Cartilage Destruction In Osteoarthritis Research Articles

Chemically modified curcumin (CMC2.24) alleviates osteoarthritis progression by restoring cartilage homeostasis and inhibiting chondrocyte apoptosis via the NF-κB/HIF-2α axis.

The disorders of cartilage homeostasis and chondrocyte apoptosis are major events in the pathogenesis of osteoarthritis (OA). Herein, we aim to assess the chondroprotective effect and underlying mechanisms of a novel chemically modified curcumin, CMC2.24, in modulating extracellular matrix (ECM) homeostasis and inhibiting chondrocyte apoptosis. Rats underwent the anterior cruciate ligament transection, and medial menisci resection was treated by intra-articular injection with CMC2.24. In an in vitro study, rat chondrocytes were pretreated with CMC2.24 before stimulation with sodium nitroprusside (SNP). Results from in vivo studies demonstrated that the intra-articular administration of CMC2.24 ameliorated osteoarthritic cartilage destruction by promoting collagen 2a1 production and inhibited cartilage degradation and apoptosis by suppressing hypoxia-inducible factor-2a (Hif-2α), matrix metalloproteinase-3, runt-related transcription factor 2, cleaved caspase-3, and vascular endothelial growth factor and the phosphorylation of IκBα and NF-κB p65. The in vitro results revealed that CMC2.24 exhibited a strong inhibitory effect on SNP-induced chondrocyte catabolism and apoptosis. The SNP-enhanced expression of Hif-2α, a catabolic and apoptotic factor, decreased in a dose-dependent manner after CMC2.24 treatment. CMC2.24 pretreatment effectively inhibited SNP-induced IκBα and NF-κB p65 phosphorylation in rat chondrocytes, whereas pretreatment with the NF-κB antagonist BMS-345541 significantly enhanced the effects of CMC2.24. Overall, these results demonstrated that CMC2.24 attenuates OA progression by modulating ECM homeostasis and chondrocyte apoptosis by suppressing the NF-κB/Hif-2α axis, thus providing a new perspective for therapeutic strategies in OA. KEY MESSAGES: • Intra-articular injection of CMC2.24 ameliorated osteoarthritic cartilage destruction. • CMC2.24 promoted cell viability and decreased SNP-induced apoptotic gene expression. • SNP-induced activation of Hif-2α is inhibited by CMC2.24. • CMC2.24 inhibits NF-κB/Hif-2α axis activation to modulate ECM homeostasis and inhibit chondrocyte apoptosis.

Interleukin-1-Interleukin-17 Signaling Axis Induces Cartilage Destruction and Promotes Experimental Osteoarthritis.

Osteoarthritis (OA), which is the most common degenerative joint disorder, has been considered a non-inflammatory disease with abnormal mechanics. Interleukin (IL)-17 is a pleiotropic cytokine involved in inflammatory diseases and their production is driven by the cytokine including IL-1 and IL-23. However, little is known about the mechanism of IL-17 in the development of OA. Here, we investigated the role of IL-17 in the pathogenesis of OA using monosodium iodoacetate (MIA)-injected IL-17 and IL-1 receptor antagonist (IL-1Ra) double-deficient mice. In MIA-injected IL-1Ra KO mice, nociceptive properties, degree of cartilage damage, and the level of inflammatory factors in articular cartilage were increased compared to MIA-injected wild-type mice. Interestingly, the intestinal architecture was impaired in IL-1Ra KO mice compared to wild-type mice and the damage was further exacerbated by MIA injection. Deficiency of IL-17 reduced nociceptive properties and cartilage destruction, as well as inflammation-related factors in MIA-injected IL-1Ra KO mice compared to MIA-injected wild-type mice. Furthermore, IL-17-treated chondrocytes from OA patients showed enhanced expression of catabolic factors that are involved in the destruction of cartilage in OA. IL-17 accelerates the destruction of cartilage and small intestine via regulation of several inflammatory mediators in an OA murine model. These results suggest that IL-17 plays a critical role in the development of OA.

