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Objectives: Investigate whether and which synoviocytes would acquire trained immunity characteristics that could exacerbate joint inflammation following a secondary Staphylococcus aureus infection. Methods: Lipopolysaccharide (LPS) and S. aureus were separately or double injected (21 days of interval) into the tibiofemoral joint cavity of male C57BL/6 mice. At different time points after these stimulations, mechanical nociception was analyzed followed by the analysis of signs of inflammation and damage in the affected joints. The trained immunity markers, including the glycolytic and mTOR pathway, were analyzed in whole tissue or isolated synoviocytes. A group of mice was treated with Rapamycin, an mTOR inhibitor before LPS or S. aureus stimulation. Results: The double LPS – S. aureus hit promoted intense joint inflammation and damage compared to single joint stimulation, including markers in synoviocyte activation, production of proinflammatory cytokines, persistent nociception, and bone damage, despite not reducing the bacterial clearance. The double LPS - S. aureus hit joints increased the synovial macrophage population expressing CX3CR1 alongside triggering established epigenetic modifications associated with trained immunity events in these cells, such as the upregulation of the mTOR signaling pathway (p-mTOR and HIF1α) and the trimethylation of histone H3. Mice treated with Rapamycin presented reduced CX3CR1+ macrophage activation, joint inflammation, and bone damage. Conclusions: There is a trained immunity phenotype in CX3CR1+ synovial macrophages that contributes to the exacerbation of joint inflammation and damage during septic arthritis caused by S. aureus. [ABSTRACT FROM AUTHOR]

Introduction/objectives: JAK/STAT signaling inhibition exerts therapeutic effects on angiogenesis in rheumatoid arthritis (RA). However, whether the inhibitory effect differs among JAK inhibitors because of differing selectivity is unknown. Therefore, we compared the inhibitory effects of tofacitinib, baricitinib, peficitinib, upadacitinib, and filgotinib on angiogenesis. Method: RA-derived fibroblast-like synoviocytes (RA-FLS) were seeded on type I collagen gel, and human umbilical vein endothelial cells (HUVECs) were directly added. The control and aforementioned JAK inhibitors were added to the medium, followed by stimulation with interleukin (IL)-6 and soluble IL-6 receptor (sIL-6R). Each JAK inhibitor's concentration was determined based on estimated blood concentrations. The vascular endothelial growth factor (VEGF) concentration was evaluated with an enzyme-linked immunosorbent assay using the medium from the first exchange. A migration assay was performed, and HUVEC migration was evaluated using CD31 fluorescence immunostaining. Results: Hematoxylin–eosin staining showed that compared with the non-JAKi treatment group, the JAKi treatment group markedly degenerated in the sub-lining and deep lining, with decreased lymphocyte infiltration and neovascularization [Rooney's score subscale, non-JAKi vs JAKi (median, 6.5 vs 2.5, p = 0.005)]. In vitro, IL-6 and sIL-6R administration increased VEGF production from RA-FLS and promoted neovascularization in HUVECs, and JAK-inhibitor administration, which decreased VEGF production from RA-FLS and suppressed HUVEC migration, inhibited neovascularization in RA-FLS and HUVEC co-cultures. Conclusions: The JAK inhibitors suppressed IL-6-induced angiogenesis via decreased VEGF production and HUVEC migration in RA-FLS and HUVEC co-cultures. No significant differences were observed among the JAK inhibitors, whose anti-angiogenic effect may be an important mechanism for RA treatment. Key Points • JAK inhibitors inhibit angiogenesis in RA by reducing VEGF production from RA-derived fibroblast-like synoviocytes. • Our study provides new insights into RA treatment by elucidating the anti-angiogenic effect of JAK inhibitors. [ABSTRACT FROM AUTHOR]

Importance: A relatively new therapeutic agent for osteoarthritis (OA), polydeoxyribonucleotide (PDRN), shows potential in treating human OA due to its regenerative and anti-inflammatory effects. However, studies on PDRN for canine OA are limited, and no study has investigated their use with mesenchymal stem cells (MSCs) conventionally used for OA treatment. Objective: This study aimed to evaluate the potential of PDRN and explore its combined effect with adipose tissue-derived MSCs (AdMSCs) in treating canine OA. Methods: To study the impact of PDRN, canine chondrocytes, synoviocytes, and AdMSCs were exposed to various PDRN concentrations, and viability was assessed using cell counting kit-8. The OA model was created by treating chondrocytes and synoviocytes with lipopolysaccharide, followed by treatment under three different conditions: PDRN alone, AdMSCs alone, and a combination of PDRN and AdMSCs. Using real-time quantitative polymerase chain reaction, the anti-inflammatory effects and mechanisms were investigated by quantitatively assessing pro-inflammatory cytokines, collagen degradation markers, adenosine A2a receptor (ADORA2A), and nuclear factor-kappa B. Results: PDRN alone and combined with AdMSCs significantly reduced the expression of pro-inflammatory cytokines and collagen degradation markers in an OA model. PDRN promoted AdMSC proliferation and upregulated ADORA2A expression. AdMSCs exhibited comprehensive anti-inflammatory effects through paracrine effects, and both substances reduced inflammatory gene expression through different mechanisms, potentially enhancing therapeutic effects. Conclusions and Relevance: The results indicate that PDRN is a safe and effective antiinflammatory material that can be used independently or as an adjuvant for AdMSCs. Although additional research is necessary, this study is significant because it provides a foundation for future research at the cellular level. [ABSTRACT FROM AUTHOR]

Abstract Background Osteoarthritis (OA) is a degenerative musculoskeletal disease that significantly impacts the quality of life. Currently, no validated biomarkers for early detection of OA are defined. The possibility of discovering OA biomarkers is the focus of this study. Methods Human primary OA synovial cells (SVs), isolated from discarded joint tissue of patients with Kellgren & Lawrence score (KL)

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BackgroundGlucose metabolism, specifically, hexokinase 2 (HK2), has a critical role in rheumatoid arthritis (RA) fibroblast-like synoviocyte (FLS) phenotype. HK2 localizes not only in the cytosol but also in the mitochondria, where it protects mitochondria against stress. We hypothesize that mitochondria-bound HK2 is a key regulator of RA FLS phenotype.MethodsHK2 localization was evaluated by confocal microscopy after FLS stimulation. RA FLSs were infected with Green fluorescent protein (GFP), full-length (FL)-HK2, or HK2 lacking its mitochondrial binding motif (HK2ΔN) expressing adenovirus (Ad). RA FLS was also incubated with methyl jasmonate (MJ; 2.5 mM), tofacitinib (1 µM), or methotrexate (1 µM). RA FLS was tested for migration and invasion and gene expression. Gene associations with HK2 expression were identified by examining single-cell RNA sequencing (scRNA-seq) data from murine models of arthritis. Mice were injected with K/BxN serum and given MJ. Ad-FLHK2 or Ad-HK2ΔN was injected into the knee of wild-type mice.ResultsCobalt chloride (CoCl2) and platelet-derived growth factor (PDGF) stimulation induced HK2 mitochondrial translocation. Overexpression of the HK2 mutant and MJ incubation reversed the invasive and migrative phenotype induced by FL-HK2 after PDGF stimulation, and MJ also decreased the expression of C-X-C Motif Chemokine Ligand 1 (CXCL1) and Collagen Type I Alpha 1 Chain (COL1A1). Of interest, tofacitinib but not methotrexate had an effect on HK2 dissociation from the mitochondria. In murine models, MJ treatment significantly decreased arthritis severity, whereas HK2FL was able to induce synovial hypertrophy as opposed to HK2ΔN.ConclusionOur results suggest that mitochondrial HK2 regulates the aggressive phenotype of RA FLS. New therapeutic approaches to dissociate HK2 from mitochondria offer a safer approach than global glycolysis inhibition.

