Synoviocyte Hyperplasia 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.

Jammed joints and constricted heart: The science of tribology and missing lubricin. A case report on camptodactyly-arthropathy-coxa vara-pericarditis syndrome

ABSTRACT An autosomal recessively inherited noninflammatory arthropathy known as camptodactyly, arthropathy, coxa vara, and pericarditis (CACP) syndrome was discovered in 1999. It is distinguished by synoviocyte hyperplasia and subcapsular fibrosis of the synovial capsule, which results in a shortage of lubricin production. The resulting lack of joint lubrication induces increased mechanical stress, causing progressive deformities that become evident with weight-bearing and heightened joint activity. Animal models with a lubricin gene knock-out display similar traits, underscoring the impact of mechanical stress on disrupting type II collagen on the articular surface. The gradual development of pericarditis and constriction often results in misdiagnosis as juvenile rheumatoid arthritis with cardiac involvement, but the defining feature remains the noninflammatory nature of the disease. Early recognition is pivotal, as interventions such as pericardiectomy and recombinant human lubricin hold promise for altering the disease’s natural course. In our familial case of CACP, two siblings exhibited distinct phenotypic variations – one with fibrosis-dominant features and pericardial constriction and the other displaying synovial hyperplasia without pericardial involvement.

Frequency of Positive Anti-CCP Antibodies in Rheumatoid Arthritis Patients with Negative Rheumatoid Factor

Background: Rheumatoid arthritis (RA) is a polyarticular autoimmune disease that affects about 1% of the adult population. The disease is characterized by synoviocyte hyperplasia, mainly synovial fibroblasts, resulting in bone and joint destruction. Recent studies have shown that cytokines and other systemic inflammation mediators have a key role in the development ofrheumatoid arthritis. Recently, Anti-Citrullinated Protein Antibodies (anti-CCP) have come into use for the diagnosis of rheumatoid arthritis (RA). It has been reported that anti-CCP has quite a high specificity for RA (98%), together with sensitivity similar to that for rheumatoid factor (RF). The study aimed to determine the frequency of positive anti-CCP antibodies in rheumatoid arthritis patients with negative rheumatoid factor.
 Methods: 91 patients (both male and females) aged 40-70years with negative RF were clinically diagnosed with rheumatoid arthritis. Patients with positive rheumatoid factor and joint injuries were disclaimed. The consultant pathologist used a second-generation enzyme-linked immunosorbent assay (ELISA) to assess the serum levels of anti-CCP antibodies, >20IU/ ml is considered positive p-value of 0.05 were considered statistically significant.
 Results: In the study, the age range was 40 to 70 years, averaging 53.44 ± 7.16 years. 59 (64.84%) of the patients were between the ages of 40 and 55. Of 91 patients, 56 (61.54%) were women, and 35 (38.46%) were men with aratio of 1.6:1 man to women. In patients with negative rheumatoid factors, 27 (40.66%) patients were found to have the levels of positive anti-CCP antibodies.
 Conclusion: Patients with negative rheumatoid factor showed the high frequency of positive anti-CCP.

GRK2 mediated degradation of SAV1 initiates hyperplasia of fibroblast-like synoviocytes in rheumatoid arthritis

Hyperplasia and migration of fibroblast-like synoviocytes (FLSs) are the key drivers in the pathogenesis of rheumatoid arthritis (RA) and joint destruction. Abundant Yes-associated protein (YAP), which is a powerful transcription co-activator for proliferative genes, was observed in the nucleus of inflammatory FLSs with unknown upstream mechanisms. Using Gene Expression Omnibus database analysis, it was found that Salvador homolog-1 (SAV1), the pivotal negative regulator of the Hippo-YAP pathway, was slightly downregulated in RA synovium. However, SAV1 protein expression is extremely reduced. Subsequently, it was revealed that SAV1 is phosphorylated, ubiquitinated, and degraded by interacting with an important serine-threonine kinase, G protein-coupled receptor (GPCR) kinase 2 (GRK2), which was predominately upregulated by GPCR activation induced by ligands such as prostaglandin E2 (PGE2) in RA. This process further contributes to the decreased phosphorylation, nuclear translocation, and transcriptional potency of YAP, and leads to aberrant FLSs proliferation. Genetic depletion of GRK2 or inhibition of GRK2 by paroxetine rescued SAV1 expression and restored YAP phosphorylation and finally inhibited RA FLSs proliferation and migration. Similarly, paroxetine treatment effectively reduced the abnormal proliferation of FLSs in a rat model of collagen-induced arthritis which was accompanied by a significant improvement in clinical manifestations. Collectively, these results elucidate the significance of GRK2 regulation of Hippo-YAP signaling in FLSs proliferation and migration and the potential application of GRK2 inhibition in the treatment of FLSs-driven joint destruction in RA.