Etoricoxib decreases subchondral bone mass and attenuates biomechanical properties at the early stage of osteoarthritis in a mouse model

Etoricoxib, a selective Cyclooxygenase-2 (COX-2) inhibitor, is commonly used in osteoarthritis (OA) for pain relief, however, little is known about the effects on subchondral bone. In the current study, OA was induced via destabilization of the medial meniscus (DMM) in C57BL/6 mice. Two days after surgery, mice were treated with different concentrations of Etoricoxib. Four weeks after treatment, micro computed tomography (Micro-CT) analysis, histological analysis, atomic force microscopy (AFM) analysis, and scanning electron microscopy (SEM) were performed to evaluate OA progression. We demonstrated that Etoricoxib inhibited osteophyte formation in the subchondral bone. However, it also reduced the bone volume fraction (BV/TV), lowered trabecular thickness (Tb.Th), and more microfractures and pores were observed in the subchondral bone. Moreover, Etoricoxib reduced the elastic modulus of subchondral bone. Exposure to Etoricoxib further increased the empty/total osteocyte ratio of the subchondral bone. Etoricoxib did not show significant improvement in articular cartilage destruction and synovial inflammation in early OA. Together, our observations suggested that although Etoricoxib can relieve OA-induced pain and inhibit osteophyte formation in the subchondral bone, it can also change the microstructures and biomechanical properties of subchondral bone, promote subchondral bone loss, and reduce subchondral bone quality in early OA mice.

SERPINA3N limits cartilage destruction in osteoarthritis by inhibiting macrophage-derived leukocyte elastase

Purpose: Interleukin-6 (IL-6) plays an important role in osteoarthritis (OA). Transcriptomic analyses (RNAseq) revealed that SerpinA3N, a serine protease inhibitor, is a key target of IL-6 in chondrocyte. This study aimed to examine the role of SerpinA3N and Leukocyte Elastase (Elane), a serine protease targeted by SerpinA3N, in cartilage destruction during OA.

Panax notoginseng saponins prevent senescence and inhibit apoptosis by regulating the PI3K‑AKT‑mTOR pathway in osteoarthritic chondrocytes.

Panaxnotoginseng saponins (PNS) are active extracts obtained from the P.notoginseng plant. PNS exhibit various anti‑inflammatory, anti‑oxidant and anti‑aging pharmacological properties in some cells. However, the effects of PNS on senescence and apoptosis in chondrocytes have not been studied to date. In the present study, whether PNS could limit tumor necrosis factor (TNF)‑α‑induced senescence and apoptosis in chondrocytes and whether they could slow down cartilage degeneration in a surgery‑induced rat osteoarthritis(OA) model by regulating the phosphatidyl inositol3 kinase(PI3K)‑protein kinaseB (AKT)‑mammalian target of rapamycin(mTOR) signaling pathway was examined. A potential mechanism underlying these effects was further elucidated. The present invitro experiments showed that PNS significantly inhibited senescence and apoptosis in OA chondrocytes and prevented a decrease in the mitochondrial membrane potential and excessive mitochondrial permeability. In addition, the expression levels of autophagy‑related proteins and the anti‑apoptotic protein Bcl‑2 were significantly increased in PNS‑treated OA chondrocytes, but the expression levels of Bax and caspase‑3 were decreased; these effects were concentration‑dependent. TNF‑α significantly increased the expression of p‑PI3K/p‑AKT/p‑mTOR in OA chondrocytes, whereas PNS reduced PI3K, AKT and mTOR phosphorylation. The results of the invivo experiments demonstrated that PNS significantly inhibited the PI3K‑AKT‑mTOR signaling pathway and collagenII degradation, as well as reduced matrix metalloproteinase (MMP)‑3 and MMP‑13 expression in chondrocytes in a rat OA model, thus attenuating cartilage destruction in OA. The results obtained in the rat model were consistent with the invitro experimental results. Furthermore, histological analyses and ultrastructural observations confirmed these results. Taken together, the results of the present study demonstrated that PNS may protect osteoarthritic chondrocytes from senescence and apoptosis by inhibiting the PI3K‑AKT pathway, thus delaying the degradation of articular cartilage.

