Invasion Of Synoviocytes Research Articles

Columbianadin ameliorates rheumatoid arthritis by attenuating synoviocyte hyperplasia through targeted vimentin to inhibit the VAV2/Rac-1 signaling pathway

IntroductionRheumatoid arthritis (RA) is an autoimmune disease pathologically characterized by synovial inflammation. The abnormal activation of synoviocytes seems to accompany the progression of RA. The role and exact molecular mechanism in RA of columbianadin (CBN) which is a natural coumarin is still unclear. ObjectivesThe present research aimed to investigate the effect of vimentin on the abnormal growth characteristics of RA synoviocytes and the targeted regulatory role of CBN. MethodsCell migration and invasion were detected using the wound healing and transwell method. Mechanistically, the direct molecular targets of CBN were screened and identified by activity-based protein profiling. The expression of relevant proteins and mRNA in cells and mouse synovium was detected by western blotting and qRT-PCR. Changes in the degree of paw swelling and body weight of mice were recorded. H&E staining, toluidine blue staining, and micro-CT were used to visualize the degree of pathological damage in the ankle joints of mice. Small interfering RNA and plasmid overexpression of vimentin were used to observe their effects on MH7A cell proliferation, migration, apoptosis, and downstream molecular signaling. ResultsThe TNF-α-induced proliferation and migration of MH7A cells could be significantly repressed by CBN (25,50 μM), and the expression of apoptosis and autophagy-associated proteins could be modulated. Furthermore, CBN could directly bind to vimentin and inhibit its expression and function in synoviocytes, thereby ameliorating foot and paw swelling and joint damage in CIA mice. Silencing and overexpression of vimentin might be involved in developing RA synovial hyperplasia and invasive cartilage by activating VAV2 phosphorylation-mediated expression of Rac-1, which affects abnormal growth characteristics, such as synoviocyte invasion and migration. ConclusionCBN-targeted vimentin restrains the overactivation of RA synoviocytes thereby delaying the pathological process in CIA mice, which provides valuable targets and insights for understanding the pathological mechanisms of RA synovial hyperplasia.

Sonic Hedgehog Regulates Proliferation, Migration and Invasion of Synoviocytes in Rheumatoid Arthritis via JNK Signaling.

Activated fibroblast-like synoviocytes (FLSs) play a central role in the formation of synovial pannus and joint destruction in rheumatoid arthritis (RA). Targeting FLSs could be a potential therapeutic strategy. The objective of this study is to explore the role of c-Jun N-terminal kinase (JNK) in proliferation, migration and invasion of FLSs promoted by the sonic hedeghog (SHH) signaling pathway in patients with RA. Activation of SHH signaling was evaluated by real-time PCR and Western Blot. Levels of phosphorylation of JNK and c-Jun were detected by Western Blot. FLSs proliferation was quantified by Cell Counting Kit-8 (CCK-8) assay and flow cytometry. Cell migration and invasion were assessed by wound healing assay and Transwell chamber assay. Invasiveness of FLSs in vivo was evaluated using a humanized synovitis animal model. We observed that treatment of SHH agonist (SAG) significantly increased the levels of phosphorylation of JNK and c-Jun, while SHH antagonist (cyclopamine) significantly decreased the expression of phospho-JNK and phospho-c-Jun in FLSs. The elevated level of phospho-c-Jun stimulated by SAG was decreased in the presence of JNK inhibitor (SP600125) (P < 0.001). FLSs proliferation, migration and invasion were promoted by SHH agonist (P < 0.05). However, the enhanced aggressiveness of FLSs was abolished in the presence of JNK inhibitor (P < 0.05). In vivo study showed that the invasion of FLSs into cartilage was increased by SHH overexpression and the excessive invasiveness was inhibited by blockade of JNK signaling (P < 0.01). These results suggest that JNK is one of the downstream molecules mediating the effect of SHH signaling in FLSs. These findings indicate that SHH-JNK signaling could be a potential therapeutic target to suppress the aggressiveness of FLSs and prevent articular damage of RA.