Piperine suppresses inflammatory fibroblast-like synoviocytes derived from rheumatoid arthritis patients Via NF-κB inhibition

Rheumatoid Arthritis (RA) is a common autoimmune disease recognized by hyperplasia of synoviocytes and chronic joint inflammation. Activation of fibroblast-like synoviocytes (FLSs) is one of the main features of RA which can trigger inflammation leading to articular cartilage and joint destruction. Aberrant activation of NF-κB signaling cascade was found to be responsible for the high proliferation and defective apoptosis of FLSs and subsequent inflammation in RA. Piperine is a principal constituent of piper species frequently used as antitumor and anti-inflammatory natural compound. In this study we aimed to assess the anti-inflammatory effect of piperine on RA-FLS through NF-κB inhibition.FLSs were isolated from 68 RA patients and 30 healthy controls and were exposed to piperine. The main assays were MTT assay, flow cytometric analysis, PI staining, reverse transcription-PCR (RT-PCR), and ELISA.Results showed that piperine can induce the apoptosis and reduce the proliferation of RA-FLSs in vitro. Moreover, piperine directly reduced the phosphorylation of NF-kB and the expression of NF-κB target genes related to RA-FLSs proliferation (c-Myc and Cycline D1), apoptosis inhibition (Bcl2 and Bcl-xl) and inflammation (COX2, IL-1β, TNF-α,IL-6, CCL5 and CXCL10) while increasing the expression of apoptosis related ones (Bax) in vitro. Piperine also reduced the protein levels of cytokines and chemokines secreted by FLSs as a result of NF-κB inhibition.In conclusion, our results provide evidence for the anti-inflammatory capacity of piperine through inhibition of NF-κB pathway in FLSs proposing this compound as a suitable alternative for chemical treatment of RA.

The Neurasthenic Nipper-Juvenile Idiopathic Arthritis

Juvenile Idiopathic Arthritis (JIA) is a heterogeneous group of paediatric, idiopathic, inflammatory arthritis exceeding >six weeks duration and commonly arising in children beneath <16 years. International League of Associations for Rheumatology (ILAR) has categorized juvenile idiopathic arthritis into distinctive subclasses as pauciarticular variant or oligoarthritis, Rheumatoid Factor (RF) positive polyarthritis, Rheumatoid Factor (RF) negative polyarthritis, systemic arthritis, psoriatic arthritis, enthesitis-related arthritis and undifferentiated arthritis. The condition is posited to arise from environmental factors, viral or bacterial infection or demonstrates a genetic predisposition. Juvenile idiopathic arthritis depicts decimated joint function with reduced Range of Motion (ROM), joint pain, morning stiffness, limping due to pain in the lower extremities, joint deformity and joint swelling commonly discerned within the knee, hand or foot, anomalous limb growth with leg length discrepancies,, uveitis, reoccurring pyrexia, cutaneous rash, myalgia, weight loss and disorders of skeletal growth. An intense, synovial infiltration of T lymphocytes, B lymphocytes, plasma cells, macrophages and dendritic cells is observed along with villous hyperplasia and hypertrophy, endothelial activation and hyperplasia and hyperplasia of synoviocytes.

Glaucocalyxin A suppresses inflammatory responses and induces apoptosis in TNF-a-induced human rheumatoid arthritis via modulation of the STAT3 pathway

The pathogenesis of rheumatoid arthritis (RA) is characterized by synoviocyte hyperplasia and proinflammatory cytokine secretion, as well as the destruction of cartilage and bone. Glaucocalyxin A (GLA) is an alkaloid derived from a Chinese medicinal plant that exhibits anti-inflammatory, anti-tumor and neuroprotective properties. We investigated the effects of GLA on RA-fibroblast-like synoviocytes (FLS cells), and collagen-induced arthritis (CIA), and further explored the underlying mechanisms. GLA inhibited TNF-a-induced RA-FLS proliferation, increased apoptotic ratios and upregulated levels of caspase-3, cleaved PARP, and Bax. GLA also inhibited the expression of IL-10, IL-1β, and IL-6 in vitro. Levels of p-STAT3 were downregulated in a dose-dependent manner. Over-expression of STAT3 partly neutralized the GLA-mediated elevation of caspase-3 and cleaved PARP levels as well as the downregulation of IL-10, IL-1B and IL-6 expression levels. This suggests that GLA inactivated the STAT3 pathway. Furthermore, the production of inflammatory cytokines in RA-FLS and a CIA rat model were inhibited effectively by GLA. Taken together, our data suggest that GLA is a potential long-term therapeutic agent for patients with RA.