Up-regulation of P21-activated kinase 1 in osteoarthritis chondrocytes is responsible for osteoarthritic cartilage destruction.

Osteoarthritis is mainly caused by a degenerative joint disorder, which is characterized by the gradual degradation of articular cartilage and synovial inflammation. The chondrocyte, the unique resident cell type of articular cartilage, is crucial for the development of osteoarthritis. Previous studies revealed that P21-activated kinase-1 (PAK1) was responsible for the initiation of inflammation. The purpose of the present study was to determine the potential role of PAK1 in osteoarthritis. The level of PAK1 expression was measured by Western blot and quantitative real-time PCR in articular cartilage from osteoarthritis model rats and patients with osteoarthritis. In addition, the functional role of aberrant PAK1 expression was detected in the chondrocytes. We found that the expression of PAK1 was significantly increased in chondrocytes treated with osteoarthritis-related factors. Increased expression of PAK1 was also observed in knee articular cartilage samples from patients with osteoarthritis and osteoarthritis model rats. PAK1 was found to inhibit chondrocytes proliferation and to promote the production of inflammatory cytokines in cartilages chondrocytes. Furthermore, we found that PAK1 modulated the production of extracellular matrix and cartilage degrading enzymes in chondrocytes. Results of the present studies demonstrated that PAK1 might play an important role in the pathogenesis of osteoarthritis.

Expression and regulation of macrophage-inducible C-type lectin in human synovial macrophages.

Recent evidence suggests that synovial macrophage activation may be involved in cartilage destruction and pain in osteoarthritis (OA). The macrophage-inducible C-type lectin (Mincle) Clec4e is expressed in macrophages and is regulated in inflammatory conditions. Given that the regulation of Mincle in synovial macrophages has not been elucidated, we investigated the expression and regulation of Mincle in human synovial tissue (ST) harvested from patients with radiographic knee OA during total knee arthroplasty. Immunohistochemical and flow cytometric analyses were used to identify cells with Mincle expression in resected tissues. CD14-positive (CD14+; macrophage-rich cell fraction) and CD14-negative (CD14-; fibroblast-rich cell fraction) cells were extracted from the ST and used to assess MINCLE mRNA expression levels. To determine the role of tumor necrosis factor alpha (TNF-α) in the regulation of MINCLE expression, TNF-α was used to stimulate cultured CD14+ cells. Immunohistochemical staining revealed Mincle-positive cells in the synovial lining layer. Flow cytometric analysis showed that CD45+CD14+ cells were Mincle positive while CD45–/CD14– cells were Mincle negative. MINCLE expression was significantly higher in CD14+ cells than in CD14– cells. Stimulation of cultured CD14+ macrophages with TNF-α significantly increased MINCLE mRNA expression, while stimulation with TNF-α neutralizing antibody significantly decreased expression. That Mincle expression was observed in synovial macrophages and its expression was induced by TNF-α suggests that Mincle might have a key role in synovial inflammation in the osteoarthritic synovium.

MicroRNA Mediate Visfatin and Resistin Induction of Oxidative Stress in Human Osteoarthritic Synovial Fibroblasts Via NF-κB Pathway

