Rheumatoid Arthritis Chondrocytes Research Articles

Isoliquiritigenin inhibits apoptosis and ameliorates oxidative stress in rheumatoid arthritis chondrocytes through the Nrf2/HO-1-mediated pathway

Rheumatoid arthritis (RA) is a chronic inflammatory condition known for its irreversible destructive impact on the joints. Chondrocytes play a pivotal role in the production and maintenance of the cartilage matrix. However, the presence of inflammatory cytokines can hinder chondrocyte proliferation and promote apoptosis. Isoliquiritigenin (ISL), a flavonoid, potentially exerts protective effects against various inflammatory diseases. However, its specific role in regulating the nuclear factor E2-associated factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway in chondrocytes in RA remains unclear. To investigate this, this study used human chondrocytes and Sprague-Dawley rats to construct in vitro and in vivo RA models, respectively. The study findings reveal that cytokines markedly induced oxidative stress, the activation of matrix metalloproteinases, and apoptosis both in vitro and in vivo. Notably, ISL treatment significantly mitigated these effects. Moreover, Nrf2 or HO-1 inhibitors reversed the protective effects of ISL, attenuated the expression of Nrf2/HO-1 and peroxisome proliferator-activated receptor gamma-coactivator-1α, and promoted chondrocyte apoptosis. This finding indicates that ISL primarily targets the Nrf2/HO-1 pathway in RA chondrocytes. Moreover, ISL treatment led to improved behavior scores, reduced paw thickness, and mitigated joint damage as well as ameliorated oxidative stress in skeletal muscles in an RA rat model. In conclusion, this study highlights the pivotal role of the Nrf2/HO-1 pathway in the protective effects of ISL and demonstrates the potential of ISL as a treatment option for RA.

Single-cell RNA sequencing reveals distinct chondrocyte states in femoral cartilage under weight-bearing load in Rheumatoid arthritis.

Rheumatoid arthritis (RA) is a common autoimmune joint disease, the pathogenesis of which is still unclear. Cartilage damage is one of the main manifestations of the disease. Chondrocytes are the main functional component of articular cartilage, which is relevant to disease progression. Mechanical loading affects the structure and function of articular cartilage and chondrocytes, but the effect of weight bearing on chondrocytes in rheumatoid arthritis is still unclear. In this paper, single-cell RNA sequencing (scRNA-seq) was performed on collected cartilage from the weight-bearing region (Fb group) and non-weight-bearing region (Fnb group) of the femur, and the differences between the Fb and Fnb groups were analyzed by cell type annotation, pseudotime analysis, enrichment analysis, cell interactions, single-cell regulatory network inference and clustering (SCENIC) for each cell type. A total of 87,542 cells were analyzed and divided into 9 clusters. Six chondrocyte subpopulations were finally identified by cellular annotation, and two new chondrocyte subtypes were annotated as immune-associated chondrocytes. The presence of each chondrocyte subpopulation and its distribution were verified using immunohistochemical staining (IHC). In this study, the atlas of femoral cartilage in knee rheumatoid arthritis and 2 new immune-related chondrocytes were validated using scRNA-seq and IHC, and chondrocytes in the weight-bearing and non-weight-bearing regions of the femur were compared. There might be a process of macrophage polarization transition in MCs in response to mechanical loading, as in macrophages. Two new immune-associated chondrocytes were identified. MCs have contrasting functions in different regions, which might provide insight into the role of immune and mechanical loading on chondrocytes in the development of knee rheumatoid osteoarthritis.

Identification of a novel cuproptosis-related gene signature for rheumatoid arthritis-A prospective study.

