Osteoarthritis Synovial Tissue Research Articles

Assessment of disease activity in patients with rheumatoid arthritis using plasma tumour M2-pyruvate kinase test.

BackgroundPyruvate kinase M2 (PKM2) is an enzyme that regulates the final process of glycolysis and exists in tetrameric and dimeric forms. The dimeric form of PKM2, also known as tumour M2-PK, increases when aerobic glycolysis is augmented, a feature observed in rheumatoid arthritis (RA). We investigated whether plasma tumour M2-PK is elevated in patients with RA and whether its levels correlate with disease activity.MethodsPlasma levels of tumour M2-PK were measured for patients with RA (n=151), those with osteoarthritis (OA) (n=37), and controls (n=37). We evaluated the association between plasma tumour M2-PK and continuous variables using Pearson’s correlation analysis, and multivariate logistic regression analysis to determine the association between plasma tumour M2-PK and disease activity status. Knee synovial tissue blocks from patients with RA and OA were subjected to real-time quantitative PCR (qPCR) using two different primers for PKM2 and tumour M2-PK immunohistochemical (IHC) staining.ResultsThe tumour M2-PK level significantly correlated with the disease activity score in 28 joints (DAS28)-erythrocyte sedimentation rate (ESR) (r=0.546, p<0.001) and DAS28-C-reactive protein (CRP) (r=0.589, p<0.001). Moreover, repeat testing of tumour M2-PK levels in 20 patients revealed a significant decline in tumour M2-PK levels after reduction in inflammation (p<0.001). Area under the receiver operating characteristic curve (AUROC) analysis demonstrated that upon incorporation of tumour M2-PK, ESR, and CRP, the area under the curve was 0.962 for distinguishing moderate/high from remission/low disease activity. Adjusted logistic regression also revealed that a tumour M2-PK >43.9 U/mL (OR 3.672, p=0.042) independently predicted moderate/high disease activity status. Furthermore, tumour M2-PK levels in patients with RA were significantly higher than in those with OA and controls (all p<0.001). However, no differences were found in PKM2 expression in RA and OA synovial tissues as assessed by qPCR, and IHC analysis revealed negligible tumour M2-PK expression in the synovial tissues.ConclusionCirculating plasma tumour M2-PK levels may be a clinically useful indicator for evaluating disease activity and RA diagnosis.

Preliminary study on immune cells in the synovium of end-stage osteoarthritis and rheumatoid arthritis patients: neutrophils and IgG4-secreting plasma cells as differential diagnosis candidates

ObjectiveImmune cell evaluation could be useful for clarifying etiopathogenesis, providing a support for formulating the diagnoses of clinically similar joint pathologies or guiding indications for possible therapeutic targets. To contribute to differential diagnosis in joint pathologies we performed an immunophenotypical profile analyzing different immune cells in synovial tissues from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). MethodsThe Krenn and immunologic synovitis (IMSYC) scores, which include the evaluation of T lymphocytes (CD3 positive), B lymphocytes (CD20), endothelial cells (CD31), macrophages (CD68) and proliferating cells (Ki-67 positive) were used to analyze the synovial tissue samples. Moreover, to corroborate immune activation, neutrophils (CD15 positive), NK cells (CD56 positive), plasma cells (CD138 positive), IgG4 and IgG4 secreting-CD138 cells were analyzed using immunohistochemical techniques. ResultsWe confirmed that all the samples had a high synovitis score according to both the Krenn and IMSYC scores. In both the RA and OA groups, we found similar scores for CD3 (T lymphocytes), CD20 (B lymphocytes), CD31 (endothelial cells), CD56 (NK cells), CD68 (macrophages) CD138 (plasma cells) and IgG4. In contrast, CD15 (neutrophils) was significantly higher in RA compared to OA. Interestingly, IgG4 secreting-CD138 cells were significantly higher in RA than OA, even if CD138 had the same score in both the RA and OA samples. ConclusionsThis study found that the scores for different immune cells were similar in both RA and OA synovial tissue with a high synovitis score. CD15 and IgG4 secreting-CD138 were the only immune cells with a higher score in RA compared to OA, suggesting a potential use for discriminating among pathologies with a high synovitis score.