Fibroblast-like synoviocytes (FLS) are mesenchymal-derived cells that play an important role in the physiology of the synovium by producing certain components of the synovial fluid and articular cartilage. In rheumatoid arthritis (RA), however, fibroblasts become a key driver of synovial inflammation and joint damage. Because of this, there has been recent interest in FLS as a therapeutic target in RA to avoid side effects such as systemic immune suppression associated with many existing RA treatments. In this review, we describe how approved treatments for RA affect FLS signaling and function and discuss the effects of investigational FLS-targeted drugs for RA.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that affects the synovial joint, which leads to inflammation, loss of function, joint destruction, and disability. The disease biology of RA involves complex interactions between genetic and environmental factors and is strongly associated with various immune cells, and each of the cell types contributes differently to disease pathogenesis. Several immunomodulatory molecules, such as cytokines, are secreted from the immune cells and intervene in the pathogenesis of RA. In immune cells, membrane proteins such as ion channels and transporters mediate the transport of charged ions to regulate intracellular signaling pathways. Ion channels control the membrane potential and effector functions such as cytotoxic activity. Moreover, clinical studies investigating patients with mutations and alterations in ion channels and transporters revealed their importance in effective immune responses. Recent studies have shown that voltage-gated potassium channels and calciumactivated potassium channels and their subtypes are involved in the regulation of immune cells and RA. Due to the role of these channels in the pathogenesis of RA and from multiple pieces of clinical evidence, they can be considered therapeutic targets for the treatment of RA. Here, we describe the role of voltage-gated and calcium-activated potassium channels and their subtypes in RA and their pharmacological application as drug targets. [ABSTRACT FROM AUTHOR]

Two new aporphine-derived alkaloids, aporaloids C and D (1 and 2), along with eight known biogenetically related alkaloids (3–10) were isolated from the stems of Fissistigma maclurei Merr. Their structures were elucidated by detailed analysis of NMR, HRESIMS, MS, IR, UV and Optical rotations data. Compounds 1 and 2 represent a rare example of N-methylol aporphine-derived alkaloids from natural sources. The inhibitory effect of all compounds on the proliferation of primary synovial cells was evaluated. Compound 3 showed potent inhibitory effect on the proliferation of synoviocytes with an IC50 value of 4.8 μM. Compounds 1, 2, 6–9 and 10 exhibited moderate inhibitory activity on synoviocytes, with IC50 values of 36.8, 37.1, 31.2 μM, 32.5, 36.3, 36.8 and 18.2 μM, respectively. [ABSTRACT FROM AUTHOR]

To observe the effect of curcumin treatment on serum inflammatory factors in rats with traumatic arthritis and explores the protective effect of articular surface cartilage in rats with traumatic arthritis. A control group (group A), a model group (group B), and a curcumin group (group C) were randomly chosen. Except for the group A, the curcumin model was established by removing the medial meniscus of the right knee in the other two groups. After the model was successfully established, the group C was intraperitoneally injected with curcumin solution 50 mg/kg, while the group A and B were injected with the same volume of dimethyl sulfoxide solution for 28 d. The levels of serum vascular endothelial growth factor and tumor necrosis factoralpha and interleukin-1 beta were measured by enzyme-linked immunosorbent assay method. The arthritis index score of the two groups was calculated, the pathological morphology of articular cartilage was observed by hematoxylin and eosin staining, and the apoptosis rate of knee chondrocytes was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Real-time polymerase chain reaction and Western blot methods were used to detect the expression of autophagy and apoptosis regulatory molecules Beclin-1, B-cell lymphoma 2, caspase-3, messenger ribonucleic acid and protein in knee cartilage. Curcumin can protect the articular surface cartilage of traumatic arthritis rats by reducing inflammatory factors in serum and regulating the apoptosis and autophagy of articular chondrocytes. Curcumin may be a potential drug for the treatment of traumatic arthritis. [ABSTRACT FROM AUTHOR]

BackgroundSynovitis (SI) is one of the most common and serious orthopedic diseases in horses of different age, breed and sex, which contributes to the development of osteoarthritis. The burden of SI includes economic loss and represents a real challenge for current veterinary health care. At the molecular level, fibroblasts-like synoviocytes (FLS) are recognized as major cell populations involved in SI pathogenesis. In the course of SI, FLSs are losing their protective and pro-regenerative cytological features, become highly proliferative and initiate various stress signaling pathways.MethodsFibroblast-like synoviocytes were treated with LPS in order to generate SI in vitro model. Mitochondria were isolated from peripheral blood derived mononuclear cells and co-cultured with FLS. After 24 h of culture, cells were subjected to RT-qPCR, western blot, cytometric and confocal microscopy analysis.ResultsMitochondrial transfer (MT) was observed in vitro studies using confocal microscopy. Further studies revealed, that MT to LPS-treated FLS reduced cell proliferation, modulated apoptosis and decreased inflammatory response. Overall, MT Resulted in the considerable recovery of recipient cells cytophysiological properties.ConclusionsPresented data provides evidence that mitochondria transfersignificantly modulate FLS proliferative and metabolic activity through improved mitochondrial biogenesis and dynamics in activated FLS. Obtained results for the first time demonstrate that horizontal MT might be considered as a therapeutic tool for synovitis treatment; however, further clinical studies are strongly required. Video abstract.

Abstract Background Juvenile Idiopathic Arthritis (JIA) induces growth disturbances in affected joints. Fibroblast-like synoviocytes (FLS) play a crucial role in JIA pathogenesis. FLS overexpress bone morphogenetic protein 4 (BMP4) and have a chondrocyte-like phenotype. FLS contribute directly to joint growth disturbances through endochondral bone formation. We investigated the ability of methotrexate to inhibit BMP4 expression and alter the hypertrophic chondrocyte-like phenotype of JIA FLS. Methods We selected primary cells from three subjects with persistent oligoarticular JIA, three subjects who eventually extended to a polyarticular disease course, which we termed extended-to-be (ETB), and three subjects who had polyarticular arthritis at time of diagnosis. We treated cells with methotrexate and two BMP4 inhibitors: noggin and chordin. We measured protein concentration from three chondrocyte cell markers: collagen II, aggrecan, and collagen X as well as BMP4. Results ColX, marker of chondrocyte hypertrophy, was significantly increased in polyarticular FLS when compared to both persistent FLS and ETB FLS, making polyarticular FLS the most like hypertrophic chondrocytes. Methotrexate caused significant decreases in BMP4 and ColX expression in persistent, ETB, and polyarticular FLS when compared to respective untreated cells. Ligand-binding BMP4 antagonists, noggin and chordin, caused significant decreases in ColX expression in FLS from all three disease courses and significant increases in collagen II protein, an early chondrocyte marker, when compared to respective untreated cells. Conclusions Methotrexate, the first-line therapy in the treatment of JIA, mimics BMP4 antagonists by effectively lowering BMP4 and ColX expression in FLS. Inhibiting FLS from undergoing hypertrophy could prevent these cells from contributing to joint growth disturbances via endochondral bone formation.