LAIR-1 shedding from human fibroblast-like synoviocytes in rheumatoid arthritis following TNF-α stimulation.

This study examined the expression of the inhibitory receptor, leucocyte-associated immunoglobulin (Ig)-like receptor-1 (LAIR-1) in fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) patients to investigate its potential role in the modulation of inflammatory cytokines, matrix metalloproteinases (MMPs) and invasiveness of synoviocytes. LAIR-1 expression in synovial tissues from RA patients, osteoarthritis patients and healthy donors was analysed by immunohistochemistry. The membrane-bound form (mLAIR-1) was detected by flow cytometry. Factors involved in inflammation and MMP activity in FLS were analysed by quantitative polymerase chain reaction (qPCR). LAIR-1 expression was higher in the synovia of the RA patients than those of the osteoarthritis patients. Co-immunostaining of vimentin/LAIR-1 demonstrated that LAIR-1 was localized mainly in FLS in the RA patients. Surprisingly, primary FLS isolated from the RA patients had low levels of mLAIR-1 expression, with cytoplasmic distribution. The extracellular domain of LAIR-1 was shed from the cell surface in response to tumour necrosis factor (TNF)-α, and thisprocess could be blocked by serine protease inhibitors. Additional experiments indicated that LAIR-1 over-expression reduced FLS invasion considerably, which reduced simultaneously the mRNA levels of interleukin (IL)-6, IL-8 and MMP-13 in the presence of TNF-α. Our study demonstrated that LAIR-1 is an anti-inflammatory molecule, and was up-regulated in FLS in the RA patients; however, cell-surface LAIR-1 could be shed from cells in the inflammatory microenvironment in RA. This may weaken the interaction of LAIR-1 with its ligand, thus reducing the anti-inflammatory effects of LAIR-1. These findings suggested that LAIR-1 may be an important factor involved in the mediation of the progressive joint destruction in RA.

A study of Sirt1 regulation and the effect of resveratrol on synoviocyte invasion and associated joint destruction in rheumatoid arthritis.

The aim of the current study was to investigate the role and mechanism of sirtuin 1 (Sirt1) in the regulation of synovial cell invasion and joint destruction in rheumatoid arthritis (RA). The Sirt1 protein and mRNA levels in fibroblast-like synoviocytes (FLS) isolated from RA synovial tissues were compared with normal tissues by western blot and reverse transcription-polymerase chain reaction. RA FLS were then treated with the Sirt1 agonist resveratrol (1, 3 and 10 µg/ml) for 48 h, and their invasiveness and expression of matrix metalloproteinase (MMP) 1 and MMP13 protein and mRNA were measured. Furthermore, a collagen-induced arthritis (CIA) rat model was established and the rats were divided into a model group, and low- and high-dose resveratrol (2.5 and 10 mg/kg/day) groups to receive an intraperitoneal injection of resveratrol for 42 consecutive days. The joint morphology, arthritis index (AI), and MMP1 and MMP13 expression in synovial tissues was monitored. The Sirt1 protein and mRNA levels in RA FLS were significantly lower compared with normal FLS (P<0.01). The resveratrol treatment significantly inhibited the invasive ability of RA FLS (P<0.01) and reduced MMP1 and MMP13 expression (P<0.01). The AI in low- and high-dose groups was significantly lower compared with the model group from day 28 (P<0.01). Resveratrol also reduced the swelling and damage and decreased MMP1 and MMP13 expression levels in CIA rats (P<0.01). The resveratrol-induced upregulation of Sirt1 in RA FLS may significantly inhibit the invasion of these cells and reduce the degree of joint damage, which may be mediated through the inhibition of MMP1 and MMP13 expression. The present results suggested a regulatory role for Sirt1 in RA pathogenesis, and demonstrated the beneficial effects of resveratrol, which may have potential as an alternative therapeutic strategy for the treatment of patients with RA.