Gene Expression Microarray Data Identify Hub Genes Involved in Osteoarthritis

The present study was performed to explore the underlying molecular mechanisms and screen hub genes of osteoarthritis (OA) via bioinformatics analysis. In total, twenty-five OA synovial tissue samples and 25 normal synovial tissue samples were derived from three datasets, namely, GSE55457, GSE55235, and GSE1919, and were used to identify the differentially expressed genes (DEGs) of OA by R language. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of DEGs were conducted using the Database for Annotation, Visualization, and Integrated Discovery (DAVID). A Venn diagram was built to show the potential hub genes identified in all three datasets. The STRING database was used for constructing the protein–protein interaction (PPI) networks and submodules of DEGs. We identified 507 upregulated and 620 downregulated genes. Upregulated DEGs were significantly involved in immune response, MHC class II receptor activity, and presented in the extracellular region, while downregulated DEGs were mainly enriched in response to organic substances, extracellular region parts, and cadmium ion binding. Results of KEGG analysis indicated that the upregulated DEGs mainly existed in cell adhesion molecules (CAMs), while downregulated DEGs were significantly involved in the MAPK signaling pathway. A total of eighteen intersection genes were identified across the three datasets. These include Nell-1, ATF3, RhoB, STC1, and VEGFA. In addition, 10 hub genes including CXCL12, CXCL8, CCL20, and CCL4 were found in the PPI network and module construction. Identification of DEGs and hub genes associated with OA may be helpful for revealing the molecular mechanisms of OA and further promotes the development of relevant biomarkers and drug targets.

Evaluation of Biomarkers and Immune Microenvironment of Osteoarthritis: Evidence From Omics Data and Machine Learning.

Objectives: This study aimed to identify novel biomarkers for osteoarthritis (OA) and explore potential pathological immune cell infiltration. Methods: We identified differentially expressed genes (DEGs) between OA and normal synovial tissues using the limma package in R, and performed enrichment analyses to understand the functions and enriched pathways of DEGs. Weighted gene co-expression network analysis (WGCNA) and distinct machine-learning algorithms were then used to identify hub modules and candidate biomarkers. We assessed the diagnostic value of the candidate biomarkers using receiver operating characteristic (ROC) analysis. We then used the CIBERSORT algorithm to analyze immune cell infiltration patterns, and the Wilcoxon test to screen out hub immune cells that might affect OA occurrence. Finally, the expression levels of hub biomarkers were confirmed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Results: We identified 102 up-regulated genes and 110 down-regulated genes. The functional enrichment analysis results showed that DEGs are enriched mainly in immune response pathways. Combining the results of the algorithms and ROC analysis, we identified GUCA1A and NELL1 as potential diagnostic biomarkers for OA, and validated their diagnosibility using an external dataset. Construction of a TF-mRNA-miRNA network enabled prediction of potential candidate compounds targeting hub biomarkers. Immune cell infiltration analyses revealed the expression of hub biomarkers to be correlated with CD8 T cells, memory B cells, M0/M2 macrophages, resting mast cells and resting dendritic cells. qRT-PCR results showed both GUCA1A and NELL1 were significantly increased in OA samples (p < 0.01). All validations are consistent with the microarray hybridization, indicating that GUCA1A and NELL1 may be involved in the pathogenesis of OA. Conclusion: The findings suggest that GUCA1A and NELL1, closely related to OA occurrence and progression, represent new OA candidate markers, and that immune cell infiltration plays a significant role in the progression of OA.

IL-6-MEDIATED OVEREXPRESSION OF HYBID (HYALURONAN-BINDING PROTEIN INVOLVED IN HYALURONAN DEPOLYMERIZATION) IN OSTEOARTHRITIC SYNOVIUM IS IMPLICATED FOR HYALURONAN DEGRADATION IN SYNOVIAL FLUIDS IN PATIENTS WITH KNEE OSTEOARTHRITIS

Purpose: Hyaluronan (HA)-binding protein involved in HA depolymerization (HYBID), alias cell migration-inducing protein (CEMIP) and KIAA1199, plays a key role in HA degradation. Although HYBID is expressed by rheumatoid synovial lining and sub-lining cells, little is known about cellular localization of HYBID in osteoarthritic synovial tissue. Transmembrane protein 2 (TMEM2), a transmembrane protein with sequence similarity to HYBID, is reportedly a hyaluronidase in mice. However, no studies have shown the expression or functional role of TMEM2 in human tissues.