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.

Background: Juvenile Idiopathic Arthritis (JIA) induces growth disturbances in affected joints. Fibroblast-like synoviocytes (FLS) play a crucial role in JIA pathogenesis. FLS overexpress bone morphogenetic protein 4 (BMP4) and have a chondrocyte-like phenotype. FLS contribute directly to joint growth disturbances through endochondral bone formation. We investigated the ability of methotrexate to inhibit BMP4 expression and alter the hypertrophic chondrocyte-like phenotype of JIA FLS. Methods: We selected primary cells from three subjects with persistent oligoarticular JIA, three subjects who eventually extended to a polyarticular disease course, which we termed extended-to-be (ETB), and three subjects who had polyarticular arthritis at time of diagnosis. We treated cells with methotrexate and two BMP4 inhibitors: noggin and chordin. We measured protein concentration from three chondrocyte cell markers: collagen II, aggrecan, and collagen X as well as BMP4. Results: ColX, marker of chondrocyte hypertrophy, was significantly increased in polyarticular FLS when compared to both persistent FLS and ETB FLS, making polyarticular FLS the most like hypertrophic chondrocytes. Methotrexate caused significant decreases in BMP4 and ColX expression in persistent, ETB, and polyarticular FLS when compared to respective untreated cells. Ligand-binding BMP4 antagonists, noggin and chordin, caused significant decreases in ColX expression in FLS from all three disease courses and significant increases in collagen II protein, an early chondrocyte marker, when compared to respective untreated cells. Conclusions: Methotrexate, the first-line therapy in the treatment of JIA, mimics BMP4 antagonists by effectively lowering BMP4 and ColX expression in FLS. Inhibiting FLS from undergoing hypertrophy could prevent these cells from contributing to joint growth disturbances via endochondral bone formation. [ABSTRACT FROM AUTHOR]

Osteoarthritis (OA) is a multifaceted disease characterized by imbalances in extracellular matrix metabolism, chondrocyte and synoviocyte senescence, as well as inflammatory responses mediated by macrophages. Although there have been notable advancements in pharmacological and surgical interventions, achieving complete remission of OA remains a formidable challenge, oftentimes accompanied by significant side effects. Mesenchymal stem cells (MSCs) have emerged as a promising avenue for OA treatment, given their ability to differentiate into chondrocytes and facilitate cartilage repair, thereby mitigating the impact of an inflammatory microenvironment induced by macrophages. This comprehensive review aims to provide a concise overview of the diverse roles played by MSCs in the treatment of OA, while elucidating the underlying mechanisms behind these contributions. Specifically, the roles include: (a) Promotion of chondrocyte and synoviocyte regeneration; (b) Inhibition of extracellular matrix degradation; (c) Attenuating the macrophage-induced inflammatory microenvironment; (d) Alleviation of pain. Understanding the multifaceted roles played by MSCs in OA treatment is paramount for developing novel therapeutic strategies. By harnessing the regenerative potential and immunomodulatory properties of MSCs, it may be possible to devise more effective and safer approaches for managing OA. Further research and clinical studies are warranted to optimize the utilization of MSCs and realize their full potential in the field of OA therapeutics.

Osteoarthritis (OA) is a progressive degenerative disease resulting in joint deterioration. Synovial inflammation is present in the OA joint and has been associated with radiographic and pain progression. Several OA risk factors, including ageing, obesity, trauma and mechanical loading, play a role in OA pathogenesis, likely by modifying synovial biology. In addition, other factors, such as mitochondrial dysfunction, damage-associated molecular patterns, cytokines, metabolites and crystals in the synovium, activate synovial cells and mediate synovial inflammation. An understanding of the activated pathways that are involved in OA-related synovial inflammation could form the basis for the stratification of patients and the development of novel therapeutics. This Review focuses on the biology of the OA synovium, how the cells residing in or recruited to the synovium interact with each other, how they become activated, how they contribute to OA progression and their interplay with other joint structures.

Rheumatoid arthritis (RA) is an immune-mediated disease affecting diarthrodial joints that remains an unmet medical need despite improved therapy. This limitation likely reflects the diversity of pathogenic pathways in RA, with individual patients demonstrating variable responses to targeted therapies. Better understanding of RA pathogenesis would be aided by a more complete characterization of the disease. To tackle this challenge, we develop and apply a systems biology approach to identify important transcription factors (TFs) in individual RA fibroblast-like synoviocyte (FLS) cell lines by integrating transcriptomic and epigenomic information. Based on the relative importance of the identified TFs, we stratify the RA FLS cell lines into two subtypes with distinct phenotypes and predicted active pathways. We biologically validate these predictions for the top subtype-specific TF RARα and demonstrate differential regulation of TGFβ signaling in the two subtypes. This study characterizes clusters of RA cell lines with distinctive TF biology by integrating transcriptomic and epigenomic data, which could pave the way towards a greater understanding of disease heterogeneity.

Abstract Background The p53 isoform Δ133p53β is known to be associated with cancers driven by inflammation. Many of the features associated with the development of inflammation in rheumatoid arthritis (RA) parallel those evident in cancer progression. However, the role of this isoform in RA has not yet been explored. The aim of this study was to determine whether Δ133p53β is driving aggressive disease in RA. Methods Using RA patient synovia, we carried out RT-qPCR and RNAScope-ISH to determine both protein and mRNA levels of Δ133p53 and p53. We also used IHC to determine the location and type of cells with elevated levels of Δ133p53β. Plasma cytokines were also measured using a BioPlex cytokine panel and data analysed by the Milliplex Analyst software. Results Elevated levels of pro-inflammatory plasma cytokines were associated with synovia from RA patients displaying extensive tissue inflammation, increased immune cell infiltration and the highest levels of Δ133TP53 and TP53β mRNA. Located in perivascular regions of synovial sub-lining and surrounding ectopic lymphoid structures (ELS) were a subset of cells with high levels of CD90, a marker of ‘activated fibroblasts’ together with elevated levels of Δ133p53β. Conclusions Induction of Δ133p53β in CD90+ synovial fibroblasts leads to an increase in cytokine and chemokine expression and the recruitment of proinflammatory cells into the synovial joint, creating a persistently inflamed environment. Our results show that dysregulated expression of Δ133p53β could represent one of the early triggers in the immunopathogenesis of RA and actively perpetuates chronic synovial inflammation. Therefore, Δ133p53β could be used as a biomarker to identify RA patients more likely to develop aggressive disease who might benefit from targeted therapy to cytokines such as IL-6.