Rheumatoid arthritis (RA) is a multifactorial systemic autoimmune disease characterized by ongoing synovial inflammation, leading to the degradation of cartilage. Cuproptosis, as a newly characterized form of cell death, may influence RA progression by regulating immune cells and chondrocytes. This study sets out to identify the hub cuproptosis-related gene (CRG) associated with the pathogenesis of RA. A series of bioinformatic analyses were performed to evaluate the expression score of CRGs and the immune infiltration landscape between RA and normal samples. The hub gene was screened through the correlation analysis of CRGs, and the interaction network between the hub gene and transcription factors (TFs) was constructed. Finally, the hub gene was validated through quantitative real-time polymerase chain reaction (qRT-PCR) of patient samples and cell experiments. Drolipoamide S-acetyltransferase (DLAT) was screened as the hub gene. Correlation analysis between the hub gene and immune microenvironment demonstrated that DLAT had the highest correlation with T follicular helper cells. Eight pairs of DLAT-TF interaction networks were constructed. Single-cell sequencing showed that CRGs were highly expressed in RA chondrocytes, and chondrocytes could be classified into three different subsets. qRT-PCR was used to validate the above results. Dlat knockdown in immortalized human chondrocytes led to significantly improved mitochondrial membrane potentials and reduced levels of intracellular reactive oxygen species (ROS), mitochondrial ROS and apoptosis. This study rudimentarily demonstrates the correlation between CRGs and immune cell infiltration in RA. The biomarker DLAT may provide comprehensive insights into the pathogenesis and drug targets of RA.

Interleukin-18 binding protein regulates the apoptosis and necroptosis of fibroblast-like synoviocytes and chondrocytes in rheumatoid arthritis.

Interleukin (IL)-18 plays a pro-inflammatory role in rheumatoid arthritis (RA), and its soluble inhibitor IL-18 binding protein (IL-18BP) has a potential therapeutic role. We investigated the role of IL-18BP on the joint destruction process of RA by accessing the effects of IL-18BP on fibroblast-like synoviocytes (FLSs) and chondrocytes. Peripheral blood mononuclear cells (PBMCs) from patients with RA and healthy controls were cultured under T cell proliferative conditions with 10, 50, or 100 ng/mL of IL-18BP. After three days of culture, flow cytometry for CD4+ T cells was performed using various IL-18BP concentrations. The apoptosis and necroptosis of FLSs and chondrocytes were measured by flow cytometry using annexin V and propidium iodide (PI) and western blot under TNF-α stimulation with IL-18BP (10, 50, and 100 ng/mL). Differentiation of CD4+ IL-17A+ and CD4+ IL-4+ cells decreased and that of CD4+ CD25high Foxp3+ and CD4+ interferon (IFN)-γ+ cells increased on addition of IL-18BP to cultured RA patient-driven PBMCs. RA-FLS migration ability was not suppressed by IL-18BP after 12 or 24 h. IL-18BP increased annexin V+ FLS level and reduced annexin V+ chondrocyte level in a dose-dependent manner, whereas PI+ annexin V- FLS and chondrocyte levels were suppressed by 50, 100 ng/mL IL-18BP in culture. The administration of IL-18BP regulated the type 17 helper T cell/ regulatory T cell imbalance and attenuated the production of pro-inflammatory cytokines. IL-18BP further increased FLS apoptosis and decreased the necroptosis of FLS/chondrocytes and apoptosis of chondrocytes suggesting the joint preservative potential of IL-18BP.

The method of Yiqi Yangyin Tongluo can attenuate the pyroptosis of rheumatoid arthritis chondrocytes through the ASIC1a/NLRP3 signaling pathway.

BackgroundBased on the ASIC1a/NLRP3 signaling pathway, we explored the specific molecular mechanism of the pyroptosis of rheumatoid arthritis (RA) chondrocytes by the method of nourishing qi, nourishing yin, and dredging collaterals to provide new ideas for the treatment of this disease.MethodsA total of 50 rats were divided into a normal group, model group, methotrexate group, Yiqi Yangyin Tongluo group, and combined group. Except for the normal control group, the other groups used Freund's complete adjuvant (FCA) to make RA rat model. The arthritis index and ankle joint swelling of rats in each group were recorded. HE staining and ELISA were used to assess the pathology of the ankle joint of each group of rats and the content of IL-1β and IL-18 in rat serum. Furthermore, immunofluorescence and qPCR methods were used to detect the protein and mRNA expression levels of NLRP3, caspase 1, ACS, and ASIC1a in the cartilage tissue of each group of rats.ResultsCompared with the normal group, the right hind foot joint of the model group was significantly swollen, the levels of IL-18 and IL-1β in the serum of rats increased significantly, and the mRNA and protein levels of NLRP3, caspase 1, ACS, and ASIC1a in the chondrocytes also increased significantly. Compared with the model group, the degree of ankle joint swelling and IL-18 and IL-1β content in rat serum in each medication group was significantly reduced, and the combined group showed the greatest reduction compared with the other groups. After 8 weeks of treatment, compared with the model group, the mRNA and protein levels of NLRP3, caspase 1, ACS, and ASIC1a in the chondrocytes of each medication group were down-regulated. HE staining found that there were large numbers of infiltrating inflammatory cells and pannus in the joint tissue of the model group, while only a small amount of inflammatory cell infiltration and pannus was seen in the joint tissue of the rats in each treatment group.ConclusionsThe method of Yiqi Yangyin Tongluo can attenuate the pyroptosis of RA chondrocytes through the ASIC1a/NLRP3 signaling pathway.