SYNOVIAL BIOMARKERS IN OSTEOARTHRITIS: A SYSTEMATIC REVIEW AND META-ANALYSIS

Purpose: Synovial cells contribute to osteoarthritis (OA) onset and progression, releasing and responding to multiple pro-inflammatory mediators and extracellular matrix (ECM) degrading enzymes. The quest for true disease status biomarkers in OA is still open and the identification of these factor combinations can have diagnostic and prognostic value. The study objective was to conduct a systematic literature review to establish if published study data, when combined in a meta-analysis, can identify synovial biomarkers which are statistically significant in human OA synovial tissue, compared to healthy/non-OA patients, thereby providing an indication of disease status.

Exosomes Derived From miR-212-5p Overexpressed Human Synovial Mesenchymal Stem Cells Suppress Chondrocyte Degeneration and Inflammation by Targeting ELF3.

Excessive chondrocyte degeneration and inflammation are the pathological features of osteoarthritis (OA), and altered miR-212-5p may contribute to meniscus and cartilage degeneration. Whether exosomes derived from miR-212-5p overexpressed synovial mesenchymal stem cells (SMSC-212-5p-Exos) could be utilized to treat degenerative chondrocytes is investigated in this study. Down-regulated miR-212-5p and up-regulated E74 Like ETS Transcription Factor 3 (ELF3) expression were detected in OA synovial tissues, which showed a negative correlation (r = −0.55, p = 0.002). miR-212-5p directly targeted ELF3 and regulated the relative expression of ELF3 in SMSCs as indicated by luciferase reporter assay and RT-PCR. The relative expression of ELF3, chondrocyte degeneration-related molecules, matrix metalloproteinase, and inflammatory molecules were detected in chondrocytes stimulated with interleukin (IL)-1β or co-incubated with SMSC-212-5p-Exos or SMSCs-derived exosomes (SMSC-Exos). IL-1β induced up-regulation of ELF3, down-regulation of degeneration molecules (Collagen II, Aggrecan, and Sox9), up-regulation of matrix metalloproteinase (MMP-1, MMP-3, and MMP-13), and up-regulation of inflammatory molecules (IL-6, MCP-1, TNF-α, COX-2, and iNOS) could be inhibited by SMSC-212-5p-Exos or SMSC-Exos administration. When compared with the SMSC-Exos, SMSC-212-5p-Exos showed more treatment benefits. All of these indicate that SMSC-212-5p-Exos could suppress chondrocyte degeneration and inflammation by targeting ELF3, which can be considered as a disease-modifying strategy.

Cordycerebroside A inhibits ICAM-1-dependent M1 monocyte adhesion to osteoarthritis synovial fibroblasts.

Osteoarthritis (OA) is represented by the accumulation and adhesion of M1 macrophages into synovium tissues in the joint microenvironment and subsequent inflammatory response. Cordycerebroside A, a cerebroside compound isolated from Cordyceps militaris, exhibits anti-inflammatory activity, but has not yet been examined in M1 macrophages during OA disease. Our results indicate higher expression of M1 macrophage markers in synovium tissue from OA patients compared with normal healthy controls. Records from the Gene Expression Omnibus (GEO) data set and our clinic samples revealed higher levels of ICAM-1 (a critical adhesion molecule during OA disease) and CD86 (a M1 macrophage marker) in OA synovial tissue than in healthy tissue. The same effects were found in rats with OA induced by anterior cruciate ligament transaction (ACLT). We also found that cordycerebroside A inhibited ICAM-1 synthesis and antagonized M1 macrophage adhesion to OA synovial fibroblasts by inhibiting the ERK/AP-1 pathway. Thus, cordycerebroside A displayed novel anti-arthritic effects. PRACTICAL APPLICATIONS: Here we report a higher level of M1 macrophage markers and ICAM-1 in synovium tissue from OA patients compared with normal healthy controls by using GEO data set and our clinic samples. The same effects were revealed in rats with OA induced by ACLT. Cordycerebroside A significantly suppressed ICAM-1 production and diminished M1 macrophage adhesion to OA synovial fibroblasts. Therefore, cordycerebroside A exhibited novel anti-OA functions.

Single-Cell Profiles of Age-Related Osteoarthritis Uncover Underlying Heterogeneity Associated With Disease Progression.