Angiogenic cytokines can reflect the synovitis severity and treatment response to biologics in rheumatoid arthritis

Angiogenesis and synoviocyte hyperplasia, called ‘pannus,’ are pathologic hallmarks of rheumatoid arthritis (RA). To determine the clinical significance of angiogenic cytokines in RA, the levels of pro-angiogenic cytokines, including VEGF, placenta growth factor (PlGF), and IL-6, were measured in the synovial fluid (SF, n = 54) and sera of RA patients (n = 157) using ELISA. Patients (n = 103) with disease activity score 28 (DAS28) > 3.2, which indicates moderate to high RA activity, underwent follow-up blood sampling at 6 months after treatment with conventional disease-modifying anti-rheumatic drugs (c-DMARD) or biologic DMARD (b-DMARD) including an anti-TNFα antibody, an anti-IL-6 antibody, and abatacept. Ultrasonography (US) was performed on affected joints to define the synovitis severity at the time of sampling. Consequently, in the SF of RA patients, PlGF and IL-6 levels correlated well with synovitis severity determined by US. In RA sera, VEGF and IL-6 levels were elevated in proportion to synovitis severity, correlating with conventional markers for disease activity, including ESR, CRP, and DAS28. In c-DMARD users (n = 53), serially monitored levels of serum VEGF, IL-6, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) all decreased in good and moderate responders but not in nonresponders. In b-DMARD users (n = 49), only serum VEGF well represented the treatment response, while CRP nonspecifically decreased irrespective of the treatment outcome. By multivariable analysis, serum ΔVEGF, but not ΔESR or ΔCRP, was an independent factor associated with good and moderate responses to DMARD. In summary, the angiogenic cytokines PlGF and VEGF represent the synovitis severity of RA assessed by US. In patients receiving b-DMARD, serum VEGF may be more valuable than CRP in reflecting the treatment response.

CircGCN1L1 promotes synoviocyte proliferation and chondrocyte apoptosis by targeting miR-330-3p and TNF-\u03b1 in TMJ osteoarthritis

Altered expression of circular RNAs (circRNAs) has been identified in various human diseases. In this study, we investigated whether circRNAs function as competing endogenous RNAs to regulate the pathological process of temporomandibular joint osteoarthritis (TMJOA). High-throughput sequencing of mRNA (RNA seq) was performed to detect the expression of circRNAs in TMJOA and control synovial tissues isolated from humans. The differentially upregulated circGCN1L1 (hsa_circ_0000448) in synoviocyte was validated in vitro and in vivo. Here we demonstrate the interactions between circGCN1L1 and both miR-330-3p and tumor necrosis factor-α (TNF-α) through bioinformatics predictions, luciferase report assays, and fluorescence in situ hybridization. mRNA expression profiles of TNF-α-stimulated synoviocyte showed that circGCN1L1 and p65 expressions were upregulated by TNF-α. Moreover, miR-330-3p was negatively correlated with TNF-α secretion. Further, we found that miR-330-3p directly targeted TNF and restrained the production of matrix-degrading enzymes (MMP3, MMP13, and ADAMTS4). Mechanistic studies unveiled that circGCN1L1 in TMJOA synovial tissues and cells may be associated with condylar chondrocyte apoptosis and synoviocyte hyperplasia. Moreover, intra-articular injection of shcircGCN1L1 alleviated TMJOA progression in rat models. Altogether, we elucidated the important roles of a novel circRNA, namely, circGCN1L1, which induced inflammation in TMJ synoviocytes and decreased anabolism of the extracellular matrix (ECM) through miR-330-3p and TNF-α gene. This circRNA may represent a potentially effective therapeutic strategy against TMJOA progression at an early stage.