Interleukin-21 induces migration and invasion of fibroblast-like synoviocytes from patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial fibroblast hyperplasia and bone erosion. Fibroblast-like synoviocytes (FLS) play a pivotal role in RA pathogenesis through aggressive migration and matrix invasion, and certain proinflammatory cytokines may affect synoviocyte invasion. Whether interleukin (IL)-21 influences this process remains controversial. Here, we evaluated the potential regulatory effect of IL-21 on the migration, invasion and matrix metalloproteinase (MMP) expression in RA-FLS. We found that IL-21 promoted the migration, invasion and MMP (MMP-2, MMP-3, MMP-9, MMP-13) production in RA-FLS. Moreover, IL-21 induced activation of the phosphoinositide 3-kinase (PI3K), signal transducer and activator of transcription-3 (STAT-3) and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) pathways, and blockage of these pathways [PI3K/protein kinase B (AKT) inhibitor LY294002, STAT-3 inhibitor STA-21 and ERK1/2 inhibitor PD98059] attenuated IL-21-induced migration and secretion of MMP-3 and MMP-9. In conclusion, our results suggest that IL-21 promotes migration and invasion of RA-FLS. Therefore, therapeutic strategies targeting IL-21 might be effective for the treatment of RA.

Phosphoinositide 3-Kinase δ Regulates Migration and Invasion of Synoviocytes in Rheumatoid Arthritis

Cartilage destruction mediated by invasive fibroblast-like synoviocytes (FLS) plays a central role in pathogenesis of rheumatoid arthritis (RA). Increased cell migration and degradation of extracellular matrix are fundamental to these processes. The class I PI3Ks control cell survival, proliferation, and migration, which might be involved in cartilage damage in RA. PI3Kδ isoform was recently identified as a key regulator of FLS growth and survival, suggesting that it could contribute to synoviocyte aggressive behavior. Therefore, we assessed the role of PI3Kδ in RA synoviocyte migration and invasion. We observed that PI3Kδ inhibition or small interfering RNA knockdown decreased platelet-derived growth factor (PDGF)-mediated migration and invasion of FLS. We then showed that PI3Kδ regulates the organization of actin cytoskeleton and lamellipodium formation during PDGF stimulation. To gain insight into molecular mechanisms, we examined the effect of PI3Kδ inhibition on Rac1/PAK, FAK, and JNK activation. Our studies suggest that Rac1/PAK is key target of PDGF-mediated PI3Kδ signaling, whereas FAK and JNK are not involved. Thus, PI3Kδ contributes to multiple aspects of the pathogenic FLS behavior in RA. These observations, together with previous findings that PI3Kδ regulates FLS growth and survival, suggest that PI3Kδ inhibition could be chondroprotective in RA by modulating synoviocyte growth, migration, and invasion.

Brucella abortus Invasion of Synoviocytes Inhibits Apoptosis and Induces Bone Resorption through RANKL Expression

Arthritis is one of the most common complications of human active brucellosis, but its pathogenic mechanisms have not been completely elucidated. In this paper, we describe the role of synoviocytes in the pathogenesis of brucellar arthritis. Our results indicate that Brucella abortus infection inhibited synoviocyte apoptosis through the upregulation of antiapoptotic factors (cIAP-2, clusterin, livin, and P21/CIP/CDNK1A). In contrast, infection did not change the expression of proteins that have been involved in apoptosis induction such as Bad, Bax, cleaved procaspase 3, CytC, and TRAIL, among others; or their expression was reduced, as occurs in the case of P-p53(S15). In addition, B. abortus infection induced upregulation of adhesion molecules (CD54 and CD106), and the adhesion of monocytes and neutrophils to infected synoviocytes was significantly higher than to uninfected cells. Despite this increased adhesion, B. abortus-infected synoviocytes were able to inhibit apoptosis induced by supernatants from B. abortus-infected monocytes and neutrophils. Moreover, B. abortus infection increased soluble and membrane RANKL expression in synoviocytes that further induced monocytes to undergo osteoclastogenesis. The results presented here shed light on how the interactions of B. abortus with synovial fibroblasts may have an important role in the pathogenesis of brucellar arthritis.