Objective: Osteoarthritis (OA) is the most common chronic degenerative joint disease, which represents the leading cause of age-related disability. Here, this study aimed to depict the intercellular heterogeneity of OA synovial tissues. Methods: Single-cell RNA sequencing (scRNA-seq) data were preprocessed and quality controlled by the Seurat package. Cell cluster was presented and cell types were annotated based on the mRNA expression of corresponding marker genes by the SingleR package. Cell-cell communication was assessed among different cell types. After integrating the GSE55235 and GSE55457 datasets, differentially expressed genes were identified between OA and normal synovial tissues. Then, differentially expressed marker genes were overlapped and their biological functions were analyzed. Results: Totally, five immune cell subpopulations were annotated in OA synovial tissues including macrophages, dendritic cells, T cells, monocytes and B cells. Pseudo-time analysis revealed the underlying evolution process in the inflammatory microenvironment of OA synovial tissue. There was close crosstalk between five cell types according to the ligand-receptor network. The genetic heterogeneity was investigated between OA and normal synovial tissues. Furthermore, functional annotation analysis showed the intercellular heterogeneity across immune cells in OA synovial tissues. Conclusion: This study offered insights into the heterogeneity of OA, which provided in-depth understanding of the transcriptomic diversities within synovial tissue. This transcriptional heterogeneity may improve our understanding on OA pathogenesis and provide potential molecular therapeutic targets for OA.

Synergistic upregulation of ADAMTS4 (aggrecanase-1) by cytokines and its suppression in knee osteoarthritic synovial fibroblasts

AbstractThe ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family includes nine members with aggrecan-degrading activity, i.e., ADAMTS1, 4, 5, 8, 9, 15, 16, 18, and 20. However, their systematic expression profile in knee osteoarthritis (OA) synovium and effects of cytokines and growth factors on the expression in OA synovial fibroblasts remain elusive. In this study, expression of all nine aggrecanolytic ADAMTS species was assessed by quantitative real-time PCR in OA and control normal synovial tissues. OA synovial fibroblasts were treated with interleukin-1α (IL-1α), IL-1β, tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β), vascular endothelial growth factor165, and heparin-binding epidermal growth factor, and analyzed for the expression of the ADAMTS species. The signaling pathways and inhibition of ADAMTS4 expression by high-molecular-weight hyaluronan, adalimumab, tocilizumab, and signaling molecule inhibitors were studied. ADAMTS1, 4, 5, 9, and 16 were expressed in OA synovium, but only ADAMTS4 expression was significantly higher in OA as compared to normal synovium. IL-1α, TNF-α, and TGF-β markedly increased ADAMTS4 expression, while their effects were minimal for the other ADAMTS species. ADAMTS4 was synergistically upregulated by treatment with IL-1α and TNF-α, IL-1α and TGF-β, or IL-1α, TNF-α and TGF-β. The signaling molecules' inhibitors demonstrated that IL-1α-induced ADAMTS4 expression is predominantly through TGF-β-associated kinase 1 (TAK1), and the TNF-α-stimulated expression is via TAK1 and nuclear factor-κB (NF-κB). The TGF-β-promoted expression was through the activin receptor-like kinase 5 (ALK5)/Smad2/3, TAK1, and non-TAK1 pathways. Adalimumab blocked TNF-α-stimulated expression. ADAMTS4 expression co-stimulated with IL-1α, TNF-α and TGF-β was abolished by treatment with adalimumab, TAK1 inhibitor, and ALK5/Smad2/3 inhibitor. These data demonstrate marked and synergistic upregulation of ADAMTS4 by IL-1α, TNF-α and TGF-β in OA synovial fibroblasts, and suggest that concurrent therapy with an anti-TNF-α drug and inhibitor(s) may be useful for prevention against aggrecan degradation in OA.