CXCL16 expression during post-traumatic osteoarthritis development: implications for mesenchymal stem cell recruitment

Purpose: Post-traumatic osteoarthritis (PTOA) develops following major joint trauma. Recently, we demonstrated that mesenchymal stem/multipotent stromal cells (MSCs) are recruited to the injured joint after ACL rupture (ACLR) in rats. While it is known that MSCs are recruited by chemokines, the temporal, injury-induced, intraarticular expression of chemokines and their specific roles in MSC recruitment remain largely undescribed. The purpose of this study was to characterize the in vivo expression of the understudied chemokine CXCL16, which can recruit MSCs via CXCR6, following ACLR. We also sought to assess whether primary fibroblast-like synoviocytes (FLS) from injured joint synovium are “reprogrammed” to exhibit differential CXCL16 expression compared to cells from a healthy joint. Finally, we assessed CXCL16- and FLS-mediated recruitment of MSCs in an in vitro chemotaxis model. Methods: With IACUC approval, ACLR was induced in adult C57BL/6 mice via tibial compression. 3- and 14-days post-injury, the synovium, menisci, and femoral and tibial epiphyses were analyzed with rtPCR for CXCL16 and ADAM10, the protease that cleaves CXCL16 from the cell membrane, (n=8 mice per time point). Further mice were injured and FLS were isolated to establish cultures (n=3 each) from injured and contralateral limbs 7-days post-injury. Cultured FLS were induced with cytokines and analyzed with rtPCR for CXCL16 expression. CXCL16 (10 and 50 ng/mL) was assessed for its role in recruitment of C3H10T1/2 cells, an MSC-like cell line, in an in vitro 3D chemotaxis system (Incucyte). Chemotaxis was quantified by automated calculation of cell-occupied area on the bottom membrane. Primary FLS were isolated from uninjured mouse synovium, induced with 10 ng/mL TNFα, co-cultured with C3H10T1/2 in the chemotaxis system, and analyzed for the ability to recruit C3H10T1/2 MSCs. Results: Histology grading shows mild PTOA and moderate synovitis in mice following ACLR, as evidenced by mild cartilage degradation, early osteophyte formation, and pronounced synoviocyte hyperplasia at 3 and 14d (Fig 1). Compared to contralateral knees, the synovium of injured limbs exhibited no upregulation of IL-17, but significant increases in TNFα (P=0.026) and IL-1β (P=0.017) at 3d and in TNFα at 14d (P=0.006). Both CXCL16 and ADAM10 are significantly upregulated by the synovium in vivo 3d after injury and remain elevated at 14d (Fig 2). The menisci of injured limbs upregulated CXCL16 at both 3d (P<0.001) and 14d (P<0.001), but there was no change in ADAM10 expression in meniscus. Results from both epiphyseal tissues showed no upregulation in chemokine expression between injured and contralateral limbs (Fig 2). Primary FLS from injured joints upregulated CXCL16 when treated with TNFα (P=0.025) and IL-1β (P=0.006), and interestingly, FLS from contralateral joints showed similar upregulation in response to TNFα (P=0.002) and IL-1β (P=0.006) (Fig 3). There was no difference in CXCL16 expression between injured and injured FLS. 3D chemotaxis assays demonstrate that 50 ng/mL CXCL16, but not 10 ng/ml, induces C3H10T1/2 chemotaxis. Further, TNFα-stimulated FLS induce C3H10T1/2 chemotaxis to a greater extent than unstimulated FLS or TNFα-containing media alone, demonstrating the ability of cytokine activated FLS to recruit MSC-like cells (Fig 4).View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT) Conclusions: Our data demonstrate that noninvasive ACLR causes mild PTOA and moderate synovitis up to 14d post-ACLR, with concurrently increased expression of cytokines. In vivo injury-induced upregulation of CXCL16 and ADAM10 by the synovium provide evidence for CXCL16’s possible role in recruiting MSCs, which highly express CXCR6. CXCL16 was upregulated by the meniscus of the injured joint at 3d with persistent upregulation at 14d. Our in vitro data demonstrate that the FLS is a contributor to CXCL16 expression, and this is mediated by both IL-1β and TNFα. Similar CXCL16 expression between FLS isolated from contralateral and injured limbs suggests that although profound inflammation is observed in vivo following ACLR, this inflammatory environment did not “reprogram” the FLS to exhibit greater CXCL16 expression in response to cytokines. Further studies will explore later time points to determine if such “stromal memory” is demonstrated by FLS in later stages of PTOA. Finally, we have established an in vitro model to study MSC recruitment and FLS-MSC interactions, as we demonstrate both CXCL16 and cytokine-induced FLS can recruit MSC. Ongoing work is employing inhibitors to assess the role of CXCL16 in FLS-mediated MSC recruitment.

