Targeted inhibition of GRK2 kinase domain by CP-25 to reverse fibroblast-like synoviocytes dysfunction and improve collagen-induced arthritis in rats

Rheumatoid arthritis (RA) is an autoimmune disease and is mainly characterized by abnormal proliferation of fibroblast-like synoviocytes (FLS). The up-regulated cellular membrane expression of G protein coupled receptor kinase 2 (GRK2) of FLS plays a critical role in RA progression, the increase of GRK2 translocation activity promotes dysfunctional prostaglandin E4 receptor (EP4) signaling and FLS abnormal proliferation. Recently, although our group found that paeoniflorin-6'-

The study of the effect of Focal adhesion kinase and p53 in rheumatoid arthritis fibroblast like synoviocytes on cell proliferation / 中华风湿病学杂志

Objective To investigate the expression of Focal adhesion kinase (FAK) and p53 in rheumatoid arthritis (RA) Fibroblast Like Synoviocytes and to explore the pathogenesis of RA.This study also studied the effect of FAK and p53 on the proliferation of Fibroblast Like Synoviocytes in RA on order to identify new target for the treatment of RA.Methods Synovial tissue from RA patients were cultured in vitro;FLS were cultured in TNF-α (10 ng/ml) and different density of protease inhibitor (MG-132) (1,5,10,20 μmol/L)for 48 h.Then the level of mRNA of both FAK and p53 in each group was tested by real time polymerase chain reaction (RT-PCR).FLS were cultured with different density of protease inhibitor (MG-132) (1,5,10,20μmol/L) for 24 h,48 h,72 h,96 h then the cell proliferation of each group were tested.ANOVA was used to compare the means between groups.Results ① After TNF-α stimulating,the level of FAK mRNA was higher than the control group [(1.48±0.09 vs(1.03±0.33),F=7.807,P＜0.05).Compared with the control group,the level of p53 mRNA decreased [(0.97:±0.03) vs (1.30±0.39),F=19.933,P＞0.05).② After stimulated with different density of MG-132,the level of p53 mRNA was higher than the control group [(3.12±0.72) vs (1.30±0.39),F=19.933,P＜0.05),and the 5μ mol/L group was the highest of them.Compared with the control group,the level of FAK mRNA decreased [(0.93±0.20) vs (1.03±0.33),F=7.807,P＞0.05).③ After stimulated with different density of MG-132,the proliferation of FLS was slower than the control group (24 h F=16.654,P＜0.05;48 hF=13.652,P＜0.05;72 h F=72.999,P＜0.05;96 h F=51.533,P＜0.05).Conclusion In vitro,after stimulated with TNF-α,the level of mRNA of Focal adhesion kinase is increased,while the level of that decreases after MG-132 stimulation.Focal adhesion kinase is involved in the pathogenesis of rheumatoid arthritis.In vitro,after MG-132 stimulating,the level of mRNA of p53 is increased.p53 inhibits the excessive proliferation of RA synovial fibroblast cells and plays an important role in the pathogenesis of rheumatoid arthritis.

Influencing factors on the primary cultur e of mouse fibroblast-like synov iocyt es / 医学研究生学报

Obej ctive Knockout mice are widely used in the studies of joint diseases .This article investigated the effects of joint processing methods , collagenase types ,and collagenase digestion time on the number of primary fibroblast -like synoviocytes (FLSs) obtained from mice. Methods The hind legs of 6 of the 12 male mice were cut open from the hip joints , but not those of the other 6.FLSs were isolated using the type-Ⅳcollagenase digestion method and purified by differential digestion .Cell morphology was observed under the inverted microscope .The type, viability, and purity of the cells were determined by flow cytometry . Rse ults Significantly fewer FLSs were obtained from the mice with the hind legs cut open ( 19 133 ±115 ) than from those without (24 933 ± 503) (P<0.05).The numbers of FLSs collected from the cell suspension at 1, 2, 3, 4, 5,6 , and 7 hours after digestion were 700 ±300 , 600 ±100 , 15 200 ±900 , 5100 ±800 ,2700 ±300 , 900 ±200, and 300 ±100, respectively, the highest at 3 hours. There were statistically significant differences in the total number of FLSs obtained by type-Ⅳ and type-Ⅱ collagenase digestions (24900 ±500v s 18 100 ±400, P<0.05). Conclusion For in virt o culture of primary mouse FLSs, it is recommended that the hip joints be not cut open, and type-Ⅳcollagenase be used with cell sus-pension at 2-6hours after digestion .