Single cell transcriptomics in human osteoarthritis synovium and in silico deconvoluted bulk RNA sequencing

To reveal the heterogeneity of different cell types of osteoarthritis (OA) synovial tissues at a single-cell resolution, and determine by novel methodology whether bulk-RNA-seq data could be deconvoluted to create in silico scRNA-seq data for synovial tissue analyses. OA scRNA-seq data (102,077 synoviocytes) were provided by 17 patients undergoing total knee arthroplasty; 9 tissues with matched scRNA-seq and bulk RNA-seq data were used to evaluate six in silico gene deconvolution tools. Predicted and observed cell types and proportions were compared to identify the best deconvolution tool for synovium. We identified seven distinct cell types in OA synovial tissues. Gene deconvolution identified three (of six) platforms as suitable for extrapolating cellular gene expression from bulk RNA-seq data. Using paired scRNA-seq and bulk RNA-seq data, an "arthritis" specific signature matrix was created and validated to have a significantly better predictive performance for synoviocytes than a default signature matrix. Use of the machine learning tool, Cell-type Identification By Estimating Relative Subsets of RNA Transcripts x (CIBERSORTx), to analyze rheumatoid arthritis (RA) and OA bulk RNA-seq data yielded proportions of T cells and fibroblasts that were similar to the gold standard observations from RA and OA scRNA-seq data, respectively. This novel study revealed heterogeneity of synovial cell types in OA and the feasibility of gene deconvolution for synovial tissue.

Antcin K inhibits VEGF-dependent angiogenesis in human rheumatoid arthritis synovial fibroblasts.

Antrodia cinnamomea is a well-known medicinal mushroom in Taiwan that exhibits anti-inflammatory biological activities. In rheumatoid arthritis (RA), chronic inflammation and angiogenesis driven by proinflammatory cytokines reflect the severity of the disease. Although biological treatments have improved the outlook for RA, no healing exists. Moreover, the available pharmacotherapies do not work for all patients and drug safety is a major consideration. Investigations into plant-based medicines hope to reveal better, more tolerable agents. We examined whether Antcin K, a phytosterol isolated from A. cinnamomea, has anti-angiogenic activity in RA. The GSE12021 gene dataset from the Gene Expression Omnibus (GEO) database was examined for levels of vascular endothelial growth factor (VEGF) expression in 10 RA and 10 osteoarthritis (OA) synovial tissue samples. In clinical samples, VEGF expression was analyzed by immunohistochemical staining and ELISA in normal and RA synovial tissue, as well as OA and RA synovial fluid. Collagen-induced arthritis (CIA) and control tissue was stained with hematoxylin and eosin (H&E) for histological changes; Safranin O/Fast Green staining examined histopathological changes and evidence of bone erosion. Human RA synovial fibroblasts (RASFs) were incubated with Antcin K and cell viability was examined by the MTT assay. VEGF mRNA expression was detected in RASFs using qPCR. Antcin K significantly inhibited VEGF expression and ameliorates endothelial progenitor cell (EPC) migration and tube formation in RASFs by downregulating the phospholipase C-γ/protein kinase C-α pathway. Antcin K also induced anti-angiogenic effects in human RASFs without cytotoxicity. PRACTICAL APPLICATIONS: Analysis of GEO dataset samples and human synovial fluids or synovial tissues revealed higher VEGF levels in rheumatoid arthritis (RA) samples compared with osteoarthritis (OA) and healthy control samples. VEGF levels were also higher in mice with collagen-induced arthritis (CIA) than in healthy controls. Antcin K markedly suppressed VEGF expression in human RA synovial fibroblasts and inhibited the migration and tube formation of epithelial progenitor cells (EPCs) by downregulating the phospholipase C-γ/protein kinase C-α pathway. Further investigations are warranted to examine the effects of Antcin K in other angiogenesis-associated disorders.

Betulin suppresses TNF-α and IL-1β production in osteoarthritis synovial fibroblasts by inhibiting the MEK/ERK/NF-κB pathway

Osteoarthritis (OA) is characterized by the infiltration of a number of proinflammatory cytokines into the joint microenvironment. Betulin is a natural analogue of betulinic acid, a lupane-type pentacyclic triterpene that displays many important biological activities, including anti-inflammatory activity, although the effects on inflammatory cytokine production in OA are unknown. Analyses of samples from the Gene Expression Omnibus (GEO) dataset and our own clinical samples revealed higher levels of tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β) in OA synovial tissue compared with normal healthy tissue. This was also the case in synovial tissue from normal healthy rats and rats subjected to anterior cruciate ligament transaction (ACLT)-induced OA. High-throughput screening of inflammatory cytokines in betulin-treated OA synovial fibroblasts (OASFs) identified TNF-α and IL-1β as potential targets of betulin. Further analyses found that betulin suppresses TNF-α and IL-1β production in OASFs by inhibiting mitogen-activated protein kinase (MEK), extracellular signal-regulated kinase (ERK) and nuclear factor kappa B (NF-κB) signaling cascades. Betulin shows promise for the treatment of OA.