Wasp Venom Possesses Potential Therapeutic Effect in Experimental Models of Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease. Wasp venom (WV), which is considered as a traditional folk medicine in Jingpo nationality in Yunnan, China, relieves rheumatoid arthritis. The current study aimed to investigate the effect of wasp venom ameliorating rheumatoid arthritis symptoms in experimental rats. We established a model of type II collagen- (CII-) induced arthritis (CIA) in SD rats and examined the inhibition of inflammation and autoimmune response. The antiarthritic effects of WV were evaluated through the paw swelling, and histopathological score and histopathology changes of the affected paw were assessed. The anti-inflammation effects were assayed by the level of IL-6, TNF-α, IL-1β, and the number of inflammatory cells in peripheral blood. The alteration of the T cell subset ratio in the spleen of rats was detected by flow cytometry, and at the same time, the viscera index and immune serum globulin levels were evaluated. The results suggested that various doses of WV (0.125, 0.25, and 0.5 mg/kg) significantly alleviated paw swelling and arthritis score in CIA rats with the untreated control (P < 0.05). WV (0.25 and 0.5 mg/kg) relieved synovial tissue lesions of ankle joints and histopathology scores of synoviocyte hyperplasia and inflammatory cell infiltration with vehicle group (P < 0.05). Regarding immunological regulation, 0.5 mg/kg WV lowered the immune serum globulin levels (P < 0.05), and we further found that WV (0.5 mg/kg) suppressed the immune response of Th cells, while enhancing the functions of Tc cells and Treg cells in spleen cells markedly (P < 0.05). The immunosuppressive action of WV displayed was analogous to its inhibitory effect on IL-1β, TNF-α, IL-8, IL-6, COX-2, and PGE2 levels in rat serum. In conclusion, these findings demonstrated that WV exhibited antiarthritic activity, which might be associated with their inhibitory effects on immunoregulation and anti-inflammatory action.

Pannus does not occur only in rheumatoid arthritis: a pathological observation of pannus of knee osteoarthritis

To compare the histopathological features of the synovium between rheumatoid arthritis (RA) and osteoarthritis (OA). We retrospectively analyzed the synovial specimens obtained after synovial surgery in 72 cases of RA and 24 cases of OA. Two independent pathologists reviewed the sections of the synovial tissues with HE staining, quantitatively scored the degree of fibroblast-like synoviocyte (FLS) hyperplasia, vascular hyperplasia, fibroplasia, and lymphocyte infiltration, and examined the presence plasma cell infiltration. The pathological morphology of the synovial tissues was evaluated in relation with the clinical data of the patients. Pannus formation was also detected in the synovium of OA patients, which showed a lesser degree of OA-FLS hyperplasia, fibrosis and lymphocyte infiltration and a significantly lower rate of plasma cell infiltration compared with the pannus in RA patients. Vascular proliferation was also milder in the pannus of OA patients than in RA pannus, but the difference was not statistically significant. In OA patients, the pannus could be observed under a microscope and was difficult to distinguish from that in RA patients. Pannus formation occurs also in the synovium of OA patients but with milder FLS hyperplasia, fibrosis and lymphocyte infiltration and a lower rate of plasma cell infiltration compared with the pannus in RA patients. These differences in the pannus between OA and RA can be of potential value in the diagnosis and treatment of the patients.

MiR-124a inhibits the proliferation and inflammation in rheumatoid arthritis fibroblast-like synoviocytes via targeting PIK3/NF-κB pathway.

Abnormal hyperplasia of fibroblast-like synoviocytes (FLS) leads to the progression of rheumatoid arthritis (RA). This study aimed to investigate the role of miR-124a in the pathogenesis of RA. The viability and cell cycle of FLS in rheumatoid arthritis (RAFLS) were evaluated by Cell Counting Kit 8 and flow cytometry assay. The expression of PIK3CA, Akt, and NF-κB in RAFLS was examined by real-time PCR and Western blot analysis. The production of tumour necrosis factor (TNF)-α and interleukin (IL)-6 was detected by ELISA. The joint swelling and inflammation in collagen-induced arthritis (CIA) mice were examined by histological and immunohistochemical analysis. We found that miR-124a suppressed the viability and proliferation of RAFLS and increased the percentage of cells in the G1 phase. miR-124a suppressed PIK3CA 3'UTR luciferase reporter activity and decreased the expression of PIK3CA at mRNA and protein levels. Furthermore, miR-124a inhibited the expression of the key components of the PIK3/Akt/NF-κB signal pathway and inhibited the expression of pro-inflammatory factors TNF-α and IL-6. Local overexpression of miR-124a in the joints of CIA mice inhibited inflammation and promoted apoptosis in FLS by decreasing PIK3CA expression. In conclusion, miR-124a inhibits the proliferation and inflammation in RAFLS via targeting PIK3/NF-κB pathway. miR-124a is a promising therapeutic target for RA.