Application of Induced Pluripotent Stem Cells in Rheumatology

Since induced pluripotent stem cell (iPSC) was first introduced by Yamanaka in 2006, it took only six years to win a Nobel Prize for his pioneering work. It is unusual to win a Nobel Prize for such recent research with a short history. Many scientists and clinicians are interested in iPSC for its potential application. Significant progression in this field has been made, while there remain many hurdles to overcome for application of iPSC technique in real clinics. In this review, the concept of reprogramming and the basic techniques of iPSC generation will be discussed for the reader's convenience, followed by discussion of recent progress, followed by the topics of "disease modeling" and "cell therapy" with iPSC in the second half of this article. Several examples of rheumatologic application of iPSC will be provided in the main text. If rheumatologists could understand the merits and potentials of iPSC, opportunities for innovative research and therapy can be expanded.

Mechanism of TWEAK on the synthesis of MMP-9 in fibroblast-like synoviocytes of rheumatoid arthritis / 中华微生物学和免疫学杂志

Objective To discuss the role of p38MAPK signal pathway in the process of TWEAK inducing rheumatoid arthritis(RA) fibroblast-like synoviocyte(FLS) to synthesize MMP-9 and look for a new target for RA treatment. Methods RA FLS were primarily cultured and stimulated with TWEAK. Western blot was used to detect the expression of p-p38MAPK and p65 in RA FLS. FLS were pretreated by SB203580 or not. ELISA was used to detect the concentration of MMP-9 in cell-cultured fluid. The mRNA expression of MMP-9 was measured by RT-PCR. Results TWEAK( 100 ng/ml) can make p38MAPK phosphorylated and increased the expression of p65 protein in the cell nucleus. SB203580 can partially inhibit the expression of MMP-9 and MMP-9 mRNA produced by RA FLS which is induced by TWEAK. Conclusion TWEAK induced RA FLS to synthesize MMP-9, in that process, the p38MAPK signal trausduction pathway was in active state, and induced the expression of NF-κB.

Study on the Apoptosis of Rheumatoid Arthritis Synoviocyte Induced by Arsenic Trioxide / 中国康复理论与实践

@# Objective To study the apoptosis of rheumatoid arthritis synoviocyte induced by arsenic trioxide.Methods Human rheumatoid arthritis fibroblast-like synoviocytes (HFLS-RA) were divided into control group and arsenic trioxide group. After 72 hours, pathology changes were observed and MTT tests were used to investigate apoptosis levels induced by arsenic trioxide.Results Arsenic trioxide can induce the apoptosis of rheumatoid arthritis synoviocyte, depending on time and dose.Conclusion Arsenic trioxide may induce apoptosis of rheumatoid arthritis synoviocyte.

beta ig-h3-Mediated Adhesion of Fibroblast-Like Synoviocytes in Rheumatoid Arthritis / 대한류마티스학회지

OBJECTIVE: beta ig-h3 is an extracellular matrix protein, which is overexpressed in synovial tissues of rheumatoid arthritis (RA) similar to adhesive glycoproteins. We sought to evaluate the compensatory role of beta ig-h3 with adhesive glycoproteins in mediating the adhesion of fibroblast- like synoviocytes (FLS) and to confirm the inhibitory effect of YH18 peptide of the 2nd fas-1 domain in beta ig-h3-mediated adhesion. METHODS: The adhesion of FLS isolated from synovial tissues of RA, was evaluated in 96 well microtiter plate coated with matrix proteins. Inhibitory effect of YH18 peptides from the 2nd and 4th fas-1 domains was estimated in beta ig-h3-mediated adhesion of FLS. RESULTS: The adhesion of FLS on beta ig-h3 was weaker than that of fibronectin and vitronectin. The beta ig-h3-mediated adhesion was enhanced by the stimulation with phorbol myristate acetate (PMA), but not by cytokines and growth factors. Combination of fibronectin with beta ig-h3 synergistically enhanced the adhesion of FLS, in contrast to the additive effect of vitronectin combined with beta ig-h3. YH18 peptide of the 2nd fas-1 domain did not block the beta ig-h3-mediated adhesion of FLS. CONCLUSION: Our results reveal that beta ig-h3 may regulate the adhesion of FLS through the interaction with adhesive glycoproteins and confirm that the essential motifs mediating adhesion on beta ig-h3 are different according to the type of cells.