Cordycerebroside A suppresses VCAM-dependent monocyte adhesion in osteoarthritis synovial fibroblasts by inhibiting MEK/ERK/AP-1 signaling

• VCAM1 and CD11b higher levels express in OA compared with healthy controls. • Cordycerebroside A suppressed VCAM-1 mediated-monocyte adhesion in human OASF. • The MEK/ERK/AP-1 signaling regulated cordycerebroside A effects. Osteoarthritis (OA) is characterized by the infiltration and adhesion of monocyte into the joint synovium. Vascular cell adhesion molecule 1 (VCAM-1) is a critical cell adhesion molecule that controls monocyte motility during OA progression. Cordycerebroside A, a cerebroside compound isolated from Cordyceps militaris , inhibits the production of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in synovial macrophages, but has not yet been investigated in OA. Gene Expression Omnibus (GEO) dataset analysis revealed higher levels of VCAM-1 and CD11b (a monocyte marker) in OA synovial tissue compared with normal healthy tissue. The same results were observed in anterior cruciate ligament transaction (ACLT)-induced OA in rats compared with normal healthy controls. Cordycerebroside A markedly suppressed VCAM-1 expression and monocyte adhesion in human OA synovial fibroblasts. The MEK, ERK and AP-1 signaling cascades regulated cordycerebroside A-induced inhibition of VCAM-1 production. Thus, cordycerebroside A is a promising agent for the treatment of OA.

Quantitative Proteomic Analysis of Synovial Tissue Reveals That Upregulated OLFM4 Aggravates Inflammation in Rheumatoid Arthritis.

Tandem mass tag (TMT)-coupled liquid chromatography coupled with tandem mass spectrometry is a powerful method to investigate synovial tissue protein profiles in patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Protein was isolated from synovial tissue samples of 22 patients and labeled with a TMT kit. Over 500 proteins were identified as the differential expression protein on comparing RA and OA synovial tissue, including 239 upregulated and 271 downregulated proteins. Data are available via ProteomeXchange with identifier PXD027703. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis showed that the majority participated in the developmental processes and protein processing in the endoplasmic reticulum. Olfactomedin 4 (OLFM4), a secreted glycoprotein, in joint inflammation of RA was explored. OLFM4 was upregulated in RA synovial tissue samples. In fibroblast-like synoviocytes (FLS), inflammation cytokines, TNF-α, interleukin (IL)-1β, and LPS can upregulate OLFM4. After OLFM4 knockdown under TNF-α stimulation, RA FLS proliferation was inhibited and the expression of CXCL9, CXCL11, and MMP-1 was decreased. Overall, the RA synovial tissue protein expression profile by proteomic analysis shows some unique targets in RA pathophysiology, and OLFM4 in FLS plays an important role in RA joint inflammation. OLFM4 can be a promising therapeutic target in RA synovial tissue.

Hyaluronic acid synthesis, degradation, and crosslinking in equine osteoarthritis: TNF-\u03b1-TSG-6-mediated HC-HA formation