Identification of differentially expressed genes and pathways crosstalk analysis in Rheumatoid and Osteoarthritis using next-generation sequencing and protein-protein networks.

Osteoarthritis occurs when protective cartilage of bones worn out. Similarlty, cartilage damage occurs mainly in the pannus cartilage in rheumatoid arthritis. It is a potentially debilitating condition, affecting women two to three times more often than men. The cause and prognosis of rheumatoid and osteoarthritis are still poorly known. However, advances in the study of disease pathogenesis have encouraged the creation of new therapeutics with improved outcomes. The purpose of this study is to investigate the differentially expressed genes potentially involved in dysregulated rheumatoid arthritis (RA) and their association to other types of arthritis, including osteoarthritis (OA). Complete RNAs were isolated for RNA expression profiling using next-generation sequencing from human primary cultured normal and RA chondrocytes. From RNA sequencing results 250 differentially expressed genes were identified using bioinformatics analysis, of which 32 were found to be significantly playing role in RA pathogenesis and its associated diseases. Molecular ontologies of the identified genes showed they are connected to Innate immune response, Protein phosphorylation, Transcription initiation from RNA polymerase II promoter, Immune response, Neoplasms of bones, as well as osteorthritis, and Rheumatoid arthritis. Among the identified genes, TRAF1, TRAF2, BAMP, STX11, MEOX2, AES, REL, FHL3, PNMA1, SGTA, LZTS2, SIAH2, PNMA1, and TFCP2 were found to be highly enriched in the protein-protein interaction network. The significant cross talks were found in Hypertrophic cardiomyopathy, Small cell lung cancer, Proteasome, p53 signaling pathway, Arrhythmogenic right ventricular cardiomyopathy, Small cell lung cancer, SNARE interactions in vesicular transport, RIG-I-like receptor signaling pathway, and Hypertrophic cardiomyopathy pathways. The results offer new opportunities for target gene control in RA and OA cartilage destruction.

Synovial fibroblast-derived exosomal microRNA-106b suppresses chondrocyte proliferation and migration in rheumatoid arthritis via down-regulation of PDK4.

Fibroblast-derived exosomes have been reported to transfer microRNAs to recipient cells, where they regulate target gene expression, which is of interest for understanding the basic biology of inflammation, tissue homeostasis, and development of therapeutic approaches. Initial microarray-based analysis carried out in this study identified the rheumatoid arthritis (RA)-related differentially expressed gene pyruvate dehydrogenase kinase 4 (PDK4). Subsequently, the upstream regulatory microRNA-106b (miR-106b) of PDK4 was predicted with bioinformatic analyses. A collagen-induced arthritis (CIA)-induced mouse model was established, and exosomes were isolated from synovial fibroblasts (SFs) and transferred into chondrocytes to identify the role of exosomes in rheumatoid arthritis (RA). We found that PDK4 was poorly expressed in RA cartilage tissues and chondrocytes, while miR-106b was highly expressed in RA SFs and SF-derived exosomes. Notably, PDK4 was confirmed as a target gene of miR-106b. Over-expression of PDK4 promoted the proliferation and migration abilities of chondrocytes and inhibited their apoptosis as well as affected the receptor activator of nuclear factor kappa B ligand (RANKL)/RANK/osteoprotegerin (OPG) system. Meanwhile, miR-106b was delivered from SFs to chondrocytes through exosomes, which suppressed chondrocyte proliferation and migration and accelerated apoptosis as well as affected the RANKL/RANK/OPG system via down-regulation of PDK4. Furthermore, in vivo results validated that miR-106b inhibition could relieve CIA-induced RA. Taken together, SF-derived exosomal miR-106b stimulates RA initiation by targeting PDK4, indicating a physiologically validated potential approach for the prevention and treatment of RA. KEY MESSAGES: PDK4 is decreased in chondrocytes of RA, while miR-106b is increased in SFBs. PDK4 promotes proliferation and migration of chondrocytes. miR-106b could target 3'UTR of PDK4 gene. SFB-exosomal miR-106b inhibits proliferation and migration of chondrocytes. Inhibition of miR-106b attenuates RA progression in a CIA mouse model.