Therapeutic Potential of Mesenchymal Cell-Derived miRNA-150-5p-Expressing Exosomes in Rheumatoid Arthritis Mediated by the Modulation of MMP14 and VEGF.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial tissue inflammation and joint destruction associated with the activation of angiogenesis. Exosomes, which play a role in cell-to-cell communication as carriers of genetic information, transfer microRNAs (miRNAs or miRs) between cells and have been studied as delivery vehicles for therapeutic molecules. The aim of the current study was to investigate the therapeutic effect of mesenchymal stem cell (MSC)-derived miR-150-5p exosomes on joint destruction in RA. The expression and secretion of miR-150-5p, matrix metalloproteinase (MMP) 14, and vascular endothelial growth factor (VEGF) in RA patients and fibroblast-like synoviocytes (FLS) were examined by quantitative RT-PCR, ELISA, and Western blotting. Immunohistochemistry was used to assess angiogenesis. MSCs were transfected with an miR-150-5p expression plasmid, and MSC-derived exosomes were harvested. The effect of MSC-derived miR-150-5p exosomes (Exo-150) on MMP14 and VEGF expression was examined. The effects of Exo-150 on cell migration and invasion in cytokine-stimulated FLS from RA patients were examined by HUVEC tube formation and transwell assays. The effect of Exo-150 in vivo was examined in a collagen-induced arthritis mouse model. Exo-150 decreased migration and invasion in RA FLS and downregulated tube formation in HUVECs by targeting MMP14 and VEGF. Injection of Exo-150 reduced hind paw thickness and the clinical arthritic scores in collagen-induced arthritis mice. Exo-150 reduced joint destruction by inhibiting synoviocyte hyperplasia and angiogenesis. Exosomes facilitate the direct intracellular transfer of miRNAs between cells and represent a potential therapeutic strategy for RA.

GREM1 Is a Key Regulator of Synoviocyte Hyperplasia and Invasiveness

To investigate the expression of Gremlin 1 (GREM1), an antagonist of bone morphogenetic protein, in rheumatoid arthritis (RA) synovia and its involvement in the hyperplasia and invasiveness of fibroblast-like synoviocytes of RA (RA-FLS). Computational analysis was introduced to identify FLS-predominant regulators. GREM1 expression was examined by immunohistochemistry, real-time PCR, and ELISA. FLS proliferation and apoptosis were determined using tetrazolium-based colorimetric assay and APOPercentage assay, respectively. FLS migration and invasion were evaluated by wound migration and Matrigel invasion assay, respectively. Expressions of Bax, Bcl2, pErk1/2, and pAkt were detected by Western blot analysis. Through global transcriptome profiling, we identified a GREM1 gene predominantly expressed in RA-FLS. Indeed, the GREM1 expression was higher in synovia, synovial fluids, and FLS of patients with RA than in those of patients with osteoarthritis, and its levels correlated well with proinflammatory cytokine concentrations. Knockdown of GREM1 transcripts using short interfering RNA (siRNA) reduced the proliferation and survival of RA-FLS along with downregulation of pErk1/2, pAkt, and Bcl2 expressions, whereas it induced Bax expression. Conversely, the addition of recombinant GREM1 to RA-FLS showed the opposite results. Moreover, GREM1 siRNA decreased the migratory and invasive capacity of RA-FLS, whereas exogenous GREM1 increased it. The GREM1-induced FLS survival, migration, and invasion were completely blocked by neutralizing antibodies to ανβ3 integrin on RA-FLS, suggesting that ανβ3 integrin mediates the antiapoptotic and promigratory effects of GREM1. GREM1 is highly expressed in RA joints, and functions as a regulator of survival, proliferation, migration, and invasion of RA-FLS.

DNA Methylome Signature in Synoviocytes From Patients With Early Rheumatoid Arthritis Compared to Synoviocytes From Patients With Longstanding Rheumatoid Arthritis.