Synovial membrane inflammation actively participate to structural damage during osteoarthritis (OA). Adipokines, miRNA, and oxidative stress contribute to synovitis and cartilage destruction in OA. We investigated the relationship between visfatin, resistin and miRNA in oxidative stress regulation, in human OA synovial fibroblasts. Cultured cells were treated with visfatin and resistin. After 24 h, we evaluated various pro-inflammatory cytokines, metalloproteinases (MMPs), type II collagen (Col2a1), miR-34a, miR-146a, miR-181a, antioxidant enzymes, and B-cell lymphoma (BCL)2 by qRT-PCR, apoptosis and mitochondrial superoxide production by cytometry, p50 nuclear factor (NF)-κB by immunofluorescence. Synoviocytes were transfected with miRNA inhibitors and oxidative stress evaluation after adipokines stimulus was performed. The implication of NF-κB pathway was assessed by the use of a NF-κB inhibitor (BAY-11-7082). Visfatin and resistin significantly up-regulated gene expression of interleukin (IL)-1β, IL-6, IL-17, tumor necrosis factor (TNF)-α, MMP-1, MMP-13 and reduced Col2a1. Furthermore, adipokines induced apoptosis and superoxide production, the transcriptional levels of BCL2, superoxide dismutase (SOD)-2, catalase (CAT), nuclear factor erythroid 2 like 2 (NRF2), miR-34a, miR-146a, and miR-181a. MiRNA inhibitors counteracted adipokines modulation of oxidative stress. Visfatin and resistin effects were suppressed by BAY-11-7082. Our data suggest that miRNA may represent possible mediators of oxidative stress induced by visfatin and resistin via NF-κB pathway in human OA synoviocytes.

Dual regulatory roles of HMGB1 in inflammatory reaction of chondrocyte cells and mice

ABSTRACT Osteoarthritis (OA) is one of the most common bone diseasesas it is reported that the impact of knee osteoarthritis symptomatic form is estimated at 240/100,000 people per year. The inflammation of articular cartilageis thought to be the pathologic drive for development of this disease. HMGB1(high mobility group box-1), a regulatory factor for gene transcription, could stimulate inflammation response. However, theexact regulatory role of HMGB1 in the inflammation of articular cartilage still need to be elucidated. In the current study, we used Quantitative Real-Time PCR(Q-PCR) to detect them RNA levels of Collagen Type II Alpha 1(Col2a1), Aggrecan, MMP3(Matrix Metallopeptidase 3), MMP13, ADAMTs4 and ADAMTs5; Enzyme-Linked Immunosorbent Assay(ELISA) was used to detect the content of IL-1β and calpain protein; Cell apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling(TUNEL) assay and flow cytometryanalysis; Western blot and immunofluorescence assays were applied to assess the expression of HMGB1; Lastly autophagic activity was mainly verified by monodansylcadaverine (MDC) staining. Our data revealed that in the early stage of chondrocyte inflammation(3 and 6 h of LPS stimulation), cytosolic HMGB1 attenuated inflammation response by facilitating cell autophagy and preventing cell apoptosis. While in the late stage (24 and 48 h of LPS stimulation), the extracellular HMGB1 stimulated inflammation reaction and contributed to the cartilage destruction in OA.

IgE-mediated mast cell activation promotes inflammation and cartilage destruction in osteoarthritis.

This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

A crucial role of arginase-2 in osteoarthritis pathogenesis

Purpose: Osteoarthritis (OA) appears to be associated with various metabolic disorders. However, a role of amino acids metabolism in OA pathogenesis has not been clearly elucidated to date. Here, we explored whether alterations in amino acid metabolism in chondrocytes regulates OA pathogenesis. Methods: Expression profiles of genes regulating amino acid metabolism in primary-culture mouse chondrocytes was examined by microarray analysis. Expression pattern of the identified genes were further characterized in mouse OA chondrocytes and OA cartilage of human and mouse models. Experimental OA in mice was induced by destabilization of the medial meniscus (DMM) or intra-articular (IA) injection of adenoviruses expressing catabolic regulators. The function of Arg2 was examined by analyzing Arg2-/- mice and those subjected to IA injection of an adenovirus encoding Arg2(Ad-Arg2). Results: Microarray analysis revealed that arginase-2 (Arg2), an arginine metabolizing enzyme, was specifically upregulated in chondrocytes under various pathological conditions and in OA cartilage from human OA patients and various mouse models. Adenovirus-mediated overexpression of Arg2 in mouse joint tissues caused OA pathogenesis, whereas genetic ablation of Arg2 in mice (Arg2-/-) abolished all manifestations of DMM-induced OA. Mechanistically, Arg2 appears to cause OA cartilage destruction at least partly by upregulating the expression of matrix-degrading enzymes (MMP3 and MMP13) in chondrocytes via the NF-κB pathway. Conclusions: Our results collectively indicate that Arg2 is a crucial catabolic regulator of OA pathogenesis, supporting the notion that Arg2 could be a therapeutic target for the treatment of OA pathogenesis.