Dual Role of Chondrocytes in Rheumatoid Arthritis: The Chicken and the Egg.

Rheumatoid arthritis (RA) is one of the inflammatory joint diseases that display features of articular cartilage destruction. The underlying disturbance results from immune dysregulation that directly and indirectly influence chondrocyte physiology. In the last years, significant evidence inferred from studies in vitro and in the animal model offered a more holistic vision of chondrocytes in RA. Chondrocytes, despite being one of injured cells in RA, also undergo molecular alterations to actively participate in inflammation and matrix destruction in the human rheumatoid joint. This review covers current knowledge about the specific cellular and biochemical mechanisms that account for the chondrocyte signatures of RA and its potential applications for diagnosis and prognosis in RA.

RETRACTED: Effects of Artesunate on chondrocyte proliferation, apoptosis and autophagy through the PI3K/AKT/mTOR signaling pathway in rat models with rheumatoid arthritis

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Editor-in-Chief. Concern was raised about the reliability of the Western blot results in Figure 5D and 5F, which appear to have a similar phenotype as contained within another publication, as detailed here: https://pubpeer.com/publications/CD4DF7B6DCA28182EC6809846F3653; and here: https://docs.google.com/spreadsheets/d/1r0MyIYpagBc58BRF9c3luWNlCX8VUvUuPyYYXzxWvgY/edit#gid=262337249. Concerns were also raised about the provenance of the flow cytometry data in Figure 7A. In addition, suspected duplications were detected in Western blots contained within Figure 5E and 5F. The journal requested the corresponding author comment on these concerns and provide raw data. The corresponding author communicated that the raw data was not available and some of the results were not repeatable and therefore not solid enough to support the conclusions. The authors requested retraction of this article. The Editor-in-Chief assessed this case and decided to retract the article.

Systematic Analysis of Differential Expression Profile in Rheumatoid Arthritis Chondrocytes Using Next-Generation Sequencing and Bioinformatics Approaches.

Cartilage destruction in rheumatoid arthritis (RA) occurs primarily in the pannus-cartilage interface. The close contact of the synovium-cartilage interface implicates crosstalk between synovial fibroblasts and chondrocytes. The aim of this study is to explore the differentially expressed genes and novel microRNA regulations potentially implicated in the dysregulated cartilage homeostasis in joint destruction of RA. Total RNAs were extracted from human primary cultured normal and RA chondrocytes for RNA and small RNA expression profiling using next-generation sequencing. Using systematic bioinformatics analyses, we identified 463 differentially expressed genes in RA chondrocytes were enriched in biological functions related to altered cell cycle process, inflammatory response and hypoxic stimulation. Moreover, fibroblast growth factor 9 (FGF9), kynureninase (KYNU), and regulator of cell cycle (RGCC) were among the top dysregulated genes identified to be potentially affected in the RA joint microenvironment, having similar expression patterns observed in arrays of clinical RA synovial tissues from the Gene Expression Omnibus database. Additionally, among the 31 differentially expressed microRNAs and 10 candidate genes with potential microRNA-mRNA interactions in RA chondrocytes, the novel miR-140-3p-FGF9 interaction was validated in different microRNA prediction databases, and proposed to participate in the pathogenesis of joint destruction through dysregulated cell growth in RA. The findings provide new perspectives for target genes in the management of cartilage destruction in RA.

