siRNA inhibition of survivin gene expression in rheumatoid arthritis synovial fibroblast proliferation and apoptosis / 中国生化药物杂志

Objective To study the targeting survivin small interfering RNA ( siRNA ) to inhibit proliferation and apoptosis survivin gene expression in rheumatoid arthritis synovial fibroblasts ( RASFs) .Methods RA patients were isolated and cultured in vitro synovial fibroblasts ( RASFs) , designed and synthesized siRNA targeting survivin and negative control, by liposome transfection RASFs cell; real-time quantitative polymerase chain reaction (PCR) and Western blot RASFs detect mRNA expression and protein levels of survivin.Tetrazolium blue (MTT) assay of cell proliferation;TUNEL assay apoptosis.Results The experimental group compared with the negative control siRNA group and control group, 48h after transfection of synovial fibroblasts survivin mRNA and protein expression levels were significantly decreased ( P<0.05 ) .The experimental group compared with the negative control siRNA group and control group, synovial fibroblast proliferation after transfection significantly decreased ( P<0.05 ) . After the experimental group transfected 24h, 48h, 72h growth inhibition rates were (11.5 ±2.6)%, (26.2 ±3.4)%, (47.6 ±4.1)%, at 72 hours after transfection most significant.The rate of apoptosis in experimental group (23.87 ±1.6)%, significantly higher than the negative control group (9.72 ± 1.15)% and the control group (8.70 ±1.09)% (all P<0.05).Conclusion siRNA targeting survivin expression levels through reducing survivin, inhibit synovial fibroblast proliferation and promotes apoptosis.

Engagement of Toll-Like Receptor 3 Induces Vascular Endothelial Growth Factor and Interleukin-8 in Human Rheumatoid Synovial Fibroblasts

BACKGROUND/AIMS: Angiogenesis, which is a critical step in the initiation and progression of rheumatoid arthritis (RA), involves pro-angiogenic factors, including interleukin (IL)-8 and vascular endothelial growth factor (VEGF). We investigated the role of Toll-like receptor 3 (TLR3) in the regulation of pro-angiogenic factors in RA fibroblast-like synoviocytes (FLS). METHODS: FLS were isolated from RA synovial tissues and stimulated with the TLR3 ligand, poly (I:C). The levels of VEGF and IL-8 in the culture supernatants were measured using enzyme-linked immunosorbent assays, and the mRNA levels were assessed by semiquantitative reverse transcription-polymerase chain reaction. The expression patterns of VEGF and IL-8 in the RA synovium and osteoarthritis (OA) synovium were compared using immunohistochemistry. RESULTS: The expression levels of TLR3, VEGF, and IL-8 were significantly higher in the RA synovium than in the OA synovium. VEGF and IL-8 production were increased in the culture supernatants of RA FLS stimulated with poly (I:C), and the genes for these proteins were up-regulated at the transcriptional level after poly (I:C) treatment. Treatment with inhibitors of nuclear factor-kappaB (NF-kappaB), i.e., pyrrolidine dithiocarbamate and parthenolide, abrogated the stimulatory effect of poly (I:C) on the production of VEGF and IL-8 in RA FLS. CONCLUSIONS: Our results suggest that the activation of TLR3 in RA FLS promotes the production of proangiogenic factors, in a process that is mediated by the NF-kappaB signaling pathway. Therefore, targeting the TLR3 pathway may be a promising approach to preventing pathologic angiogenesis in RA.

Induction of Macrophage Migration Inhibitory Factor in ConA-Stimulated Rheumatoid Arthritis Synovial Fibroblasts through the P38 MAP Kinase-Dependent Signaling Pathway

BACKGROUND/AIMS: This study was undertaken to identify the intracellular signaling pathway involved in induction of macrophage migration inhibitory factor (MIF) in human rheumatoid arthritis (RA) synovial fibroblasts. METHODS: Human RA synovial fibroblasts were treated with concanavalin A (ConA), various cytokines, and inhibitors of signal transduction molecules. The production of MIF by synovial fibroblasts was measured in culture supernatants by ELISA. The expression of MIF mRNA was determined using reverse transcriptase polymerase chain reaction (RT-PCR) and real-time PCR. Phosphorylation of p38 mitogen-activated protein (MAP) kinase in synovial fibroblasts was confirmed using Western blotting. The expression of MIF and p38 MAP kinase in RA synovium was determined using dual immunohistochemistry. RESULTS: The production of MIF by RA synovial fibroblasts increased in a dose-dependent manner after ConA stimulation. MIF was also induced by interferon-gamma, CD40 ligand, interleukin-15, interleukin-1beta, tumor necrosis factor-alpha, and transforming growth factor-beta. The production of MIF by RA synovial fibroblasts was significantly reduced after inhibition of p38 MAP kinase. The expression of MIF and p38 MAP kinase was upregulated in the RA synovium compared with the osteoarthritis synovium. CONCLUSIONS: These results suggest that MIF production was induced through a p38 MAP-kinase-dependent pathway in RA synovial fibroblasts.