Fibroblast‐like synoviocytes accumulation, proliferation and activation, and the subsequent inflammatory mediators production play a key role in the progression of rheumatoid arthritis (RA). It is well established that Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signaling triggers inflammation, and induces cytokine levels in RA. Ohwia caudata have long been used against many disorders. However, in RA, the effects of O. caudata have not been elucidated. In the current study, synoviocytes were used to evaluate the suppressive effects of O. caudate extract (OCE) on the pro‐inflammatory cytokines production. In vitro, the underlying mechanisms by which OCE inhibits inflammatory response through regulation of suppressors of cytokine signaling 3 (SOCS3) and JAK2/STAT3 expression in IL‐17A‐treated HIG‐82 synoviocytes were investigated. The results demonstrated that the proliferation of IL‐17A‐challenged cells were increased in comparison with non‐stimulated control cells. The synoviocyte proliferation was decreased significantly of OCE concentrations in dose dependent manner. The p‐JAK2, p‐STAT3, interleukin (IL)‐1β, and IL‐6 were reduced in IL‐17A‐challenged cells treated with OCE. Furthermore, AZD1480 (a JAK2‐specific inhibitor) or WP1066 (a STAT3‐specific inhibitor) affected the inflammatory mediators production in IL‐17A‐challenged synoviocytes, and OCE failed to mitigate the IL‐17A‐induced inflammatory mediators and SOCS3, acting as a feedback inhibitor of the JAK/STAT3 pathway, in the presence of SOCS3 siRNA, indicating that the beneficial effects of OCE on the regulation of inflammatory response homeostasis were dependent on SOCS3 and the JAK2/STAT3 signaling pathway. Our study also showed that SOCS3 was markedly activated by OCE in RA fibroblast‐like synoviocytes, thereby decreasing the JAK/STAT3 pathway, and the IL‐1β, and IL‐6 activation. Thus, O. caudate should be further investigated as a candidate anti‐inflammatory and anti‐arthritic agent. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA) is a chronic immune-mediated disease that primarily affects the synovium of diarthrodial joints. During the course of RA, the synovium transforms into a hyperplastic invasive tissue that causes destruction of cartilage and bone. Fibroblast-like synoviocytes (FLS), which form the lining of the joint, are epigenetically imprinted with an aggressive phenotype in RA and have an important role in these pathological processes. In addition to producing the extracellular matrix and joint lubricants, FLS in RA produce pathogenic mediators such as cytokines and proteases that contribute to disease pathogenesis and perpetuation. The development of multi-omics integrative analyses have enabled new ways to dissect the mechanisms that imprint FLS, have helped to identify potential FLS subsets with distinct functions and have identified differences in FLS phenotypes between joints in individual patients. This Review provides an overview of advances in understanding of FLS biology and highlights omics approaches and studies that hold promise for identifying future therapeutic targets.

The pathological hallmark of rheumatoid arthritis (RA) is a synovial pannus that comprises proliferating and invasive fibroblast-like synoviocytes, infiltrating inflammatory cells, and an associated neoangiogenic response. Animal models have been established to study these pathological features of human RA. Spontaneous and induced animal models of RA primarily reflect inflammatory aspects of the disease. Among various induced animal models, collagen-induced arthritis (CIA) and collagen antibody-induced arthritis (CAIA) models are widely used to study the pathogenesis of RA. Improved transplantation techniques for severe combined immunodeficiency (SCID) mouse models of RA can be used to evaluate the effectiveness of potential therapeutics in human tissues and cells. This review provides basic information on various animal models of RA, including CIA and CAIA. In addition, we describe a SCID mouse coimplantation model that can measure the long-distance migration of human RA synoviocytes and cartilage destruction induced by these cells.

Abstract Background Synovitis (SI) is one of the most common and serious orthopedic diseases in horses of different age, breed and sex, which contributes to the development of osteoarthritis. The burden of SI includes economic loss and represents a real challenge for current veterinary health care. At the molecular level, fibroblasts-like synoviocytes (FLS) are recognized as major cell populations involved in SI pathogenesis. In the course of SI, FLSs are losing their protective and pro-regenerative cytological features, become highly proliferative and initiate various stress signaling pathways. Methods Fibroblast-like synoviocytes were treated with LPS in order to generate SI in vitro model. Mitochondria were isolated from peripheral blood derived mononuclear cells and co-cultured with FLS. After 24 h of culture, cells were subjected to RT-qPCR, western blot, cytometric and confocal microscopy analysis. Results Mitochondrial transfer (MT) was observed in vitro studies using confocal microscopy. Further studies revealed, that MT to LPS-treated FLS reduced cell proliferation, modulated apoptosis and decreased inflammatory response. Overall, MT Resulted in the considerable recovery of recipient cells cytophysiological properties. Conclusions Presented data provides evidence that mitochondria transfersignificantly modulate FLS proliferative and metabolic activity through improved mitochondrial biogenesis and dynamics in activated FLS. Obtained results for the first time demonstrate that horizontal MT might be considered as a therapeutic tool for synovitis treatment; however, further clinical studies are strongly required. Video abstract

Dual‐specificity phosphatase 5 (DUSP5) is a novel anti‐inflammatory modulator in many inflammatory diseases. However, the role of DUSP5 in fibroblast‐like synoviocytes (FLS) of rheumatoid arthritis (RA) remains unknown. In this study, we aimed to explore the biological function and regulation of DUSP5 in FLS. We found that lower DUSP5 expression level was detected in collagen‐induced arthritis (CIA) and synoviocyte MH7A. Overexpression of DUSP5 markedly decreased the proliferation, migration, and invasion of MH7A, which correlated with suppressing the phosphorylation of extracellular signal‐regulated kinase (ERK). Moreover, DUSP5 was identified as a novel target gene of miR‐216a‐3p, which was upregulated in FLS. Therefore, DUSP5 expression was negatively regulated by miR‐216a‐3p, and the effect of DUSP5 overexpression on FLS was reversed by miR‐216a‐3p mimics. Overall, our study demonstrates that DUSP5 is a miR‐216a‐3p target gene and its anti‐inflammatory function in FLS via inactivation of ERK. These results revealed that the miR‐216a‐3p/DUSP5 pathway may play a crucial role in the malignant behavior of FLS, which may serve as a new target for the treatment of RA. [ABSTRACT FROM AUTHOR]

Accumulation of senescent cells is the prominent risk factor for osteoarthritis (OA), accelerating the progression of OA through a senescence-associated secretory phenotype (SASP). Recent studies emphasized the existence of senescent synoviocytes in OA and the therapeutic effect of removing senescent synoviocytes. Ceria nanoparticles (CeNP) have exhibited therapeutic effects in multiple age-related diseases due to their unique capability of ROS scavenging. However, the role of CeNP in OA remains unknown. Our results revealed that CeNP could inhibit the expression of senescence and SASP biomarkers in multiple passaged and hydrogen-peroxide-treated synoviocytes by removing ROS. In vivo, the concentration of ROS in the synovial tissue was remarkably suppressed after the intra-articular injection of CeNP. Likewise, CeNP reduced the expression of senescence and SASP biomarkers as determined by immunohistochemistry analysis. The mechanistic study showed that CeNP inactivated the NFκB pathway in senescent synoviocytes. Finally, safranin O–fast green staining showed milder destruction of articular cartilage in the CeNP-treated group compared with the OA group. Overall, our study suggested that CeNP attenuated senescence and protected cartilage from degeneration via scavenging ROS and inactivating the NFκB signaling pathway. This study has potentially significant implications in the field of OA as it provides a novel strategy for OA treatment. [ABSTRACT FROM AUTHOR]

Rheumatoid synovitis is infiltrated by immune cells that interact with synoviocytes, leading to the pannus formation. Inflammation or cell interaction effects are mainly evaluated with cytokine production, cell proliferation or migration. Few studies interest on cell morphology. Here, the purpose was to deepen some morphological changes of synoviocytes or immune cells under inflammatory conditions. Inflammatory cytokines, IL-17 and TNF that are largely involved in RA pathogenesis, induced a change in synoviocyte morphology, inducing a retracted cell with higher number of pseudopodia. Several morphological parameters decreased in inflammatory conditions: cell confluence, area and motility speed. The same impact on cell morphology was observed in co-culture of synoviocytes and immune cells in inflammatory/non-inflammatory conditions or with cell activation (miming the in vivo situation), affecting both cell types: synoviocytes were retracted and inversely immune cells proliferated, indicating that cell activation induced a morphological change of cells. In contrast, with RA but not control synoviocytes, cell interactions were not sufficient to affect PBMC and synoviocyte morphology. The morphological effect came only from the inflammatory environment. These findings reveal that the inflammatory environment or cell interactions induced massive changes in control synoviocytes, with cell retraction and increase of pseudopodia number, leading to better interactions with other cells. Except in the case of RA, the inflammatory environment was absolutely required for such changes.