BackgroundTNF-α-stimulated gene 6 (TSG-6) protein, a TNF-α-responsive hyaladherin, possesses enzymatic activity that can catalyze covalent crosslinks of the polysaccharide hyaluronic acid (HA) to another protein to form heavy chain-hyaluronic acid (HC-HA) complexes in pathological conditions such as osteoarthritis (OA). Here, we examined HA synthase and inflammatory gene expression; synovial fluid HA, TNF-α, and viscosity; and TSG-6-mediated HC-HA complex formation in an equine OA model. The objectives of this study were to (1) evaluate the TNF-α-TSG-6-HC-HA signaling pathway across multiple joint tissues, including synovial membrane, cartilage, and synovial fluid, and (2) determine the impact of OA on synovial fluid composition and biophysical properties.MethodsHA and inflammatory cytokine concentrations (TNF-α, IL-1β, CCL2, 3, 5, and 11) were analyzed in synovial fluid from 63 OA and 25 control joints, and HA synthase (HAS1-3), TSG-6, and hyaluronan-degrading enzyme (HYAL2, HEXA) gene expression was measured in synovial membrane and cartilage. HA molecular weight (MW) distributions were determined using agarose gel electrophoresis and solid-state nanopore measurements, and HC-HA complex formation was detected via immunoblotting and immunofluorescence. SEC-MALS was used to evaluate TSG-6-mediated HA crosslinking, and synovial fluid and HA solution viscosities were analyzed using multiple particle-tracking microrheology and microfluidic measurements, respectively.ResultsTNF-α concentrations were greater in OA synovial fluid, and TSG6 expression was upregulated in OA synovial membrane and cartilage. TSG-6-mediated HC-HA complex formation was greater in OA synovial fluid and tissues than controls, and HC-HA was localized to both synovial membrane and superficial zone chondrocytes in OA joints. SEC-MALS demonstrated macromolecular aggregation of low MW HA in the presence of TSG-6 and inter-α-inhibitor with concurrent increases in viscosity.ConclusionsSynovial fluid TNF-α concentrations, synovial membrane and cartilage TSG6 gene expression, and HC-HA complex formation were increased in equine OA. Despite the ability of TSG-6 to induce macromolecular aggregation of low MW HA with resultant increases in the viscosity of low MW HA solutions in vitro, HA concentration was the primary determinant of synovial fluid viscosity rather than HA MW or HC-HA crosslinking. The TNF-α-TSG-6-HC-HA pathway may represent a potential therapeutic target in OA.

Identification of differential key biomarkers in the synovial tissue between rheumatoid arthritis and osteoarthritis using bioinformatics analysis.

Rheumatoid arthritis (RA) and osteoarthritis (OA) are two common joint diseases with similar clinical manifestations. Our study aimed to identify differential gene biomarkers in the synovial tissue between RA and OA using bioinformatics analysis and validation. GSE36700, GSE1919, GSE12021, GSE55235, GSE55584, and GSE55457 datasets were downloaded from the Gene Expression Omnibus database. A total of 57 RA samples and 46 OA samples were included. The differentially expressed genes (DEGs) were identified. The Gene Ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were also performed. Protein-protein interaction (PPI) network of DEGs and the hub genes were constructed and visualized via Search Tool for the Retrieval of Interacting Genes/Proteins, Cytoscape, and R. Selected hub genes were validated via reverse transcription-polymerase chain reaction. A total of 41 DEGs were identified. GO functional enrichment analysis showed that DEGs were enriched in immune response, signal transduction, regulation of immune response for biological process, in plasma membrane and extracellular region for cell component, and antigen binding and serine-type endopeptidase activity for molecular function. KEGG pathway analysis showed that DEGs were enriched in cytokine-cytokine receptor interaction and chemokine signaling pathway. PPI network analysis established 70 nodes and 120 edges and 15 hub genes were identified. The expression of CXCL13, CXCL10, and ADIPOQ was statistically different between RA and OA synovial tissue. Differential expression of CXCL13, CXCL10, and ADIPOQ between RA and OA synovial tissue may provide new insights for understanding the RA development and difference between RA and OA. Key Points • Bioinformatics analysis was used to identify the differentially expressed genes in the synovial tissue between rheumatoid arthritis and osteoarthritis. • CXCL13, CXCL10, and ADIPOQ might provide new insight for understanding the differences between RA and OA.

HTR2B and SLC5A3 Are Specific Markers in Age-Related Osteoarthritis and Involved in Apoptosis and Inflammation of Osteoarthritis Synovial Cells.

Objective: Osteoarthritis (OA) is a heterogeneous age-related disease, which is badly difficult to cure due to its complex regulatory networks of pathogenesis. This study explored OA-specific genes in synovial tissues and validated their roles on apoptosis and inflammation of OA synovial cells. Methods: Weighted correlation network analysis (WGCNA) was employed to explore OA-related co-expression modules in the GSE55235 and GSE55457 datasets. Then, this study screened OA-specific genes. After validation of these genes in the GSE12021 and GSE32317 datasets, HTR2B and SLC5A3 were obtained. Their expression was detected in human OA and healthy synovial tissues by RT-qPCR and western blot. OA rat models were constructed by anterior cruciate ligament transection (ACLT) operation. In OA synovial cells, HTR2B and SLC5A3 proteins were examined via western blot. After transfection with sh-HTR2B or sh-SLC5A3, apoptosis and inflammation of OA synovial cells were investigated by flow cytometry and western blot. Results: A total of 17 OA-specific DEGs were identified, which were significantly enriched in inflammation pathways. Among them, HTR2B and SLC5A3 were highly expressed in end-than early-stage OA. Their up-regulation was validated in human OA synovial tissues and ACLT-induced OA synovial cells. Knockdown of HTR2B and SLC5A3 restrained apoptosis and increased TGF-β and IL-4 expression as well as reduced TNF-α and IL-1β expression in OA synovial cells. Conclusion: Collectively, this study identified two OA-specific markers HTR2B and SLC5A3 and their knockdown ameliorated apoptosis and inflammation of OA synovial cells.

