Modulation of platelet-derived microparticles to adhesion and motility of human rheumatoid arthritis fibroblast-like synoviocytes.
Ontology highlight
ABSTRACT:
SUBMITTER: Wang W
PROVIDER: S-EPMC5509257 | biostudies-other | 2017
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Fibroblast-like synoviocytes (FLS) were cultured from synovial tissues from 19 RA patients. RNA was isolated using the Fast Track 2.0 kit (Invitrogen, Carlsbad, California)from the FLS cultures. Fluorescent cDNA probes were prepared from 1 ug experimental mRNA by oligo dT-primed polymerization using Superscript II reverse transcriptase in the presence of Cy5 labeled dCTP. A common reference mRNA sample (CRE) that consists of a mixture of mRNAs isolated from 11 different cell-lines, was labeled with Cy3. The Cy5 and Cy3 labeled cDNAs were pooled and hybridized to a 24,000 cDNA microarray. Set of arrays that are part of repeated experiments Biological Replicate Using regression correlation
2006-01-18 | E-GEOD-4061 | biostudies-arrayexpress
Project description:Fibroblast-like synoviocytes (FLS) were cultured from synovial tissues from 19 RA patients. RNA was isolated using the Fast Track 2.0 kit (Invitrogen, Carlsbad, California)from the FLS cultures. Fluorescent cDNA probes were prepared from 1 ug experimental mRNA by oligo dT-primed polymerization using Superscript II reverse transcriptase in the presence of Cy5 labeled dCTP. A common reference mRNA sample (CRE) that consists of a mixture of mRNAs isolated from 11 different cell-lines, was labeled with Cy3. The Cy5 and Cy3 labeled cDNAs were pooled and hybridized to a 24,000 cDNA microarray. Set of arrays that are part of repeated experiments Keywords: Biological Replicate
2006-01-19 | GSE4061 | GEO
Project description:Epigenetics contributes to the pathogenesis of immune-mediated diseases like rheumatoid arthritis (RA). Here we show the first comprehensive epigenomic characterization of RA fibroblast-like synoviocytes (FLS), including histone modifications (H3K27ac, H3K4me1, H3K4me3, H3K36me3, H3K27me3, and H3K9me3), open chromatin, RNA expression and whole-genome DNA methylation. To address complex multidimensional relationship and reveal epigenetic regulation of RA, we perform integrative analyses using a novel unbiased method to identify genomic regions with similar profiles. Epigenomically similar regions exist in RA cells and are associated with active enhancers and promoters and specific transcription factor binding motifs. Differentially marked genes are enriched for immunological and unexpected pathways, with "Huntington's Disease Signaling" identified as particularly prominent. We validate the relevance of this pathway to RA by showing that Huntingtin-interacting protein-1 regulates FLS invasion into matrix. This work establishes a high-resolution epigenomic landscape of RA and demonstrates the potential for integrative analyses to identify unanticipated therapeutic targets.
| S-EPMC5953939 | biostudies-literature
Project description:Fibroblast-like synoviocytes (FLS) play an important pathological role in persistent inflammatory joint diseases such as rheumatoid arthritis (RA). These cells have primarily been characterized in the RA synovial membrane. Here we aim to phenotypically and functionally characterize cultured synovial fluid-derived FLS (sfRA-FLS). Paired peripheral blood mononuclear cells (PBMC) and sfRA-FLS from patients with RA were obtained and monocultures of sfRA-FLS and autologous co-cultures of sfRA-FLS and PBMC were established. The in situ activated sfRA-FLS were CD34-, CD45-, Podoplanin+, Thymocyte differentiation antigen-1+. SfRA-FLS expressed uniform levels of NFкB-related pathway proteins and secreted several pro-inflammatory cytokines dominated by IL-6 and MCP-1. In a co-culture model with autologous PBMC, the ICAM-1 and HLA-DR expression on sfRA-FLS and secretion of IL-1β, IL-6, and MCP-1 increased. In vivo, human sfRA-FLS were cartilage invasive both at ipsilateral and contralateral implantation site. We conclude that, sfRA-FLS closely resemble the pathological sublining layer FLS subset in terms of surface protein expression, cytokine production and leukocyte cross-talk potential. Further, sfRA-FLS are comparable to tissue-derived FLS in their capabilities to invade cartilage at implantation sites but also spread tissue destruction to a distant site. Collectively, sfRA-FLS can serve as a an easy-to-obtain source of pathological sublining FLS in RA.