Liver X Receptor Regulates Rheumatoid Arthritis Fibroblast-like Synoviocyte Invasiveness, Matrix Metalloproteinase 2 Activation, Interleukin-6 and CXCL10

Fibroblast-like synoviocyte (FLS) invasiveness correlates with articular damage in rheumatoid arthritis (RA), yet little is known about its regulation. In this study we aimed to determine the role of the nuclear receptor liver X receptor (LXR) in FLS invasion. FLS were isolated from synovial tissues obtained from RA patients and from DA rats with pristane-induced arthritis. Invasion was tested on Matrigel-coated chambers in the presence of the LXR agonist T0901317, or control vehicle. FLS were cultured in the presence or absence of T0901317, and supernatants were used to quantify matrix metalloproteinase 1 (MMP-1), MMP-2, MMP-3, interleukin-6 (IL-6), tumor necrosis factor-α and C-X-C motif chemokine ligand 10 (CXCL10). Nuclear factor-κB (NF-κB) (p65) and Akt activation, actin cytoskeleton, cell morphology and lamellipodia formation were also determined. The LXR agonist T0901317 significantly reduced DA FLS invasion by 99% (P ≤ 0.001), and RA FLS invasion by 96% (P ≤ 0.001), compared with control. T0901317-induced suppression of invasion was associated with reduced production of activated MMP-2, IL-6 and CXCL10 by RA FLS, and with reduction of actin filament reorganization and reduced polarized formation of lamellipodia. T0901317 also prevented both IL-1β-induced and IL-6-induced FLS invasion. NF-κB (p65) and Akt activation were not significantly affected by T0901317. This is the first description of a role for LXR in the regulation of FLS invasion and in processes and pathways implicated both in invasion as well as in inflammatory responses. These findings provide a new rationale for considering LXR agonists as therapeutic agents aimed at reducing both inflammation and FLS-mediated invasion and destruction in RA.

Nitric Oxide-Driven Hypoxia Initiates Synovial Angiogenesis, Hyperplasia and Inflammatory Lesions in Mice

BackgroundRheumatoid arthritis (RA) is an inflammatory articular disease with cartilage and bone damage due to hyperplasic synoviocyte invasion and subsequent matrix protease digestion. Although monoclonal antibodies against tumor necrosis factor alpha (TNFα) have been approved for clinical use in patients with RA, desired therapeutic regimens suitable for non-responders are still unavailable because etiological initiators leading to RA remain enigmatic and unidentified.Methodology/Principal FindingsBacteria-induced arthritis (BIA) that simulates collagen-induced arthritis (CIA) is developed in mice upon daily live bacterial feeding. The morphological lesions of paw erythema and edema together with the histological alterations of synovial hyperplasia and lymphocytic infiltration emerge as the early-phase manifestations of BIA and CIA. Bacteria- or collagen-mediated global upregulation of pro-inflammatory cytokines is accompanied by the burst of nitric oxide (NO). Elevation of the serum NO level is correlated with decline of the blood oxygen saturation percentage (SpO2), reflecting a hypoxic consequence during development towards arthritis. NO-driven hypoxia is further evident from a positive relationship between NO and lactic acid (LA), an end product from glycolysis. Upregulation of hypoxia inducible factor 1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF) validates hypoxia-induced angiogenesis in the inflamed synovium of modeling mice. Administration of the NO donor compound sodium nitroprusside (SNP) causes articular inflammation by inducing synovial hypoxia. Anti-bacteria by the antibiotic cefotaxime and/or the immunosuppressant rapamycin or artesunate that also inhibits nitric oxide synthase (NOS) can abrogate NO production, mitigate hypoxia, and considerably ameliorate or even completely abort synovitis, hence highlighting that NO may serve as an initiator of inflammatory arthritis.Conclusions/SignificanceLike collagen, bacteria also enable synovial lesions via upregulating pro-inflammatory cytokines, triggering NO production, driving hypoxic responses, and inducing synovial angiogenesis and hyperplasia, suggesting that sustained infection might be, in part, responsible for the onset of synovitis and arthritis in mice.