Ligustilide attenuates nitric oxide-induced apoptosis in rat chondrocytes and cartilage degradation via inhibiting JNK and p38 MAPK pathways.

Ligustilide (LIG) is the main lipophilic component of the Umbelliferae family of pharmaceutical plants, including Radix angelicae sinensis and Ligusticum chuanxiong. LIG shows various pharmacological properties associated with anti‐inflammation and anti‐apoptosis in several kinds of cell lines. However, the therapeutic effects of LIG on chondrocyte apoptosis remain unknown. In this study, we investigated whether LIG had an anti‐apoptotic activity in sodium nitroprusside (SNP)‐stimulated chondrocyte apoptosis and could delay cartilage degeneration in a surgically induced rat OA model, and elucidated the potential mechanisms. In vitro studies revealed that LIG significantly suppressed chondrocyte apoptosis and cytoskeletal remodelling, which maintained the nuclear morphology and increased the mitochondrial membrane potential. In terms of SNP, LIG treatment considerably reduced the expression levels of cleaved caspase‐3, Bax and inducible nitric oxide synthase and increased the expression level of Bcl‐2 in a dose‐dependent manner. The LIG‐treated groups presented a significantly suppressed expression of activating transcription factor 2 and phosphorylation of Jun N‐terminal kinase (JNK) and p38 mitogen‐activated protein kinase (MAPK). The inhibitory effect of LIG was enhanced by the p38 MAPK inhibitor SB203580 or the JNK inhibitor SP600125 and offset by the agonist anisomycin. In vivo studies demonstrated that LIG attenuated osteoarthritic cartilage destruction by inhibiting the cartilage chondrocyte apoptosis and suppressing the phosphorylation levels of activating transcription factor 2, JNK and p38 MAPK. This result was confirmed by histological analyses, micro‐CT, TUNEL assay and immunohistochemical analyses. Collectively, our studies indicated that LIG protected chondrocytes against SNP‐induced apoptosis and delayed articular cartilage degeneration by suppressing JNK and p38 MAPK pathways.

Targeted Inhibition of Aggrecanases Prevents Articular Cartilage Degradation and Augments Bone Mass in the STR/Ort Mouse Model of Spontaneous Osteoarthritis.

Cartilage destruction in osteoarthritis (OA) is mediated mainly by matrix metalloproteinases (MMPs) and ADAMTS. The therapeutic candidature of targeting aggrecanases has not yet been defined in joints in which spontaneous OA arises from genetic susceptibility, as in the case of the STR/Ort mouse, without a traumatic or load-induced etiology. In addition, we do not know the long-term effect of aggrecanase inhibition on bone. We undertook this study to assess the potential aggrecanase selectivity of a variant of tissue inhibitor of metalloproteinases 3 (TIMP-3), called [-1A]TIMP-3, on spontaneous OA development and bone formation in STR/Ort mice. Using the background of STR/Ort mice, which develop spontaneous OA, we generated transgenic mice that overexpress [-1A]TIMP-3, either ubiquitously or conditionally in chondrocytes. [-1A]TIMP-3 has an extra alanine at the N-terminus that selectively inhibits ADAMTS but not MMPs. We analyzed a range of OA-related measures in all mice at age 40 weeks. Mice expressing high levels of [-1A]TIMP-3 were protected against development of OA, while those expressing low levels were not. Interestingly, we also found that high levels of [-1A]TIMP-3 transgene overexpression resulted in increased bone mass, particularly in females. This regulation of bone mass was at least partly direct, as adult mouse primary osteoblasts infected with [-1A]TIMP-3 in vitro showed elevated rates of mineralization. The results provide evidence that [-1A]TIMP-3-mediated inhibition of aggrecanases can protect against cartilage degradation in a naturally occurring mouse model of OA, and they highlight a novel role that aggrecanase inhibition may play in increased bone mass.

Proteoglycans isolated from the bramble shark cartilage show potential anti-osteoarthritic properties.