Epigenetics can contribute to pathogenic mechanisms in autoimmunity. We recently identified an imprinted DNA methylation pattern in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) involving multiple genes in pathways implicated in cell migration, matrix regulation and immune responses.1,2 To understand when alterations in DNA methylation occur in RA and the specificity of the methylation changes in RA, we compared differentially methylated loci (DMLs) of early RA (ERA), juvenile idiopathic arthritis (JIA) to longstanding RA (LRA) and osteoarthritis (OA). Genomic DNA from fibroblast-like synoviocytes (FLS) was isolated from 4 ERA (1–13 months of symptoms; all female; average age 52) as previously described. 1, 2, 3 All ERA patients were anti-citrullinated peptide antibody positive. Two were treated with methotrexate alone and one was treated with methotrexate and abatacept. One patient was not taking anti-rheumatic drugs. Patients met the 2010 classification criteria for RA and samples were obtained at the time of arthroscopic biopsy.4, 5 FLS from patients with JIA (n=3; 2 female, one male; average age 31) was isolated from synovium obtained at the time of arthroplasty. Previously reported 11 LRA and 11 OA were used as controls.5 DNA methylation level was measured using the Illumina Infinium HumanMethylation450 chip across 485,512 loci and the methylation level at individual locus was reported as β values (see Supplementary Methods). Small but statistically significant global hypomethylation in LRA compared with ERA (average β value per CpG loci = 0.163 in LRA and 0.168 in ERA; p value=0.0046) was detected by summing β values in gene promoter region. Unbiased hierarchical clustering and principal component analysis (PCA) revealed relationships of ERA and JIA to LRA and OA FLS lines (Figure 1A and B, respectively) based on previously identified 15,220 DMLs between 11 LRA and 11 OA2. ERA and JIA clustered with LRA and segregated from OA. ERA and LRA patterns partially overlapped, which could be consistent with a transition of ERA to LRA methylation. FLS from JIA also segregated with the LRA group but formed a separate subgroup. With methylome comparisons between LRA and ERA using Welch’s t-test, we identified 20,776 DMLs (8,059 hypomethylated and 12,717 hypermethylated in LRA compared with ERA) in 5,469 DMGs (average differences of β values > 0.1 and q values < 0.05). Pathway enrichment analysis evaluated by Ingenuity pathway analysis (IPA) identified five potentially enriched pathways (Wnt/β-catenin signaling, enrichment ratio (ER) = 0.249; Integrin signaling, ER = 0.233; RAR activation, ER = 0.233; PDGF signaling, ER = 0.286; Superpathway of D-myo-inositol (1,4,5)-trisphosphate metabolism, ER = 0.417) with hypomethylated DMGs in LRA (BH-adjusted p values < 0.1). In addition, 340 CpG loci that most distinguished ERA from LRA were identified by comparing the entire methylation datasets by random forest method and confirmed that ERA and LRA could be distinguished from each other (Supplementary Figure 1). Figure 1 Analysis of DNA methylation patterns in early and longstanding rheumatoid arthritis. (A) Hierarchical clustering of longstanding RA, (LRA), early RA (ERA), juvenile idiopathic arthritis (JIA) and osteoarthritis (OA) based on differentially methylated ... Inflammatory arthritis genome-wide DNA methylation patterns differed from OA and clustered together, suggesting that there are common epigenetic elements of these diseases. However, the FLS formed subgroups implying that the variations are disease specific. Of interest, the methylome of ERA demonstrated a distinctive pattern compared with long-standing disease. These data indicate that differential methylation of RA FLS might occur early and evolve over time. Differentially methylated pathways between ERA and LRA involved cell migration, differentiation, and adhesion, which raises the intriguing possibility that the transition to chronic RA involves epigenetic changes that alter synoviocyte hyperplasia and aggressive behavior. One obvious limitation of the study is that the number of samples is small, in part because obtaining cell lines from ERA biopsies is challenging. Despite that, these unique marks can potentially provide information on mechanisms of disease and how RA evolves over time as well as identifying therapeutic targets based on the duration and type of synovitis.

Enthesitis as a component of dactylitis in psoriatic juvenile idiopathic arthritis: histology of an established clinical entity

ContextImaging of dactylitis in adult psoriatic arthritis suggests a pathophysiological role for enthesitis. However, histological definition of the dactylitic digit is unavailable.ObjectiveWe evaluated the role of enthesitis in dactylitis associated with psoriatic juvenile idiopathic arthritis (psJIA) in a child who underwent detailed imaging and histologic evaluation.DesignRadiographs, ultrasound and high-resolution magnetic resonance imaging were employed to define the locus of disease in two dactylitic digits from a 14-year-old girl with psJIA. Biopsies were stained with hematoxylin and eosin, CD3, CD20, CD4, CD8 and CD117 and examined via light microscopy.ResultsRadiologic features of dactylitis included enhanced signal at digital entheses without accompanying synovitis or tenosynovitis. Histologically, finger and toe tissue exhibited hypervascular tenosynovium with a fibromyxoid expansion of fibrous tissue. This was accompanied by sparse to moderate perivascular lymphocytic inflammation consisting predominantly of T cells, with occasional admixed B cells and mast cells. Neutrophils and plasma cells were absent. Fibrocartilage exhibited reactive features including increased extracellular myxoid matrix, binucleation, and focal necrosis, without cellular inflammation. Fibrinous synovitis and mild reactive synoviocyte hyperplasia were seen.ConclusionsDactylitis in psJIA bears a radiographic resemblance to the condition in adult psoriatic arthritis. Histologic hallmarks include an expanded mast cell-populated extracellular myxoid matrix, cartilage degeneration, and a T cell-rich perivascular inflammatory infiltrate. These findings help to define enthesitis as a clinicopathologic entity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12969-015-0003-2) contains supplementary material, which is available to authorized users.