Abstract 3258: Gene expression and mutation analysis in human osteosarcoma xenograft models associated with combination activity of lenvatinib with etoposide and ifosfamide

Abstract Objectives: Lenvatinib mesilate (lenvatinib) is a selective inhibitor of VEGFR1-3, and other proangiogenic and oncogenic pathway-related receptor tyrosine kinases (RTKs) including fibroblast growth factor receptors (FGFR1-4), the platelet-derived growth factor receptor (PDGFR) α, KIT, and RET. Lenvatinib showed antitumor activity against various tumor types mainly through its inhibition of angiogenesis. The triple combination of lenvatinib, ifosfamide, and etoposide was effective against human pediatric osteosarcoma xenografts in nude mice. The objective of the study was to identify genes and pathways associated with tumor response to combination of Lenvatinib with etoposide (ETP) and ifosfamide (IFM). Methods: Five human osteosarcoma cell lines (143B, HuO9, G292 clone A141B1, Saos-2, HOS) were grown in vitro and were used for DNA isolation and subsequent whole exome sequencing to determine their mutational profiles. Thirty tissue samples from tumor xenografts in nude mice were used for RNA isolation and subsequent transcriptional profiling using RNA sequencing. Each tumor xenograft model was represented by 6 samples isolated from 6 different mice. Results: We performed mutation and gene expression profiling of 5 human pediatric osteosarcoma xenograft models using whole exome and RNA sequencing. Three of the models (G292, 143B and HOS) showed enhanced antitumor activity when lenvatinib was combined with IFM and ETP. We identified genes and signaling pathways associated with tumor response to the combination treatment. Top three pathways identified were hepatic fibrosis (p = 1.7*10-7), molecular mechanism of cancer (p = 1.3*10-4) and role of osteoblasts, osteoclasts and chondrocytes in rheumatoid arthritis (p = 3.3*10-4). Genes from the pathways were related to cell differentiation and RTK. Hierarchical clustering of the models based on genes from the FGF pathways showed different expression patterns between models. A large fraction of genes from the FGF pathway showed statistically significant differences between the models. For example FGFR1 showed highest expression in G292, FGFR2 in Saos2, FGFR4 in 143B and HOS and FGFR3 in Hu09 and Saos2. The sequencing analysis identified 27 genes with previously reported mutations in TCGA with unique mutation or wild type status in the models with enhanced anti-tumor activity for combination of lenvatinib with IFM and ETP combination. Conclusions: Gene expression analysis by RNAseq and whole exome sequencing showed variation between the xenograft models. Based on the differences we were able to identify genes and signaling pathways associated with anti-tumor activity of combination treatment of lenvatinib with IFM and ETP compared to combination of IFM and ETP. Citation Format: Zoltan Dezso, Sergei I. Agoulnik, Crystal MacKenzie, Kaoru Mitsuhashi, Kiyoshi Okamoto, Junji Matsui, Yasuhiro Funahashi. Gene expression and mutation analysis in human osteosarcoma xenograft models associated with combination activity of lenvatinib with etoposide and ifosfamide. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3258.

Genetic association analysis of radiographic hip osteoarthritis with established loci for bone mineral density: data from the osteoarthritis initiative

Genetic association analysis of radiographic hip osteoarthritis with established loci for bone mineral density: data from the osteoarthritis initiative

Combination of High-Molecular-Weight Hyaluronic Acid and Cytokine Inhibitor Potently Inhibits Expression of Joint-Damage-Related Genes Induced By Synovial Fluid of RA Patients