| S-EPMC8590001 | biostudies-literature
Project description:Large-conductance calcium-activated potassium channel (KCa1.1; BK, Slo1, MaxiK, KCNMA1) is the predominant potassium channel expressed at the plasma membrane of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs) isolated from the synovium of patients with RA. It is a critical regulator of RA-FLS migration and invasion and therefore represents an attractive target for the therapy of RA. However, the molecular mechanisms by which KCa1.1 regulates RA-FLS invasiveness have remained largely unknown. Here, we demonstrate that KCa1.1 regulates RA-FLS adhesion through controlling the plasma membrane expression and activation of β1 integrins, but not α4, α5, or α6 integrins. Blocking KCa1.1 disturbs calcium homeostasis, leading to the sustained phosphorylation of Akt and the recruitment of talin to β1 integrins. Interestingly, the pore-forming α subunit of KCa1.1 coimmunoprecipitates with β1 integrins, suggesting that this physical association underlies the functional interaction between these molecules. Together, these data outline a new signaling mechanism by which KCa1.1 regulates β1-integrin function and therefore invasiveness of RA-FLSs.-Tanner, M. R., Pennington, M. W., Laragione, T., Gulko, P. S., Beeton, C. KCa1.1 channels regulate β1-integrin function and cell adhesion in rheumatoid arthritis fibroblast-like synoviocytes.
| S-EPMC5503717 | biostudies-literature
Project description:ObjectiveSince previous studies indicate that metabolism is altered in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS), we undertook this study to determine if changes in the genome-wide chromatin and DNA states in genes associated with nutrient transporters could help to identify activated metabolic pathways in RA FLS.MethodsData from a previous comprehensive epigenomic study in FLS were analyzed to identify differences in genome-wide states and gene transcription between RA and osteoarthritis. We utilized the single nearest genes to regions of interest for pathway analyses. Homer promoter analysis was used to identify enriched motifs for transcription factors. The role of solute carrier transporters and glutamine metabolism dependence in RA FLS was determined by small interfacing RNA knockdown, functional assays, and incubation with CB-839, a glutaminase inhibitor. We performed 1 H nuclear magnetic resonance to quantify metabolites.ResultsThe unbiased pathway analysis demonstrated that solute carrier-mediated transmembrane transport was one pathway associated with differences in at least 4 genome-wide states or gene transcription. Thirty-four transporters of amino acids and other nutrients were associated with a change in at least 4 epigenetic marks. Functional assays revealed that solute carrier family 4 member 4 (SLC4A4) was critical for invasion, and glutamine was sufficient as an alternate source of energy to glucose. Experiments with CB-839 demonstrated decreased RA FLS invasion and proliferation. Finally, we found enrichment of motifs for c-Myc in several nutrient transporters.ConclusionOur findings demonstrate that changes in the epigenetic landscape of genes are related to nutrient transporters, and metabolic pathways can be used to identify RA-specific targets, including critical solute carrier transporters, enzymes, and transcription factors, to develop novel therapeutic agents.
| S-EPMC9246826 | biostudies-literature
Project description:ObjectiveUp-regulation of glucose metabolism has been implicated not only in tumor cell growth but also in immune cells upon activation. However, little is known about the metabolite profile in rheumatoid arthritis (RA), particularly in fibroblast-like synoviocytes (FLS). This study was undertaken to evaluate whether changes in glucose metabolism in RA FLS could play a role in inflammation and joint damage.MethodsSynovium and FLS were obtained from patients with RA and patients with osteoarthritis (OA). The rate of glycolysis after stimulation of FLS with lipopolysaccharide and platelet-derived growth factor BB was measured using glycolysis stress test technology. FLS function was evaluated using a glycolysis inhibitor, 2-deoxy-d-glucose (2-DG). After stimulation of the FLS, a migration scratch assay, MTT assay, and enzyme-linked immunosorbent assay were performed to measure the effect of 2-DG on FLS migration, viability of the FLS, and cytokine secretion, respectively. IRDye 800CW 2-DG was used to assess glucose uptake in the arthritic joints and stromal cells of mice after K/BxN mouse serum transfer. The mice were injected daily, intraperitoneally, with 3-bromopyruvate (BrPa; 5 mg/kg) to assess the effect of inhibition of glycolysis in vivo.ResultsCompared to human OA FLS, the balance between glycolysis and oxidative phosphorylation was shifted toward glycolysis in RA FLS. Glucose transporter 1 (GLUT1) messenger RNA (mRNA) expression correlated with baseline functions of the RA FLS. Glucose deprivation or incubation of the FLS with glycolytic inhibitors impaired cytokine secretion and decreased the rate of proliferation and migration of the cells. In a mouse model of inflammatory arthritis, GLUT1 mRNA expression in the synovial lining cells was observed, and increased levels of glucose uptake and glycolytic gene expression were detected in the stromal compartment of the arthritic mouse joints. Inhibition of glycolysis by BrPa, administered in vivo, significantly decreased the severity of arthritis in this mouse model.ConclusionTargeting metabolic pathways is a novel approach to understanding the mechanisms of disease. Inhibition of glycolysis may directly modulate synoviocyte-mediated inflammatory functions and could be an effective treatment strategy for arthritis.