Osteoarthritis (OA) is the leading cause of joint failure, yet the underlying mechanisms remain elusive, and no approved therapies that slow progression exist. Dysregulated integrin function was previously implicated in OA pathogenesis. However, the roles of integrin αVβ3 and the integrin-associated receptor CD47 in OA remain largely unknown. Here, transcriptomic and proteomic analyses of human and murine osteoarthritic tissues revealed dysregulated expression of αVβ3, CD47, and their ligands. Using genetically deficient mice and pharmacologic inhibitors, we showed that αVβ3, CD47, and the downstream signaling molecules Fyn and FAK are crucial to OA pathogenesis. MicroPET/CT imaging of a mouse model showed elevated ligand-binding capacities of integrin αVβ3 and CD47 in osteoarthritic joints. Further, our in vitro studies demonstrated that chondrocyte breakdown products, derived from articular cartilage of individuals with OA, induced αVβ3/CD47-dependent expression of inflammatory and degradative mediators, and revealed the downstream signaling network. Our findings identify a central role for dysregulated αVβ3 and CD47 signaling in OA pathogenesis and suggest that activation of αVβ3 and CD47 signaling in many articular cell types contributes to inflammation and joint destruction in OA. Thus, the data presented here provide a rationale for targeting αVβ3, CD47, and their signaling pathways as a disease-modifying therapy.

ObjectiveWe aimed to understand the role of the tyrosine phosphatase PTPN14-which in cancer cells modulates the Hippo pathway by retaining YAP in the cytosol-in fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA).MethodsGene/protein expression levels were measured by quantitative PCR and/or Western blotting. Gene knockdown in RA FLS was achieved using antisense oligonucleotides. The interaction between PTPN14 and YAP was assessed by immunoprecipitation. The cellular localisation of YAP and SMAD3 was examined via immunofluorescence. SMAD reporter studies were carried out in HEK293T cells. The RA FLS/cartilage coimplantation and passive K/BxN models were used to examine the role of YAP in arthritis.ResultsRA FLS displayed overexpression of PTPN14 when compared with FLS from patients with osteoarthritis (OA). PTPN14 knockdown in RA FLS impaired TGFβ-dependent expression of MMP13 and potentiation of TNF signalling. In RA FLS, PTPN14 formed a complex with YAP. Expression of PTPN14 or nuclear YAP-but not of a non-YAP-interacting PTPN14 mutant-enhanced SMAD reporter activity. YAP promoted TGFβ-dependent SMAD3 nuclear localisation in RA FLS. Differences in epigenetic marks within Hippo pathway genes, including YAP, were found between RA FLS and OA FLS. Inhibition of YAP reduced RA FLS pathogenic behaviour and ameliorated arthritis severity.ConclusionIn RA FLS, PTPN14 and YAP promote nuclear localisation of SMAD3. YAP enhances a range of RA FLS pathogenic behaviours which, together with epigenetic evidence, points to the Hippo pathway as an important regulator of RA FLS behaviour.

Metabolomic studies show that rheumatoid arthritis (RA) is associated with metabolic disruption that may be therapeutically targetable. Among them, glucose metabolism and glycolytic intermediaries seem to have an important role in fibroblast-like synoviocytes (FLS) phenotype and might contribute to early stage disease pathogenesis. RA FLS are transformed from quiescent to aggressive and metabolically active cells and several works have shown that glucose metabolism is increased in activated FLS. Glycolytic inhibitors reduce not only FLS aggressive phenotype in vitro but also decrease bone and cartilage damage in several murine models of arthritis. Essential glycolytic enzymes, including hexokinase 2 (HK2) and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB) enzymes, have important roles in FLS behavior. Of interest, HK2 is an inducible enzyme present only in the inflamed rheumatic tissues compared to osteoarthritis synovium. It is a contributor to glucose metabolism that could be selectively targeted without compromising systemic homeostasis as a novel approach for combination therapy independent of systemic immunosuppression. More information about metabolic targets that do not compromise global glucose metabolism in normal cells is needed.

Osteoarthritis (OA) is a chronic, progressive, severely debilitating, and multifactorial joint disease that is recognized as the most common type of arthritis. During the last decade, it shows an incremental global rise in prevalence and incidence. The interaction between etiologic factors that mediate joint degradation has been explored in numerous studies. However, the underlying processes that induce OA remain obscure, largely due to the variety and complexity of these mechanisms. During synovial joint dysfunction, the osteochondral unit undergoes cellular phenotypic and functional alterations. At the cellular level, the synovial membrane is influenced by cartilage and subchondral bone cleavage fragments and extracellular matrix (ECM) degradation products from apoptotic and necrotic cells. These "foreign bodies" serve as danger-associated molecular patterns (DAMPs) that trigger innate immunity, eliciting and sustaining low-grade inflammation in the synovium. In this review, we explore the cellular and molecular communication networks established between the major joint compartments—the synovial membrane, cartilage, and subchondral bone of normal and OA-affected joints. [ABSTRACT FROM AUTHOR]

Osteoarthritis (OA) is hallmarked as a silent progressive rheumatic disease of the whole joint. The accumulation of inflammatory and catabolic factors such as IL6, TNFα, and COX2 drives the OA pathophysiology into cartilage degradation, synovia inflammation, and bone destruction. There is no clinical available OA treatment. Although traditional ayurvedic medicine has been using Boswellia serrata extracts (BSE) as an antirheumatic treatment for a millennium, none of the BSE components have been clinically approved. Recently, β boswellic acid (BBA) has been shown to reduce in vivo OA-cartilage loss through an unknown mechanism. We used computational pharmacology, proteomics, transcriptomics, and metabolomics to present solid evidence of BBA therapeutic properties in mouse and primary human OA joint cells. Specifically, BBA binds to the innate immune receptor Toll-like Receptor 4 (TLR4) complex and inhibits both TLR4 and Interleukin 1 Receptor (IL1R) signaling in OA chondrocytes, osteoblasts, and synoviocytes. Moreover, BBA inhibition of TLR4/IL1R downregulated reactive oxygen species (ROS) synthesis and MAPK p38/NFκB, NLRP3, IFNαβ, TNF, and ECM-related pathways. Altogether, we present a solid bulk of evidence that BBA blocks OA innate immune responses and could be transferred into the clinic as an alimentary supplement or as a therapeutic tool after clinical trial evaluations. [ABSTRACT FROM AUTHOR]