Background: Blockade of inflammatory cytokines such as IL-1β, IL-6, and TNF-α inhibits the progression of joint damage in hands and feet in Rheumatoid Arthritis (RA). However, it has been recently reported that TNF inhibitors have little effect on the large weight-bearing joints once joint destruction has started. On the other hand, intraarticular injection of high-molecular-weight sodium Hyaluronate (HA) prevents cartilage degeneration in the large weight-bearing joints in osteoarthritis. Objectives: We examined the effects of combinations of cytokine inhibitor and HA on the expression of ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs), MMPs (matrix metalloproteinases), and RANKL (receptor activator of nuclear factor κB ligand), which all play important roles in joint damage, in human RA fibroblastlike synoviocytes (RA-FLS) and chondrocytes. Methods: RA-FLS and chondrocytes were cultured for 24 h in the presence of synovial fluid from active RA patients (CRP>12 mg/dL, n=5) with or without HA, IL-1 receptor antagonist, soluble TNF receptor-Fc (etanercept), and anti-IL-6 receptor antibody (tocilizumab). After culture, the levels of ADAMTS-4, MMP-3, and RANKL mRNA were measured by real-time PCR. Results: RA synovial fluids induced ADAMTS-4,MMP-3, and RANKL mRNA expressions in RA-FLS and chondrocytes. Cytokine inhibitors partially inhibited ADAMTS-4,MMP-3, and RANKL mRNA expressions, showing that several cytokines are involved in the expression of joint destruction factors. The RA synovial fluids contained IL-1β, IL-6, soluble IL-6 receptor, and TNF-α. HA also partially inhibited ADAMTS-4,MMP-3, and RANKL mRNA expressions. Use of each cytokine inhibitor in combination with HA inhibited the production of these factors further than monotherapy with either. Conclusions: Our results indicate that HA may be useful in preventing joint damage in large weight-bearing joints in RA patients. Furthermore, the concomitant use of cytokine inhibitors and HA might be of greater benefit for preserving cartilage function in RA patients.

Identification of homogeneous genetic architecture of multiple genetically correlated traits by block clustering of genome-wide associations

Genome-wide association studies (GWAS) using high-density genotyping platforms offer an unbiased strategy to identify new candidate genes for osteoporosis. It is imperative to be able to clearly distinguish signal from noise by focusing on the best phenotype in a genetic study. We performed GWAS of multiple phenotypes associated with fractures [bone mineral density (BMD), bone quantitative ultrasound (QUS), bone geometry, and muscle mass] with approximately 433,000 single-nucleotide polymorphisms (SNPs) and created a database of resulting associations. We performed analysis of GWAS data from 23 phenotypes by a novel modification of a block clustering algorithm followed by gene-set enrichment analysis. A data matrix of standardized regression coefficients was partitioned along both axes--SNPs and phenotypes. Each partition represents a distinct cluster of SNPs that have similar effects over a particular set of phenotypes. Application of this method to our data shows several SNP-phenotype connections. We found a strong cluster of association coefficients of high magnitude for 10 traits (BMD at several skeletal sites, ultrasound measures, cross-sectional bone area, and section modulus of femoral neck and shaft). These clustered traits were highly genetically correlated. Gene-set enrichment analyses indicated the augmentation of genes that cluster with the 10 osteoporosis-related traits in pathways such as aldosterone signaling in epithelial cells, role of osteoblasts, osteoclasts, and chondrocytes in rheumatoid arthritis, and Parkinson signaling. In addition to several known candidate genes, we also identified PRKCH and SCNN1B as potential candidate genes for multiple bone traits. In conclusion, our mining of GWAS results revealed the similarity of association results between bone strength phenotypes that may be attributed to pleiotropic effects of genes. This knowledge may prove helpful in identifying novel genes and pathways that underlie several correlated phenotypes, as well as in deciphering genetic and phenotypic modularity underlying osteoporosis risk.

Hyaluronan inhibits matrix metalloproteinase‐13 in human arthritic chondrocytes via CD44 and P38

We investigated the effects of hyaluronan (HA) on interleukin-1β (IL-1β)-stimulated matrix metalloproteinase (MMP)-13 production in human chondrocytes from patients with osteoarthritis (OA) or rheumatoid arthritis (RA). Secreted levels of MMP-13 in conditioned media were detected by immunoblotting, while intracellular MMP-13 synthesis in articular cartilage was evaluated by immunofluorescence microscopic analysis. Mitogen-activated protein kinases (MAPKs), p38, extracellular signal-regulated kinases (ERK), and c-jun NH2-terminal kinase (JNK) were assessed by Western blotting. IL-1β (2 ng/ml) stimulates the secretion of MMP-13 in both OA and RA chondrocytes. Inhibition studies using specific MAPK inhibitors revealed that IL-1β induced MMP-13 via p38 in both OA and RA chondrocytes. HA down-regulates IL-1β-stimulated MMP-13 and phosphorylated p38 (p-p38) in a dose-dependent manner (0.1, 1, 2, and 4 mg/ml). When used at 4 mg/ml, HA inhibits p-p38 phosphorylation by more than 60%. In response to IL-1β, RA chondrocytes express a higher level of p-p38 than that of OA chondrocytes. Inhibition of CD44, using a blocking antibody, significantly reversed the inhibitory effect of HA on both MMP-13 and p-p38. Our study clearly shows that HA inhibits IL-1β-induced MMP-13 via its principal receptor, CD44, and subsequent intracellular p38 MAPK signaling in OA and RA chondrocytes.