The infrapatellar fat pad produces interleukin-6-secreting T cells in response to a proteoglycan aggrecan peptide and provides dominant soluble mediators different from that present in synovial fluid.

The purpose of this study was to investigate the effects of osteoarthritis (OA) peripheral blood mononuclear cell (PBMC) -stimulating proteoglycan aggrecan peptides on T cells present in infrapatellar fat pads (IPFPs) and synovial tissues, and to correlate these findings with mediators present in synovial fluid of OA patients. We tested for interleukin-6 (IL-6) -producing T cells in IPFPs of patients with knee OA using ELISPOT. Cytokine and cytotoxic mediator production from OA PBMCs, IPFPs, synovial tissues, and synovial fluids in response to proteoglycan aggrecan peptides were quantified by cytometric bead array. Patterns of cytokine and cytotoxic mediator production were analyzed and compared. T cells from IPFPs elicited strong responses towards the p263-280 peptide by secreting IL-6. In addition, there was a trend that the p263-280 peptide stimulated higher production of cytokines/cytotoxic mediators than other proteoglycan aggrecan peptides, although this was not statistically significant. In patients with knee OA, a group of cytotoxic mediators (sFas, perforin, granzyme A, and granulysin) and IL-6 were detectable at high levels from the synovial fluid. In addition, inflammation in patients with knee OA was more pronounced in joint-surrounding tissues than levels in circulating peripheral blood. Our data suggest that T cells responding to the p263-280 peptide contribute to the secretion of various soluble mediators that are found within the synovial fluid. We also identified potential new candidates that may serve as biomarkers of knee OA.

Identification of Disease-Specific Hub Biomarkers and Immune Infiltration in Osteoarthritis and Rheumatoid Arthritis Synovial Tissues by Bioinformatics Analysis.

Background Osteoarthritis (OA) and rheumatoid arthritis (RA) are well-known cause of joint disability. Although they have shown the analogous clinical features involving chronic synovitis that progresses to cartilage and bone destruction, the pathogenesis that initiates and perpetuates synovial lesions between RA and OA remains elusive. Objective This study is aimed at identifying disease-specific hub genes, exploring immune cell infiltration, and elucidating the underlying mechanisms associated with RA and OA synovial lesion. Methods Gene expression profiles (GSE55235, GSE55457, GSE55584, and GSE12021) were selected from Gene Expression Omnibus for analysis. Differentially expressed genes (DEGs) were identified by the “LIMMA” package in Bioconductor. The DEGs were identified by Gene Ontology (GO) and KEGG pathway analysis. A protein-protein interaction network was constructed to identify candidate hub genes by using STRING and Cytoscape. Hub genes were identified by validating from GSE12021. Furthermore, we employed the CIBERSORT website to assess immune cell infiltration between OA and RA. Finally, we explored the correlation between the levels of hub genes and relative proportion of immune cells in OA and RA. Results We identified 68 DEGs which were mainly enriched in immune response and chemokine signaling pathway. Six hub genes with a cutoff of AUC > 0.80 by ROC analysis and relative expression of P < 0.05 were identified successfully. Compared with OA, the RA synovial tissues consisted of a higher proportion of 7 immune cells, whereas 4 immune cells were found in relatively lower proportion (P < 0.05). In addition, the levels of 6 hub genes were closely associated with relative proportion of 11 immune cells in OA and RA. Conclusions We used bioinformatics analysis to identify hub genes and explored immune cell infiltration of immune microenvironment in synovial tissues. Our results should offer insights into the underlying molecular mechanisms of synovial lesion and provide potential target for immune-based therapies of OA and RA.