Ferroalloy has shown potential as implant materials, but little attention has been paid to their effects on synovial tissue ferroptosis. This study aimed to examine the mechanical properties, degradability and biocompatibility of Fe–30Mn–0.6N alloy and effects of it on synovial tissue ferroptosis. Tensile testing showed that Fe–30Mn–0.6N alloys exhibited tensile strength of 487 ± 18 MPa, yield strength of 221 ± 10 MPa, elongation of 16.9 ± 0.3% and Young's modulus of 37.7 ± 1.3 GPa. In vivo experiments, the cross‐sectional area of the Fe–30Mn–0.6N alloys decreased by 73.32 ± 12.73% after 8 weeks of implantation. The results of scanning electron microscopy (SEM) and surface elemental analysis (EDS) showed that the Fe–30Mn–0.6N alloys had more Ca, O, C and P element deposition (p <.05). After 2, 4 and 8 weeks of implantation, no inflammatory response was observed in peri‐implant synovial tissue of Fe–30Mn–0.6N and Ti–6Al–4V alloys, and Fe–30Mn–0.6N alloys did not affect the expression of the ferroptosis inhibitory gene Glutathione peroxidase 4 (GPX4). Compared with the control group, 30% Fe–30Mn–0.6N alloy extracts did not affect the cell viability (p >.05) in vitro, and intracellular Fe2+ and the reactive oxygen species (ROS) was significantly reduced (p <.05). WB and PCR results showed that the 30% extracts increased the protein activity and mRNA expression of GPX4, FTH1 and SLC7A11 in synoviocytes, but had no effect on PTGS2 and p53. It is concluded that Fe–30Mn–0.6N had degradability and biocompatibility in peri‐implant synovial tissue, and did not induce significantly ferroptosis in synoviocytes. [ABSTRACT FROM AUTHOR]

ObjectivesRecent studies indicate that glucose metabolism is altered in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). Hexokinases (HKs) catalyse the first step in glucose metabolism, and HK2 constitutes the principal HK inducible isoform. We hypothesise that HK2 contributes to the synovial lining hypertrophy and plays a critical role in bone and cartilage damage.MethodsHK1 and HK2 expression were determined in RA and osteoarthritis (OA) synovial tissue by immunohistochemistry. RA FLS were transfected with either HK1 or HK2 siRNA, or infected with either adenovirus (ad)-GFP, ad-HK1 or ad-HK2. FLS migration and invasion were assessed. To study the role of HK2 in vivo, 108 particles of ad-HK2 or ad-GFP were injected into the knee of wild-type mice. K/BxN serum transfer arthritis was induced in HK2F/F mice harbouring Col1a1-Cre (HK2Col1), to delete HK2 in non-haematopoietic cells.ResultsHK2 is particular of RA histopathology (9/9 RA; 1/8 OA) and colocalises with FLS markers. Silencing HK2 in RA FLS resulted in a less invasive and migratory phenotype. Consistently, overexpression of HK2 resulted in an increased ability to migrate and invade. It also increased extracellular lactate production. Intra-articular injection of ad-HK2 in normal knees dramatically increased synovial lining thickness, FLS activation and proliferation. HK2 was highly expressed in the synovial lining after K/BxN serum transfer arthritis. HK2Col1 mice significantly showed decreased arthritis severity, bone and cartilage damage.ConclusionHK2 is specifically expressed in RA synovial lining and regulates FLS aggressive functions. HK2 might be an attractive selective metabolic target safer than global glycolysis for RA treatment.

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.

Transient receptor potential (TRP) ankyrin repeat 1 (TRPA1), which is involved in inflammatory pain sensation, is activated by endogenous factors, such as intracellular Zn2+ and hydrogen peroxide, and by irritant chemical compounds. The synthetic compound JT010 potently and selectively activates human TRPA1 (hTRPA1) among the TRPs. Therefore, JT010 is a useful tool for analyzing TRPA1 functions in biological systems. Here, we show that JT010 is a potent activator of hTRPA1, but not mouse TRPA1 (mTRPA1) in human embryonic kidney (HEK) cells expressing hTRPA1 and mTRPA1. Application of 0.3–100 nM of JT010 to HEK cells with hTRPA1 induced large Ca2+ responses. However, in HEK cells with mTRPA1, the response was small. In contrast, both TRPA1s were effectively activated by allyl isothiocyanate (AITC) at 10–100 μM. Similar selective activation of hTRPA1 by JT010 was observed in electrophysiological experiments. Additionally, JT010 activated TRPA1 in human fibroblast-like synoviocytes with inflammation, but not TRPA1 in mouse dorsal root ganglion cells. As cysteine at 621 (C621) of hTRPA1, a critical cysteine for interaction with JT010, is conserved in mTRPA1, we applied JT010 to HEK cells with mutations in mTRPA1, where the different residue of mTRPA1 with tyrosine at 60 (Y60), with histidine at 1023 (H1023), and with asparagine at 1027 (N1027) were substituted with cysteine in hTRPA1. However, these mutants showed low sensitivity to JT010. In contrast, the mutation of hTRPA1 at position 669 from phenylalanine to methionine (F669M), comprising methionine at 670 in mTRPA1 (M670), significantly reduced the response to JT010. Moreover, the double mutant at S669 and M670 of mTRPA1 to S669E and M670F, respectively, induced slight but substantial sensitivity to 30 and 100 nM JT010. Taken together, our findings demonstrate that JT010 potently and selectively activates hTRPA1 but not mTRPA1. [ABSTRACT FROM AUTHOR]

The morphology of fibroblast-like synoviocytes (FLS) issued from the synovial fluid (SF) of patients suffering from osteoarthritis (OA), rheumatoid arthritis (RA), or from healthy subjects (H), as well as the ultrastructure and mechanical properties of the FLS-secreted extracellular vesicles (EV), were analyzed by confocal microscopy, transmission electron microscopy, atomic force microscopy, and tribological tests. EV released under healthy conditions were constituted of several lipid bilayers surrounding a viscous inner core. This "gel-in" vesicular structure ensured high mechanical resistance of single vesicles and good tribological properties of the lubricant. RA, and to a lesser extent OA, synovial vesicles had altered morphology, corresponding to a "gel-out" situation with vesicles surrounded by a viscous gel, poor mechanical resistance, and poor lubricating qualities. When subjected to inflammatory conditions, healthy cells developed phenotypes similar to that of RA samples, which reinforces the importance of inflammatory processes in the loss of lubricating properties of SF. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA) is an autoimmune and chronic inflammatory disease that results in joint destruction and disability in the adult population. RA is characterized by the accumulation and proliferation of fibroblast‐like synoviocytes. Many pro‐inflammatory mediators are associated with RA, such as interleukin (IL)‐1β, IL‐6, IL‐17, cyclooxygenase‐2 (COX‐2), and nuclear factor kappa B (NF‐κB). Furthermore, IL‐17 upregulates the production of other pro‐inflammatory mediators, including IL‐1β and IL‐6, and promotes the recruitment of neutrophils in RA. Artemisia argyi, a traditional Chinese herbal medicine, is used for the treatment of diseases associated with inflammation and microbial infections. In this study, synoviocytes (HIG‐82) were treated with varying doses of A. argyi extract (AAE) following IL‐17A stimulation. Proliferation of the IL‐17A‐stimulated cells was increased compared to that of the non‐stimulated control cells. However, cell proliferation decreased significantly in a dose‐dependent manner following AAE treatment. Treatment of IL‐17A‐stimulated cells with AAE resulted in decreased levels of phosphorylated (p)‐NF‐κB, p‐IκB‐α, and COX‐2. Enzyme‐linked immunosorbent assay results showed that IL‐1β and IL‐6 levels were increased in the IL‐17A‐stimulated group but decreased in the AAE treatment group. Additionally, we found that AAE facilitated nuclear factor erythroid 2‐related factor 2 (Nrf2) expression and promoted its nuclear translocation, thereby inducing the expression of heme oxygenase‐1. Moreover, AAE did not attenuate IL‐17A‐induced inflammatory mediator production in the presence of ML385, an Nrf2‐specific inhibitor. These results suggest that the downregulation of expression of pro‐inflammatory cytokines and the transcription factor NF‐κB by AAE may be a potential therapeutic strategy for reducing inflammation associated with RA. [ABSTRACT FROM AUTHOR]