Expression of CD147 (EMMPRIN) on neutrophils in rheumatoid arthritis enhances chemotaxis, matrix metalloproteinase production and invasiveness of synoviocytes

The occurrence of neutrophils at the pannus-cartilage border is an important phenomenon for understanding the pathogenesis of rheumatoid arthritis (RA). Matrix metalloproteinases (MMPs) are predominant enzymes responsible for the cartilage degradation. The present article studied the expression of CD147 on neutrophils and its potential role in neutrophil chemotaxis, MMPs production and the invasiveness of fibroblast-like synoviocytes (FLS). The results of flow cytometry revealed that the mean fluorescence intensity of CD147 expression on neutrophils of peripheral blood from RA patients was higher than that in healthy individual. The potential role of CD147 in cyclophilin A (CyPA)-mediated cell migration was studied using chemotaxis assay and it was found that the addition of anti-CD147 antibody significantly decreased the chemotactic index of the neutrophils. Significantly elevated release and activation of MMPs were seen in the co-culture of neutrophil and FLS compared with cultures of the cells alone. An increased number of cells invading through the filters in the invasion assays were also observed in the co-cultured cells. The addition of anti-CD147 antibody had some inhibitory effect, not only on MMP production but also on cell invasion in the co-culture model. Our study demonstrates that the increased expression of CD147 on neutrophils in RA may be responsible for CyPA-mediated neutrophil migration into the joints, elevated MMPs secretion and cell invasion of synoviocytes, all of which may contribute to the cartilage invasion and bone destruction of RA. Better knowledge of these findings will hopefully provide a new insight into the pathogenesis of RA.

CD147 overexpression on synoviocytes in rheumatoid arthritis enhances matrix metalloproteinase production and invasiveness of synoviocytes

Macrophage-like synoviocytes and fibroblast-like synoviocytes (FLS) are known as the most active cells of rheumatoid arthritis (RA) and are close to the articular cartilage in a position enabling them to invade the cartilage. Macrophage-like synoviocytes and FLS expression of matrix metalloproteinases (MMPs) and their interaction has aroused great interest. The present article studied the expression of CD147, also called extracellular matrix metalloproteinase inducer, on monocytes/macrophages and FLS from RA patients and its potential role in enhancing MMPs and the invasiveness of synoviocytes. Expression of CD147 on FLS derived from RA patients and from osteoarthritis patients, and expression of CD147 on monocytes/macrophages from rheumatic synovial fluid and healthy peripheral blood were analyzed by flow cytometry. The levels of CD147, MMP-2 and MMP-9 mRNA in FLS were detected by RT-PCR. The role of CD147 in MMP production and the cells' invasiveness in vitro were studied by the co-culture of FLS with the human THP-1 cell line or monocytes/macrophages, by gel zymography and by invasion assay. The results showed that the expression of CD147 was higher on RA FLS than on osteoarthritis FLS and was higher on monocytes/macrophages from rheumatic synovial fluid than on monocytes/macrophages from healthy peripheral blood. RT-PCR showed that the expressions of CD147, MMP-2 and MMP-9 mRNA was higher in RA FLS than in osteoarthritis FLS. A significantly elevated secretion and activation of MMP-2 and MMP-9 were observed in RA FLS co-cultured with differentiated THP-1 cells or RA synovial monocytes/macrophages, compared with those co-cultured with undifferentiated THP-1 cells or healthy control peripheral blood monocytes. Invasion assays showed an increased number of invading cells in the co-cultured RA FLS with differentiated THP-1 cells or RA synovial monocytes/macrophages. CD147 antagonistic peptide inhibited the MMP production and the invasive potential. Our studies demonstrated that the CD147 overexpression on monocytes/macrophages and FLS in RA patients may be responsible for the enhanced MMP secretion and activation and for the invasiveness of synoviocytes. These findings suggest that CD147 may be one of the important factors in progressive joint destruction of RA and that CD147 may be a potential therapeutic target in RA treatment.

Invasion of synoviocytes is inhibited by gene transfer of TNF-BP or IL10 in an in vitro invasion model

Invasion of synoviocytes is inhibited by gene transfer of TNF-BP or IL10 in an in vitro invasion model