Osteoarthritis (OA) causes articular cartilage destruction, initiating pain and inflammation in the joints, resulting in joint disability. Medications are available to manage these symptoms; however, their effects on the disease progression are limited. Loss of proteoglycans (PGs) was reported to contribute articular cartilage destruction in OA. Therapeutics approaches were previously studied in the animal models of OA. In the present study, we investigated the oral efficacy of four dosages of PGs (25mg/kg, 50mg/kg, 100mg/kg and 200mg/kg), isolated from the bramble shark cartilage, in an animal model of OA. Indomethacin was used as a bioequivalent formulation. Primarily, the mass spectrum analysis of the purified PGs obtained from bramble shark cartilage revealed the presence of two unique peptides including AGWLSDGSVR and LDGNPINLSK, that showed sequence similarity with aggrecan core-protein and epiphycan, respectively. The levels of C-reactive protein and uric acid in the OA rats were reduced when treated with PGs. Histopathology analysis displayed less cartilage erosion and neovascularization in OA rats treated with PGs. The X-ray imaging presented higher bone density with 200mg/kg dosage of PG treatment in OA rats. The expressions of the inflammatory modulators including TNF-α, IL-1β, MMP13, NOS2, IL-10 and COX-2 were found to be moderated with PG treatment. In addition, PG treatment maintained the activities of antioxidant enzymes, including SOD and catalase in the joint tissues with a higher GSH content, in a dose-dependent manner. Taken together, our preliminary findings report the anti-osteoarthritic properties of PGs and recommend to evaluate its efficacy and safety in randomized trials.

BATF regulates collagen-induced arthritis by regulating T helper cell differentiation

BackgroundWe recently demonstrated that BATF, a member of the activator protein-1 (AP-1) family, regulates osteoarthritic cartilage destruction. Here, we explored the roles and regulatory mechanisms of BATF in collagen-induced arthritis (CIA) in mice.MethodsCIA and K/BxN serum transfer were used to generate inflammatory arthritis models in wild-type (WT) and Batf−/− mice. RA manifestations were determined by examining CIA incidence, clinical score, synovitis, synovial hyperplasia, angiogenesis in inflamed synovium, pannus formation, bone erosion, and cartilage destruction. Immune features in RA were analyzed by examining immune cell populations and cytokine production.ResultsBATF was upregulated in the synovial tissues of joints in which inflammatory arthritis had been caused by CIA or K/BxN serum transfer. The increases in CIA incidence, clinical score, and autoantibody production in CIA-induced WT mice were completely abrogated in the corresponding Batf−/− DBA/1 J mice. Genetic ablation of Batf also inhibited CIA-induced synovitis, synovial hyperplasia, angiogenesis in synovial tissues, pannus formation, bone erosion, and cartilage destruction. Batf knockout inhibited the differentiation of T helper (Th)17 cells and the conversion of CD4+Foxp3+ cells to CD4+IL-17+ cells. However, BATF did not modulate the functions of fibroblast-like synoviocytes (FLS), including the expressions of chemokines, matrix-degrading enzymes, vascular endothelial growth factor, and receptor activator of NF-κB ligand (RANKL).ConclusionOur findings indicate that BATF crucially mediates CIA by regulating Th cell differentiation without directly affecting the functions of FLS.

MTOR-mediated inactivation of 4E-BP1, an inhibitor of translation, precedes cartilage degeneration in rat osteoarthritic knees.