Adiponectin‐mediated changes in effector cells involved in the pathophysiology of rheumatoid arthritis

Rheumatoid arthritis (RA) is associated with increased production of adipokines, which are cytokine-like mediators that are produced mainly in adipose tissue but also in synovial cells. Since RA synovial fibroblasts (RASFs), lymphocytes, endothelial cells, and chondrocytes are key players in the pathophysiology of RA, this study was undertaken to analyze the effects of the key adipokine adiponectin on proinflammatory and prodestructive synovial effector cells. Lymphocytes were activated in part prior to stimulation. All cells were stimulated with adiponectin, and changes in gene and protein expression were determined by Affymetrix and protein arrays. Messenger RNA and protein levels were confirmed using semiquantitative reverse transcription-polymerase chain reaction (PCR), real-time PCR, and immunoassays. Intracellular signal transduction was evaluated using chemical signaling inhibitors. Adiponectin stimulation of human RASFs predominantly induced the secretion of chemokines, as well as proinflammatory cytokines, prostaglandin synthases, growth factors, and factors of bone metabolism and matrix remodeling. Lymphocytes, endothelial cells, and chondrocytes responded to adiponectin stimulation with enhanced synthesis of cytokines and various chemokines. Additionally, chondrocytes released increased amounts of matrix metalloproteinases. In RASFs, adiponectin-mediated effects were p38 MAPK and protein kinase C dependent. Our previous findings indicated that adiponectin was present in inflamed synovium, at sites of cartilage invasion, in lymphocyte infiltrates, and in perivascular areas. The findings of the present study indicate that adiponectin induces gene expression and protein synthesis in human RASFs, lymphocytes, endothelial cells, and chondrocytes, supporting the concept of adiponectin being involved in the pathophysiologic modulation of RA effector cells. Adiponectin promotes inflammation through cytokine synthesis, attraction of inflammatory cells to the synovium, and recruitment of prodestructive cells via chemokines, thus promoting matrix destruction at sites of cartilage invasion.

Hyaluronan inhibits p38 mitogen-activated protein kinase via the receptors in rheumatoid arthritis chondrocytes stimulated with fibronectin fragment

This study was aimed to examine the inhibitory mechanism of high molecular weight hyaluronan (HA) on nitric oxide (NO) production by NH2-terminal heparin-binding fibronectin fragment (FN-f) in rheumatoid arthritis (RA) chondrocytes. When the RA cartilage explants or the isolated RA chondrocytes in monolayer were incubated with FN-f, the fragment stimulated NO production with induction of inducible nitric oxide synthase (iNOS) and activation of p38 mitogen-activated protein kinase. Pretreatment with 2,700 kDa HA resulted in significant suppression of FN-f-stimulated NO production in RA cartilage as well as in chondrocyte monolayer cultures in association with iNOS down-regulation. Inhibition studies with p38 inhibitor indicated the requirement of p38 for FN-f-induced NO production. HA suppressed p38 activation by the FN-f, leading to a decrease in NO production. Immunofluorescence cytochemistry revealed HA association with intercellular adhesion molecule-1 (ICAM-1) and CD44. While the individual antibody to ICAM-1 or CD44 partially reversed HA effect on the FN-f action, both antibodies in combination completely blocked the HA effect. The present study clearly demonstrated that the high molecular weight of HA suppressed the FN-f-activated p38 via ICAM-1 and the CD44 in RA chondrocytes. HA could down-regulate the catabolic action of FN-f in RA joints through the mechanism demonstrated in this study.