IL-10 Inihibits VEGF Production in the Synovial Fibroblasts of RA Patients via Down-regulation of the ERK and AP-1 Pathways / 대한류마티스학회지

OBJECTIVE: Interleukin (IL)-10 has been demonstrated to have anti-inflammatory and anti-tumour activity. Because aberrant angiogenesis is a significant pathogenic component of tumor growth and chronic inflammation, we investigated the effect of IL-10 on the production of vascular endothelial growth factor (VEGF) by the synovial fibroblasts derived from the patients with rheumatoid arthritis (RA). METHODS: Fibroblast-like synoviocytes (FLS) were cultured with transforming growth factor (TGF-beta) alone or with IL-10. The level of VEGF was measured by RT-PCR and enzyme-linked immunosorbent assay (using the 24, 48 and 72 h culture supernatants). The FLSs were cultured with TGF-b for 48 hr in the presence of PD98059 (an ERK inhibitor), curcumin and SP600125 (a JNK and Ap-1 inhibitor, respectively). The level of VEGF in the supernatants was measured by ELISA. Cell viability was assessed using MTT assay. The expressions of VEGF, ERK, AP-1 and IL-10 in the synovial tissue were quantified by immunohistochemistry. RESULTS: IL-10 exhibited the inhibitory effect on VEGF production when the FLSs were stimulated with TGF-beta. ERK and AP-1 inhibitors inhibited the TGF-beta induced VEGF production. Moreover, TGF-beta increased the phosphorylation of ERK and C-Jun, which was significantly inhibited by the IL-10. CONCLUSION: IL-10 may exert an antiangiogenic effect by inhibiting the ERK- and AP-1 mediated VEGF expression in rheumatoid synovial fibroblasts.

The Effects of Cartilage Extract on the Rheumatoid Synovial Cells / 대한정형외과연구학회지

PURPOSE: Rheumatoid arthritis is a chronic multisystemic disease involving joints. It has been difficult to explain why the inflammatory responses of the rheumatoid arthritis (RA) are mostly limited to the joints. To explain this localizing phenomenon, we hypothetized that the cartilage, which exists in the joints, maintaines the activated status of synovial membrane and provides local specific environment for the maintenance of inflammatory response of arthritis. MATERIALS AND METHODS: Synovial fibroblast were proliferated with and without the addition of cartilage tissues, chondrocytes, type II collagens to the growth media respectively. MTS assay (CellTiter 96(R) AQ One Solution Cell Proliferation Assay) was done at 1st, 3rd, 4th, 5th, 6th, 7th, 9th and 11th days to evaluate the proliferation of the cells. The data were assessed with one-way ANOVA and unpaired t-test. Results : In a group where 0.1 g/ml of cartilage tissues were added to the growth media, the synovial fibroblasts from the normal people and rhematoid arthritic patients showed higher amount of proliferation from the 3rd day compared to the control group. When 0.02 g/ml of cartilage tissues were added, the synovial fibroblasts from the normal people showed higher amount of proliferation from the 4th day and those from RA patients from the 3rd day compared to the control group (p< 0.05). There was no significant statistical difference according to the concentration of the cartilage tissues.In a group where chondrocyte extract was added to the growth media, the synovial fibroblasts from the normal people showed higher amount of proliferation from the 4th day and those from RA patients from the 3rd day compared to the control group (p< 0.05). In a group where type II collagens were added to the growth media, the synovial fibroblasts from normal people and RA patients showed no significant statistical differences compared to the control group. CONCLUSION: The cartilage tissue is important in maintenance of synovial fibroblast proliferation. Of the cartilage tissue, chondrocyte, not extracellular type II collagen, played a major role. But further study is needed about what component of the chondrocyte extract maintaines the proliferation of synovial fibroblast.

Selective Susceptibility of Synovial Fibroblasts Isolated from Patients with Rheumatoid Arthritis to TRAIL -induced Cell Death / 체질인류학회지

TNF -related apoptosis inducing ligand (TRAIL) is a member of TNF ligand superfamily. TRAIL transduces death signal through two distinct receptors, TRAILR -1I and TRAILR -2, while the engagement of TRAILR -3 and TRAILR -4 interferes with TRAIL -induced apoptosis. The profile of TRAILR expression has been reported to be a mechanism by which transformed cells undergo apoptosis in response to TRAIL while normal cells do not. Rheumatoid arthritis (RA) is an inflammatory autoimmune disease which is characterized by the hyperplasia of synovial membrane. The dysregulation of apoptosis in synoviocytes has been suggested to contribute to synovial hyperplasia. Synovial fibroblasts obtained from patients with RA have been reported to exhibit several semi - transformed aspects. To investigate whether RA synovial fibroblasts acquire the susceptibility to TRAIL -induced apoptosis, synovial fibroblast lines obtained from 2 RA patients and two osteoarthritis (OA) patients were cultured in the presence of recombinant human TRAIL and followed by MTT assay. TRAIL treatment resulted in a significant decrease in the viability of both lines of RA cells, indicating TRAIL -induced cell death of RA synovial fibroblasts, whereas OA synovial fibroblasts and normal human dermal fibroblasts were either resistant or less sensitive to TRAIL as compared with RA synovial fibroblasts. In RT -PCR analyses, the expression levels of TRAILR 4 in RA synovial fibroblasts were lower than in OA synovial fibroblasts, while other receptors in both cell lines were expressed at comparable levels. Immunohistochemical studies showed that in RA synovial tissues TRAILR -3cells were mainly leukocyte infiltrates, implying that such leukocyte infiltrates play a role in the perpetuation of the disease. Taken together, these results suggest that RA synovial fibroblasts acquire the susceptibility to TRAIL -induced cell death during disease progression and this death signal may be regulated by, at least in part, differential expression of TRAILR -4 molecule.