| S-EPMC4963240 | biostudies-literature
Project description:The pathway of Janus tyrosine kinases (JAKs) has a central role in the pathogenesis of Rheumatoid Arthritis (RA) by regulating multiple immune functions and cytokine production. The JAK inhibitor tofacitinib is effective in RA patients not responding to methotrexate or TNF-inhibitors. Since hyperactive autophagy has been associated with impaired apoptosis of RA fibroblast-like synoviocytes (FLS), we aimed to investigate the role of tofacitinib in modulating autophagy and apoptosis in these cells. FLS isolated from RA biopsies were cultured with tofacitinib in presence of autophagy inducer rapamycin and in serum deprivation condition. Levels of autophagy, apoptosis, and citrullinated proteins were analyzed by western blot, flow cytometry, immunocytofluorescence, and Real-Time PCR. Rapamycin induced an increase in RA-FLS autophagy while the levels of autophagy marker LC3-II were reduced after in vitro treatment with tofacitinib. The analysis of autophagic flux by specific fluorescence dye confirmed the reduction of autophagy in RA FLS. The treatment with tofacitinib did not influence apoptosis of RA FLS. Modulation of the autophagic process by tofacitinib did not significantly change citrullination. The results of this study demonstrate that tofacitinib is able to modulate autophagy of FLS contributing to its effectiveness in RA patients.
| S-EPMC8928732 | biostudies-literature
Project description:ObjectiveRheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) derived from hip and knee have distinctive DNA methylation and transcriptome patterns in interleukin (IL)-6 signaling and Janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathways. To determine the functional effects of these joint-specific signatures, we evaluated how RA hip and knee FLS differ in their response to IL-6.MethodsHip or knee RA FLS were obtained after arthroplasty. Previously published datasets on epigenetic landscape of FLS were mined to identify joint-specific IL-6-related epigenomic differences. RNA sequencing was performed on five RA hip and five knee FLS treated with or without IL-6. Differential gene expression was determined using edgeR software. STAT3 phosphorylation was measured using bead assays. Sensitivity to tofacitinib was evaluated by measuring CCL2 inhibition using quantitative polymerase chain reaction.ResultsAssay for Transposase-Accessible Chromatin sequencing and histone chromatin immunoprecipitation sequencing datasets from RA FLS were analyzed to identify epigenomic differences between hip and knee. Differential chromatin accessibility was associated with IL-6,IL-6R, and JAK1 genes. H3K27ac was also differentially marked at other JAK-STAT-related genes, including STAT3-STAT5A region. Principal component analysis of RNA sequencing data confirmed segregation between RA hip and knee FLS under basal conditions, that persisted following IL-6 treatment. STAT3 phosphorylation after IL-6 was significantly higher in knee than hip FLS and was highly correlated with JAK1 protein levels. Knee FLS were less sensitive to the JAK inhibitor tofacitinib than hip FLS.ConclusionRA hip and knee FLS have distinct transcriptomes, epigenetic marks, and STAT3 activation patterns in the IL-6 pathway. These joint-specific differences might contribute to a differential clinical response in individual joints to targeted therapies such as JAK inhibitors.
| S-EPMC6917316 | biostudies-literature
Project description:Fibroblast-like synoviocytes (FLS) play a pathogenic role in rheumatoid arthritis (RA). STAT3 signaling is activated in FLS of RA patients (RA-FLS), which in turn causes RA-FLS hyperproliferation. RL is a traditional remedy for treating inflammatory diseases in China. It comprises Rosae Multiflorae Fructus and Lonicerae Japonicae Flos. A standardized ethanolic extract of RL (RLE) has been shown to exert anti-arthritic effects in collagen-induced arthritis (CIA) rats. Some constituents of RLE were reported to inhibit JAK2/STAT3 signaling in rat FLS. Here, we determined whether RLE inhibits FLS hyperproliferation, and explored the involvement of STAT3 signaling in this inhibition. In joints of CIA rats, RLE increased apoptotic FLS. In IL-6/sIL-6R-stimulated RA-FLS, RLE reduced cell viability and evoked cell apoptosis. In synovial tissues of CIA rats, RLE lowered the protein level of phospho-STAT3. In IL-6/sIL-6R-stimulated RA-FLS, RLE inhibited activation/phosphorylation of STAT3 and JAK2, decreased the nuclear localization of STAT3, and downregulated protein levels of Bcl-2 and Mcl-1. Over-activation of STAT3 diminished RLE's anti-proliferative effects in IL-6/sIL-6R-stimulated RA-FLS. In summary, RLE inhibits hyperproliferation of FLS in rat and cell models, and suppression of STAT3 signaling contributes to the underlying mechanisms. This study provides further pharmacological groundwork for developing RLE as a modern anti-arthritic drug.
| S-EPMC7886911 | biostudies-literature