Regulation of betaig-h3 Production in Rheumatoid Synovitis by Inflammatory Mediators / 대한류마티스학회지

OBJECTIVE: To investigate the expression pattern of transforming growth factor-beta-inducible gene-h3 (betaig-h3) within rheumatoid synovial tissue and the regulation of betaig-h3 synthesis in fibroblast-like synoviocyte (FLS). METHODS: Synovial tissues obtained from patients with rheumatoid arthritis and osteoarthritis were obtained during joint replacement surgery. betaig-h3 expression was evaluated with immunohistochemical stain. FLS was isolated from synovial tissues and stimulated with cytokines including TGF-beta, TNF-alpha, IL-1beta, IFN-gamma, IL-6, IL-4, and IL-10. betaig-h3 synthesis was measured using semiquantitative RT-PCR, ELISA, immunofluorescence stain, and flow cytometry. RESULTS: Expression of betaig-h3 was diffuse and abundant in both lining and sublining layers of rheumatoid synovium, which was more prominent than those of osteoarthritis. Production of betaig-h3 in FLS was regulated by TGF-beta1 in a dose-dependent manner and was highest at 5 ng/mL of TGF-beta1. TNF-alpha and IL-1beta upregulated the production of betaig-h3 from FLS synergistically with TGF-beta1 but other cytokines such as IL-4, IL-6, IL-10 did not affect. betaig-h3 synthesis was efficiently inhibited by dexamethasone at higher dose (100 nM) but not by cyclosporine-A. CONCLUSION: Production of betaig-h3, which is highly upregulated in rheumatoid synovitis, is differentially regulated by inflammatory cytokines.

Reciprocal Activation of Fibroblast-like Synoviocyte and Type II Collagen-reactive T cell in Rheumatoid Arthritis / 대한류마티스학회지

OBJECTIVE: To investigate the interaction between type II collagen (CII)-reactive T cell and fibroblast-like synoviocyte in rheumatoid arthritis (RA). METHODS: Peripheral blood T cells from RA patients were cultured with bovine CII and analyzed by flow cytometry. After co-culture with CII-reactive T cells and fibroblast-like synoviocytes (FLS), the expression of cytokines (IL-15 and TNF-alpha from FLS, IFN-gamma and IL-17 from CII-reactive T cells) were determined by ELISA and RT-PCR. RESULTS: CII-reactive T cells expressed CD69, one of the early activation markers, and produced significant amount of IFN-gamma, and proliferated. IL-15 and TNF-alpha expression from FLS were significantly elevated when co-culture with CII-reactive T cells and inhibited by physical interruption of cell-to-cell contact or anti-CD40 antibody. IFN-gamma and IL-17 expression from CII-reactive T cells were also significantly elevated when co-culture with FLS and inhibited by anti-IL-15 monoclonal antibody. CONCLUSIONS: CII-reactive T cells can activate FLS to secret proinflammatoy cytokines and interactions between these two cells drive further activation of each other. These data suggest that CII-reactive T cell may play a important role in pathogenesis of RA.

In Vitro Effects of Ultrasound on Fibroblast Like Synoviocytes from Rheumatoid Arthritis

OBJECTIVE: Ultrasound has been therapeutically applied for pain control in rheumatoid arthritis although little physiologic effects of sonication on rheumatoid tissue were known. This investigation was conducted to determine the effects of sonication on the cell proliferation and matrix metalloproteinase (MMP) production of cultured fibroblast like synoviocytes (FLS) derived from synovial tissues of rheumatoid arthritis. METHOD: Pulsed ultrasound (1.0 MHZ, 20 msec on, 80 msec off) with varying intensities (0, 0.1, 0.25, 0.5, 0.75, 1.0 W/cm2) was applied to experimental cell groups growing as monolayers in culture plates for varying durations (0, 30, 90, 180 seconds) in the presence and absence of interleukin-1beta (IL-1beta). RESULTS: There were no significant differences in thymidine incorporation between 0, 30, 90 and 180 second sonication groups with 0.5 W/cm2 after 1 day and 2 days. There were no significant differences in thymidine incorporation between 0, 0.1, 0.25, 0.5, 0.75, 1.0 W/cm2 sonication groups 1 day and 2 days after 90 second sonication. There were significant increase in MMP-1 (p=0.025) and MMP-3 production (p=0.000) of FLS after sonication in the absence of IL-1beta but there were no significant differences in MMP-1 and MMP-3 production in the presence of IL-1beta. And MMP-1 and MMP-3 production were increased significantly in the presence of IL-1beta but not than in the absence of IL-1beta. CONCLUSION: While comparisons made between a limited number of FLS cell lines must be open to question, the overall consistency of the findings suggest sonication with nonthermal effect is not the contraindication in rheumatoid arthritis treatment but further study is needed in vivo in animal and in clinical studies.