An increasing number of studies show how changes in intracellular metabolic pathways alter tumor and immune cell function. However, little information about metabolic changes in other cell types, including synovial fibroblasts, is available. In rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) are the most common cell type at the pannus-cartilage junction and contribute to joint destruction through their production of cytokines, chemokines, and matrix-degrading molecules and by migrating and invading joint cartilage. In this review, we show that these cells differ from healthy synovial fibroblasts, not only in their marker expression, proto-oncogene expression, or their epigenetic changes, but also in their intracellular metabolism. These metabolic changes must occur due to the stressful microenvironment of inflamed tissues, where concentrations of crucial nutrients such as glucose, glutamine, and oxygen are spatially and temporally heterogeneous. In addition, these metabolic changes will increase metabolite exchange between fibroblast and other synovial cells, which can potentially be activated. Glucose and phospholipid metabolism as well as bioactive lipids, including sphingosine-1-phosphate and lysophosphatidic acid, among others, are involved in FLS activation. These metabolic changes likely contribute to FLS involvement in aspects of immune response initiation or abnormal immune responses and strongly contribute to joint destruction.

To investigate the expression and roles of TAM (Tyro3/Axl/Mer) receptor tyrosine kinases (TK) in synovial fluid and synovial tissue of patients with rheumatoid arthritis (RA). The expression of TAM TKs in the synovial fluid and synovial tissues of RA and osteoarthritis (OA) patients was measured by ELISA and immunohistochemistry. The relationships between soluble TAM TKs (sTAM TKs) levels and the clinical features, laboratory parameters and disease activity were analyzed in RA. The concentrations of sTAM TK in the synovial fluids of RA patients were increased in comparison to those of OA patients. Compared with OA patients, the expression of membrane Tyro3 TK (mTyro3 TK) and mMer TK in RA patient synovial tissue were significantly increased, which may partly explain the possible mechanism of elevated levels of sTAM TK in RA patient synovial fluid. sAxl TK levels were decreased in RA patients under sulfasalazine treatment and elevated in patients under Iguratimod treatment. Furthermore, sTyro3 TK levels were positively correlated with erythrocyte sedimentation rate (ESR) and negatively correlated with white blood cells (WBCs), red blood cells (RBCs), and hemoglobin (HB) in RA patients. The levels of sMer TK were positively associated with disease duration and rheumatoid factor (RF) and negatively correlated with HB, complement 3 (C3), and C4. Taken together, TAM TKs might be involved in RA synovial tissue inflammation. [ABSTRACT FROM AUTHOR]

Introduction: Rheumatoid and psoriatic arthritis are both characterised by synovial destruction associated with a higher turnover of the extracellular matrix. In both conditions, inflammatory processes create hypoxic environments which destabilise members of the plasminogen activating system. Aim: Comparing the effect of bioactive concentrations of urokinase (uPA) and serpine (PAI-1) on cellular survival of human fibroblast-like-synoviocytes (HFLS) in rich and hypoxic growth media. Material and methods: Monocultures of HFLS were exposed to bioactive uPA and PAI-1 concentrations in both media conditions for 24, 48 and 72 h. Cellular survival was evaluated with a cell viability assay by spectrum absorbance of formazan reduced WST-8. Results: PAI-1 at 0.1 and 1 μg/ml was found to stimulate cell viability under hypoxic stress at 48 and 72 h of incubation, with the effect increasing from 48 to 72 h. uPA increased cell viability in rich medium at 48 and 72 h of incubation between 5 and 40 ng/l, but was found to reduce viability at 80 ng/l at 24 and 48 h. PAI-1 increased cell viability in the hypoxic stress model, while high concentrations of uPA decreased cell viability in rich medium. Conclusions: The alternative modes of function at extreme concentrations provide a novel description of PAI-1 and uPA activity based on their colocalization and mutual buffering capacity, helping to place these molecules more accurately in the context of arthritic synovial deterioration. [ABSTRACT FROM AUTHOR]

Introduction: Rheumatoid 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., Objectives: The present research aimed to investigate the effect of vimentin on the abnormal growth characteristics of RA synoviocytes and the targeted regulatory role of CBN., Methods: Cell 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., Results: The 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., Conclusion: CBN-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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Production and hosting by Elsevier B.V.)

Background: Osteoarthritis (OA) is the most common joint disease worldwide. Routine treatment options are limited, and total knee replacement surgeries often come with complications. In recent years, the use of biologics, such as Wharton's jelly (Wj) derived from the umbilical cord (UC), has gained popularity. While mesenchymal stem cells (MSCs) derived from Wj show promise in restoring articular cartilage, they also have some limitations. Recent studies have indicated that exosomes isolated from acellular Wj may offer advantages under certain conditions., Objective: To investigate the anti-inflammatory properties of exosomes isolated from Wj in synoviocytes., Methods: Decellularization of Wj was performed using sterile umbilical cords obtained from patients. Next, the exosomes were isolated from Wj using ultracentrifugation. After characterizing the exosomes, they were co-cultured with inflammatory synovial fibroblast cells (HIG-82) for 24 hours. Then, the gene expression levels and protein contents of some important inflammatory mediators including metalloproteinase-13 (MMP-13), cyclooxygenase-2 (COX2) and inducible nitric oxide synthase (iNOS) were measured in the cells using real-time PCR and ELISA tests, respectively., Results: The expression levels of MMP-13, COX-2, and iNOS genes were significantly reduced in the cultured cells treated with exosomes compared to untreated cells. Moreover, the content of MMP-13, COX-2, and iNOS proteins were significantly lower in the supernatant of the cultured cells compared to the control., Conclusion: Wj-derived exosomes exhibit notable anti-inflammatory properties, which can help mitigate inflammation in the synovial environment of joints. However, further research is required to fully understand their benefits and potential applications in treating osteoarthritis.