Experimentally induced lameness in turkeys inoculated with a newly emergent turkey reovirus.

Newly emergent turkey arthritis reoviruses (TARVs) have been isolated from cases of lameness in male turkeys over 10 weeks of age. In a previous study, experimental inoculation of TARV in one-week-old turkey poults produced lymphocytic tenosynovitis at four weeks post inoculation but without causing clinical lameness. This study was undertaken to determine if TARV infection at an early age can lead to clinical lameness in birds as they age. One-week-old male turkeys were inoculated orally with a TARV (strain TARV-O’Neil) and monitored for the development of gait defects until 16 weeks of age. At 4, 8, 12 and 16 weeks of age, a subset of birds was euthanized followed by the collection of gastrocnemius tendon, digital flexor tendon, and intestines for virus detection by rRT-PCR and for histologic inflammation scoring. Clinical lameness was first displayed in TARV-infected turkeys at 8 weeks of age and ruptured gastrocnemius tendons with progressive lameness were also seen at 12–16 weeks of age. The virus was detected in gastrocnemius tendon of 4- 8- and 12-week-old turkeys but not in 16-week-old turkeys. Histologic inflammation scores of tendons at each of the four time points were significantly higher in the virus-inoculated group than in the control group (p < 0.01). Lesions began as lymphocytic tenosynovitis with mild synoviocyte hyperplasia at four weeks of age and progressed to fibrosis as the birds aged. These results demonstrate the potential of TARV to infect young turkeys and to produce subclinical tenosynovitis that becomes clinically demonstrable as the turkeys age.

FRI0360 Histone Deacetylase One Contributes to the Auto-Aggressive Phenotype of Rheumatoid Arthritis Synovial Fibroblasts

BackgroundRheumatoid arthritis (RA) is a chronic, autoimmune, inflammatory disease that affects synovial joints. A key characteristic of RA is hyperplasia of fibroblast-like synoviocytes (FLS). These FLS are known to develop...

Small Ruminant Lentivirus–Induced Arthritis

We describe the clinicopathologic features of an arthritis outbreak in sheep induced by small ruminant lentivirus (SRLV), linked to the presence of a new SRLV isolate phylogenetically assigned to caprine arthritis encephalitis virus-like subgroup B2. Thirteen SRLV seropositive Rasa Aragonesa adult ewes were selected from 5 SRLV highly infected flocks (mean seroprevalence, 90.7%) for presenting uni- or bilateral chronic arthritis in the carpal joint. A complete study was performed, including symptomatology, histopathology, immunocytochemistry, immunohistochemistry, in situ hybridization, and microbiology. The carpus was the joint almost exclusively affected, with 10 sheep (76%) showing a moderate increase in carpal joint size (diameter range, 18-20 cm; normal range, 15-16 cm) without signs of locomotion problems and with 3 ewes (23%) showing severe inflammation with marked increase in diameter (21-24 cm), pain at palpation, and abnormal standing position. Grossly, chronic proliferative arthritis was observed in affected joints characterized by an increased thickness of the synovial capsule and synovial membrane proliferation. Microscopically, synovial membrane inflammation and proliferation and hyperplasia of synoviocytes were observed. More positive cases of SLRV infection were detected by immunocytochemistry of articular fluid than of bronchoalveolar lavage fluid. Immunohistochemistry and in situ hybridization also detected positive cells in the subsynovial connective tissue, lung, mediastinal lymph node, mammary gland, and mammary lymph node. All animals were negative for the presence of Mycoplasma or other bacteria in the articular space. The present outbreak likely represents an adaptation of a caprine virus to sheep. Our results underline the importance of the arthritis induced by SRLV in sheep, a clinical form that might be underestimated.