) or synovial fluid (SF). Anti-GM-CSF neutralizing antibody blocked the MLN51 expression even though the FLSs were