Sarcoma samples from 33 dogs, 25 subcutaneous and 8 articular, were submitted for cytokeratin immunohistochemistry. Eight of the 25 subcutaneous sarcomas (32%) expressed cytokeratin in 1% to 50% of the neoplastic cells. Of the 7 articular sarcomas evaluated, 1 (14%) expressed cytokeratin in 10% of neoplastic cells. The Kaplan-Meier survival analysis showed that the mean overall survival of dogs with subcutaneous sarcomas (28.1 months [confidence interval [CI]:17.8, 38.4]) did not significantly differ from those with articular sarcomas (24.8 months [CI = 0.5, 29.0]). Overall survival of dogs with sarcomas (both locations combined) immunoreactive for cytokeratin (31.2 months [CI = 17.8, 44.6]) did not differ from those not immunoreactive for cytokeratin (22.0 months [CI = 8.4, 35.6]). Therefore, cytokeratin expression does not indicate synovial origin ( P = .64) and neither sarcoma location ( P = .76) nor cytokeratin expression ( P = .53) affects patient overall survival in this small study. The use of cytokeratin immunohistochemistry is not helpful to determine synovial origin of sarcomas in dogs., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Importance: A relatively new therapeutic agent for osteoarthritis (OA), polydeoxyribonucleotide (PDRN), shows potential in treating human OA due to its regenerative and anti-inflammatory effects. However, studies on PDRN for canine OA are limited, and no study has investigated their use with mesenchymal stem cells (MSCs) conventionally used for OA treatment., Objective: This study aimed to evaluate the potential of PDRN and explore its combined effect with adipose tissue-derived MSCs (AdMSCs) in treating canine OA., Methods: To study the impact of PDRN, canine chondrocytes, synoviocytes, and AdMSCs were exposed to various PDRN concentrations, and viability was assessed using cell counting kit-8. The OA model was created by treating chondrocytes and synoviocytes with lipopolysaccharide, followed by treatment under three different conditions: PDRN alone, AdMSCs alone, and a combination of PDRN and AdMSCs. Using real-time quantitative polymerase chain reaction, the anti-inflammatory effects and mechanisms were investigated by quantitatively assessing pro-inflammatory cytokines, collagen degradation markers, adenosine A2a receptor (ADORA2A), and nuclear factor-kappa B., Results: PDRN alone and combined with AdMSCs significantly reduced the expression of pro-inflammatory cytokines and collagen degradation markers in an OA model. PDRN promoted AdMSC proliferation and upregulated ADORA2A expression. AdMSCs exhibited comprehensive anti-inflammatory effects through paracrine effects, and both substances reduced inflammatory gene expression through different mechanisms, potentially enhancing therapeutic effects., Conclusions and Relevance: The results indicate that PDRN is a safe and effective anti-inflammatory material that can be used independently or as an adjuvant for AdMSCs. Although additional research is necessary, this study is significant because it provides a foundation for future research at the cellular level., Competing Interests: The authors declare no conflicts of interest., (© 2024 The Korean Society of Veterinary Science.)

Osteoarthritis (OA) affects more than 300 million people worldwide and it is about to become the first disabling disease. OA is characterized by the progressive degradation of the articular cartilage but is a disease of the whole joint. Articular innate immune responses (IIR) associated with tissue degradation contribute to its progression. However, no treatment is available to block these IIRs. Through data text mining and computational pharmacology, we identified two clinical available drugs, naloxone, and thalidomide, with potential inhibitory properties on toll-like receptor 4 (TLR4), a major activator of these IIR. Proteome analysis confirmed that activation of this receptor or the IL1 receptor generated OA-like and gout-like proteomic changes in human primary chondrocytes. Both compounds were found to block TLR4 complex and inhibit TLR4 and IL1R-mediated IIR in OA chondrocytes, osteoblasts, and synoviocytes. Furthermore, naloxone and thalidomide inhibitory effects involved the downregulation of the NLRP3 inflammasome pathway, which is downstream of TLR4/IL1R signaling. We demonstrated that these compounds, within a therapeutic range of concentrations, exhibited anti-inflammatory and anti-catabolic properties in joint primary OA cells without any toxic effect. This data underpins naloxone & thalidomide repurpose to treat OA-associated inflammatory responses.

Apple polyphenols (AP) have strong antioxidant and anti-inflammatory properties. We examined the effects of AP on the progression of osteoarthritis (OA) AP was administered to surgically-induced OA model rats for 4 or 8 weeks. This treatment suppressed inflammation and oxidative stress in the synovium, resulting in a decrease in the OA severity score, and the expression of tumor necrosis factor (TNF)-α and matrix metalloproteinase (MMP)-13 in the synovium. It was suggested that long-term administration of AP may be effective for the treatment of OA. In addition, superoxide dismutase (SOD) activity was enhanced in serum samples by AP. AP or its constituent procyanidin B2 (PC) were added to HIG-82 synoviocytes. The results showed that AP enhanced cell proliferation and hyaluronan production. This indicates that AP may improve synovial conditions in OA and suppress OA progression. These effects may be attributed to the antioxidant and anti-inflammatory properties of AP. • Apple polyphenols (AP) have strong antioxidant and anti-inflammatory properties. • The effects of AP on the progression of osteoarthritis (OA) is studied. • AP suppresses inflammation and oxidative stress in the synovium. • Administration of AP may be effective to treat OA. • Superoxide dismutase (SOD) activity in serum was enhanced by AP. [ABSTRACT FROM AUTHOR]

Objective·To construct a low toxicity and high-efficiency joint synovial siRNA transfection vector OPEI, and inhibit tumor necrosis factor receptor-associated factor 6 (TRAF6) to rescue the chondrogenic ability of bone marrow mesenchymal stem cells (BMSCs) under inflammatory conditions. Methods·The osteoarthritis (OA) models of SD rats (n=20) were established by medial meniscectomy of knee. Another sham operation group (n=10) was established, and the meniscus remained intact. The cartilage and synovium were collected 3 months after surgery. The expression of TRAF6 was detected by immunohistochemistry, and Western blotting was used to detect the expression of MMP13, TRAF6 and p-p65 in primary rat synovial cells induced by interleukin-1β (IL-1β). Further, the small molecule polyethylenimine (PEI) derivative OPEI was synthesized in anhydrous anaerobic environment, and the ability of OPEI to encapsulate siRNA was detected by agarose gel electrophoresis. The particle size and Zeta potential of OPEI/siRNA complex were measured by dynamic light scattering. The cytotoxicity of the formed complex to rat primary synovial cells was detected by MTT method. The effect of the complex on synovial cell apoptosis was analyzed by flow cytometry. The transfection efficiency of siRNA by OPEI in synovial cells in vivo and in vitro was detected by laser confocal technique and fluorescence microscopy. The proteoglycan content of chondrocyte matrix was detected by alcian blue staining. Results·Compared with the sham operation group, TRAF6 was highly expressed in synovium and cartilage of the rat OA models, and inhibition of TRAF6 could significantly reduce the expression of MMP13 and p-p65 in IL-1β-stimulated primary synovial cells. The siRNA transfection efficiency of OPEI in the rat primary synovial cells was as high as 99.33%. A large number of synovial cells ingested siRNA on the 3rd and the 7th day after injection of OPEI / Cy3-siRNA into rat knee joints. Two days after OPEI / siTRAF6 complex with different w/w ratios was transfected into rat primary synovial cells, the results of TRAF6 protein expression showed that the knockout efficiency of TRAF6 gene in the OPEI / siTRAF6 group was 49.05%, 74.61% and 83.18% respectively when the mass ratio was 3∶1, 4∶1 and 5∶1. After TRAF6 gene was silenced by OPEI/siTRAF6, compared with the control group (OPEI/siNC), alcian blue staining of chondrocytes was significantly enhanced under IL-1β stimulation. Conclusion·OPEI is a low toxicity and high efficiency siRNA transfection vector, silencing TRAF6 in synovial cells could promote OA cartilage regeneration. [ABSTRACT FROM AUTHOR]