Comparison of hyaluronan effects among normal, osteoarthritis, and rheumatoid arthritis cartilages stimulated with fibronectin fragment

High molecular weight hyaluronan (HA) is widely used in the treatment of osteoarthritis (OA) and rheumatoid arthritis (RA) by intra-articular injection. However, comparative studies of HA actions on catalytically activated cartilages in different pathologic conditions have rarely been investigated. This study was aimed to compare the inhibitory effects of HA on nitric oxide (NO) production by COOH-terminal heparin-binding fibronectin fragment (HBFN-f) between normal and diseased cartilages. When articular cartilage explants from normal, OA, or RA joints were incubated with HBFN-f, the RA and OA cartilages produced higher levels of NO compared with normal cartilage. Pretreatment with 2700 kDa HA resulted in significant suppression of HBFN-f-stimulated NO production in OA and RA cartilages. While CD44 was up-regulated in OA and RA cartilages, anti-CD44 antibody reversed HA inhibition of HBFN-f action in those cartilages. The present results clearly demonstrated that HA blocked HBFN-f actions in OA and RA cartilages through interaction with CD44. HA, which targets CD44 highly expressed on OA and RA chondrocytes, could suppress catabolic actions by fibronectin fragments like HBFN-f in diseased cartilage.

Targeting Growth Factors in Arthritis: A Rational for Restoring the IGF-I Response in Chondrocytes

The role of growth factors in the pathogenesis of arthritis has received considerably less attention as compared to proinflammatory cytokines in recent years. This is understandable as it is evident that current anti-tumor necrosis factor-α (TNFα) therapy is efficacious in at least in 60% of the rheumatoid arthritis (RA) patients and when given in combination with methotrexate stops radiological progression of bone erosion. Other proinflammatory cytokines, such as interleukin-1 (IL-1) and IL-17 have also been implicated in the dysregulation of bone and cartilage remodelling characteristic of rheumatoid arthritis (RA) and/or osteoarthritis (OA). The direct effects of anti-cytokine treatment on cartilage pathology are not that easy to determine in humans and it remains to be seen if amelioration of cartilage destruction or better cartilage repair has been accomplished with anti-TNF therapy. A complicating factor is that chondrocytes in RA and OA become non-responsive to insulin-like growth factor-I (IGF-I). IGF-I is the main growth factor for stimulation of chondrocyte proteoglycan and collagen type II synthesis in order to maintain the cartilage matrix integrity. This article reviews our current understanding of the role of IGF-I on cartilage extracellular matrix production and the mechanisms underlying the cause of IGF-I non-responsiveness, and in particular the inhibition of IGF-receptor signalling by suppressor of cytokine signalling (SOCS) proteins. The epigenetic and cytokine-dependent regulation of SOCS expression may give an explanation for the loss of IGF-I signalling in chondrocytes in ageing-related diseases such as OA and RA. Whether IGF-1 is replaceable by other growth factors to maintain the cartilage extracellular matrix will be discussed. It is concluded that in addition to the current use of anti-cytokine therapy in arthritis the IGF-I response of chondrocytes must be restored in order to achieve cartilage matrix repair.

Inhibition of nuclear factor-κB by hyaluronan in rheumatoid chondrocytes stimulated with COOH-terminal heparin-binding fibronectin fragment

The aim of this study was to examine the inhibitory effect of high molecular weight hyaluronan (HA) on nuclear factor (NF)-kappaB activation by COOH-terminal heparin-binding fibronectin fragment (HBFN-f) in rheumatoid arthritis (RA) chondrocytes. When RA chondrocytes in monolayer or cartilage explants were cultured with HBFN-f, the fragment stimulated the phosphorylation and nuclear translocation of NF-kappaB, leading to nitric oxide (NO) production in association with inducible form of NO synthase (iNOS) up-regulation. Inhibition studies with NF-kappaB inhibitors indicated the requirement of NF-kappaB for HBFN-f-induced NO production. Pretreatment with 2700 kDa HA resulted in significant suppression of NF-kappaB activation by HBFN-f. HA also inhibited HBFN-f-stimulated NO production with down-regulation of iNOS. The present study clearly demonstrated that high molecular weight HA suppressed HBFN-f-activated NF-kappaB in RA chondrocytes. HA could down-regulate the catabolic action of fibronectin fragments like HBFN-f in RA joints as a potent NF-kappaB inhibitor.