Proper control of protein synthesis is vital for tissue homeostasis and its deregulation is characteristic of many disorders including osteoarthritis (OA). The objectives of this work were to analyze and correlate changes in activity of the translation apparatus associated with cartilage degeneration in an animal model of OA. Osteoarthritis was induced surgically in rats by anterior cruciate ligament transection (ACLT). Using a modified Mankin scoring system and analysis of protein expression we demonstrated, that mechanistic target of rapamycin complex 1 (mTORC1)-mediated 4E-BP1 phosphorylation was detected significantly earlier than other mTORC1-mediated modifications, such as p70S6K and ULK1 phosphorylation. 4E-BP1 is an inhibitor of cap-dependent translation those functions are inhibited by mTORC1 mediated phosphorylation. This signaling event not only preceded prominent signs of cartilage degeneration but also the increase in global protein synthesis rate. These results suggest that abnormal mTORC1 activity is one of the primary dysregulations observed in OA cartilage. Importantly, it is distributed disproportionately between targets, with 4E-BP1 being phosphorylated earlier than other downstream targets. Thus, our work provides new insights into the sequence of molecular events leading to cartilage destruction in OA and identifies translational control as an important regulatory hub involved in initiating OA. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2728-2735, 2018.

Mithramycin A Alleviates Osteoarthritic Cartilage Destruction by Inhibiting HIF-2α Expression.

Osteoarthritis (OA) is the most common and increasing joint disease worldwide. Current treatment for OA is limited to control of symptoms. The purpose of this study was to determine the effect of specificity protein 1 (SP1) inhibitor Mithramycin A (MitA) on chondrocyte catabolism and OA pathogenesis and to explore the underlying molecular mechanisms involving SP1 and other key factors that are critical for OA. Here, we show that MitA markedly inhibited expressions of matrix-degrading enzymes induced by pro-inflammatory cytokine interleukin-1β (IL-1β) in mouse primary chondrocytes. Intra-articular injection of MitA into mouse knee joint alleviated OA cartilage destruction induced by surgical destabilization of the medial meniscus (DMM). However, modulation of SP1 level in chondrocyte and mouse cartilage did not alter catabolic gene expression or cartilage integrity, respectively. Instead, MitA significantly impaired the expression of HIF-2α known to be critical for OA pathogenesis. Such reduction in expression of HIF-2α by MitA was caused by inhibition of NF-κB activation, at least in part. These results suggest that MitA can alleviate OA pathogenesis by suppressing NF-κB-HIF-2α pathway, thus providing insight into therapeutic strategy for OA.

A biomarker of hypertrophic chondrocytes, type X collagen, completely distinguishes between OA and RA

Purpose: Osteoarthritis (OA) is partly characterized by cartilage deterioration and cellular changes leading to chondrocyte hypertrophy. Hypertrophy of chondrocytes is a key event in cartilage calcification and the thickening of the subchondral bone, of which type X collagens is a well-known marker. The purpose was to develop a specific immunoassay to detect and quantify in serum/plasma a newly identified neo-epitope of type X collagen to assess the diagnostic value of this neo-epitope for radiographic knee OA and to compare that to the cartilage destruction in OA. Methods: An analysis for naturally occurring peptides in human urine from OA patients was carried out by LC-MS/MS. A novel cleavage site in type X collagen was identified. Monoclonal antibody against the first 10aa (479GIATKGLNGP) of the free N-terminus generated by this cleavage was produced. To identify the enzyme responsible for the cleavage, articular cartilage from patients who underwent knee replacement was cleaved by numerous proteases in vitro. Immunohistochemical detection of this neo-epitope was performed on human OA cartilage from formal condyle. A specific enzyme-linked immunoassay was developed by employing the neo-epitope antibody and quantified in plasma samples of two clinical studies: the NUY I study at baseline (21 non-OA healthy controls, 146 symptomatic OA with K/L 0-4and 36 RA; 1 non-OA healthy control, 4 OA and 2 RA samples were unavailable for measurement in the current study) and the C4Pain-003 study ( 281 subjects with K/L 0-4; only 253 plasma samples were available for measurement in the current study). Col10neo was log transformed and analyzed with One-way ANOVA and ANCOVA. ROC curve analysis was carried out to evaluate the discriminative power of Col10neo between OA and RA. Results: A monoclonal antibody 2F4 targeting 479GIATKG neo-epitope was produced. Cathepsin K among 9 tested proteases including MMPs, ADAMTSs and other Cathepsins could generate this neo-epitope when incubated with human OA cartilage. In sections with mild or moderate cartilage degradation stained with Safranin O/Fast green, GIATKG neo-epitope was localized to the pericellular matrix of chondrocytes, while in sections with advanced proteoglycan degradation, its presence was extended to the territorial matrix of chondrocyte clusters and more prominent in superficial fibrillation. A technical performing immunoassay, Col10neo, detecting GIATKG neo-epitope was developed (Intra-assay CV% 4, Inter-assay CV% 9.7). In the NUY I study, log10_Col10neo was statistically higher in OA than control (p=0.0002) or RA (p<0.0001), and no significant difference was seen between control and RA (Fig. 1A). When adjusted for age and gender, Col10neo remains significantly higher in OA compared to control (p=0.003) and RA (p<0.0001). However, the difference between Col10neo in control and RA became significant (p=0.0084) after adjustment. ROC curve analysis revealed area under curve (AUC) was 0.875 (95% confidence interval 0.806-0.9427; P˂0.0001)(Fig 1.B).In the C4Pain study, there was a trend toward higher level of Col10neo in subjects with greater K/L score (Fig. 1C). The greatest percent of subjects with K/L3-4 was in the highest title of Col10neo (Fig. 1D). Conclusions: Our findings indicate that a neo-epitope of type X collagen, a marker of hypertrophic chondrocyte, is a potential biochemical marker for burden of disease in knee OA. Interesting, these data also suggest for the first time that cartilage destruction may be mediated by different pathological mechanisms in RA and OA.

E74-Like Factor (ELF3) and Leptin, a Novel Loop Between Obesity and Inflammation Perpetuating a Pro-Catabolic State in Cartilage

Background/Aims: The E74-like factor 3 (ELF3) is an inflammatory mediator that participates in cartilage destruction in osteoarthritis. Leptin and other adipokines negatively impact articular cartilage, triggering catabolic and inflammatory responses in chondrocytes. Here, we investigated whether leptin induces ELF3 expression in chondrocytes and the signaling pathway involved in this process. Methods: We determined mRNA and protein levels of ELF3 by RT-qPCR and Western blotting using cultured human primary chondrocytes and the human T/C-28a2 chondrocyte cell line. Further, we measured luciferase activities of different reporter constructs, and we assessed the contribution of leptin to the induction of ELF3 mRNA by knocking down hLEPR gene expression using siRNA technology. Results: Leptin synergizes with IL-1β in inducing ELF3 expression in chondrocytes. We also found that PI3K, p38, and JAK2 signaling pathways are at play in the leptin-driven induction of ELF3. Moreover, we confirm the participation of NFΚB in the leptin/IL-1β synergistic induction of ELF3. Conclusion: Here we show, for the first time, the regulation of ELF3 expression by leptin, suggesting that this transcription factor likely mediates the inflammatory responses triggered by leptin in articular chondrocytes.

Interferon regulatory factor 5 (IRF5) regulates the expression of matrix metalloproteinase-3 (MMP-3) in human chondrocytes

Matrix metalloproteinase-3 (MMP-3) plays a pivotal role in the destruction of articular cartilage in osteoarthritis (OA). The regulation of gene expression of MMP-3 is complicated. Interferon regulatory factor 5 (IRF5) is a member of the interferon regulatory factor family of transcription factors. Little information regarding the biological function of IRF5 on chondrocytes and the pathogenesis of OA has been reported. In the current study, for the first time, we report that IRF5 is expressed in human primary chondrocytes and human chondrosarcoma cell line SW1353 cells. In addition, IRF5 is upregulated in response to TNF-α treatment in a dose dependent manner. Interestingly, IRF5 is significantly higher in chondrocytes from OA patients compared to those from normal subjects. Notably, IRF5 mediates TNF-α- induced expression of MMP-3 in chondrocytes. Overexpression of IRF5 promotes the expression of MMP-3, however, knockdown of IRF5 reduces the expression of MMP-3. Mechanistically, IRF5 is able to enhance the transcription of MMP-3 by binding to its promoter. Also, we found that NF-κB was involved in the effects of IRF-5 on MMP-3 expression. These findings suggest that IRF5 might be a novel pharmacological target for the treatment of OA and RA.