Beneficial Impact of Eicosapentaenoic Acid on the Adverse Effects Induced by Palmitate and Hyperglycemia on Healthy Rat Chondrocyte.

Osteoarthritis (OA) is the most prevalent form of arthritis and a major cause of pain and disability. The pathology of OA involves the whole joint in an inflammatory and degenerative process, especially in articular cartilage. OA may be divided into distinguishable phenotypes including one associated with the metabolic syndrome (MetS) of which dyslipidemia and hyperglycemia have been individually linked to OA. Since their combined role in OA pathogenesis remains to be elucidated, we investigated the chondrocyte response to these metabolic stresses, and determined whether a n-3 polyunsaturated fatty acid (PUFA), i.e., eicosapentaenoic acid (EPA), may preserve chondrocyte functions. Rat chondrocytes were cultured with palmitic acid (PA) and/or EPA in normal or high glucose conditions. The expression of genes encoding proteins found in cartilage matrix (type 2 collagen and aggrecan) or involved in degenerative (metalloproteinases, MMPs) or in inflammatory (cyclooxygenase-2, COX-2 and microsomal prostaglandin E synthase, mPGES) processes was analyzed by qPCR. Prostaglandin E2 (PGE2) release was also evaluated by an enzyme-linked immunosorbent assay. Our data indicated that PA dose-dependently up-regulated the mRNA expression of MMP-3 and -13. PA also induced the expression of COX-2 and mPGES and promoted the synthesis of PGE2. Glucose at high concentrations further increased the chondrocyte response to PA. Interestingly, EPA suppressed the inflammatory effects of PA and glucose, and strongly reduced MMP-13 expression. Among the free fatty acid receptors (FFARs), FFAR4 partly mediated the EPA effects and the activation of FFAR1 markedly reduced the inflammatory effects of PA in high glucose conditions. Our findings demonstrate that dyslipidemia associated with hyperglycemia may contribute to OA pathogenesis and explains why an excess of saturated fatty acids and a low level in n-3 PUFAs may disrupt cartilage homeostasis.

The infrapatellar fat pad induces inflammatory and degradative effects in articular cells but not through leptin or adiponectin.

OBJECTIVES: Based on a novel approach suggesting a role of adipose tissue in osteoarthritis (OA), we aimed to determine whether the infrapatellar fat pad (IFP) may affect joint cell functions through adipokines. METHODS: The conditioned media of IFP and subcutaneous adipose tissue from OA patients were used to determine the production of leptin and adiponectin, and to stimulate chondrocytes and fibroblast-like synoviocytes (FLS). Blocking experiments were carried out to evaluate the contribution of adipokines to IFP effects. The gene expression of inflammatory and degradative proteins, growth factors and components of the extracellular matrix, and the production of inflammatory mediators and metalloproteases were determined to evaluate cell response to fat-conditioned media. RESULTS: IFP releases elevated amounts of leptin and adiponectin independently of the body mass index and the gender. The conditioned media from IFP strongly induce the expression of inflammatory genes in both articular cells and the expression of degradative genes in chondrocytes, but remain ineffective in regulating the expression of aggrecan, type 2 collagen or growth factors. Blocking leptin or adiponectin does not change the cell response to IFP. A great variability between patients is found when compared the inflammatory activity of paired samples of IFP and subcutaneous adipose tissue. CONCLUSIONS: IFP may trigger both cartilage destruction and inflammation of the synovium, but not through leptin or adiponectin. The data suggest also that IFP may have specific inflammatory phenotypic features independent from the general phenotype found in obesity.

Synovial fluid levels of adipokines in osteoarthritis: Association with local factors of inflammation and cartilage maintenance.

The role of body weight in the pathogenesis of osteoarthritis (OA) - previously considered the sole factor in the association between obesity and OA - is being re-evaluated as the contribution of adiposity to the cartilage degenerative process becomes clearer. The current study has been undertaken to better understand the role of adipose-derived proteins, namely adipokines, in OA. For this purpose, we investigated in patients with OA the relationships between the joint levels of leptin, adiponectin and resistin and those of factors involved in inflammation and cartilage maintenance. The sandwich enzyme-linked immunosorbent assays were used to determine in the synovial fluid (SF) from 35 OA patients, the concentrations of adipokines, interleukin-6 (IL-6) and transforming growth factor-ß (TGF-ß). The soluble form of leptin receptor (sOb-R) was also examined to evaluate the biological active free form of leptin. Correlation analysis indicate that IL-6 levels are positively related to the levels of resistin and adiponectin. Surprisingly, the free form of leptin, but not the total leptin, is negatively associated with IL-6. Beside, adiponectin is the single adipokine that is correlated with TGF-ß. Interestingly, a sexual dimorphism is observed in the study as correlations between adipokines and IL-6 or TGF-ß are found only with female OA patients. Taken together, these findings suggest that only adiponectin may contribute to the metabolic changes associated with OA. The three adipokines may also be involved in inflammation, but with opposite effects. Both resistin and adiponectin may exhibit pro-inflammatory activity while the free form of leptin may down-regulate the inflammation.

Association between the chondrocyte phenotype and the expression of adipokines and their receptors: evidence for a role of leptin but not adiponectin in the expression of cartilage-specific markers.

Although extensive evidence support the key role of adipokines in cartilage homeostasis, contradictory data have been found for their expression and their effects in chondrocytes. This study was then undertaken to determine whether a phenotypic modulation may affect the expression of adipokines and their receptors in human chondrocytes. The expression of leptin, adiponectin and their receptors, as well as cartilage-specific genes was examined in chondrocytes obtained from patients with osteoarthritis either directly after cells harvest or after culture in monolayer or in alginate beads. The results showed major changes in the gene expression pattern after culture in monolayer with a shift from the adipokines to their receptors. Interestingly, this downregulation of adipokines was associated with a loss of chondrocyte phenotype, and chondrocytes recovered a cartilage-like expression profile of leptin and adiponectin when cultured in a tridimensional chondrocyte phenotype-inducing system, but ceased expressing their receptors. Further experiments clearly showed that leptin but not adiponectin promoted the expression of cartilage-specific markers through mitogen-activated protein kinase, Janus kinase and phosphatidylinositol-3 kinase signaling pathways. In conclusion, our data indicate that any phenotypic modulation could affect chondrocyte responsiveness to leptin or adiponectin, and provide evidence for an important role for leptin in regulating the expression of cartilage-specific markers.

Obesity affects the chondrocyte responsiveness to leptin in patients with osteoarthritis.

INTRODUCTION: Increasing evidence support the regulatory role of leptin in osteoarthritis (OA). As high circulating concentrations of leptin disrupt the physiological function of the adipokine in obese individuals, the current study has been undertaken to determine whether the elevated levels of leptin found in the joint from obese OA patients also induce changes in the chondrocyte response to leptin. METHODS: Chondrocytes isolated from OA patients with various body mass index (BMI) were treated with 20, 100 or 500 ng/ml of leptin. The expression of cartilage-specific components (aggrecan, type 2 collagen), as well as regulatory (IGF-1, TGFbeta, MMP-13, TIMP 2) or inflammatory (COX-2, iNOS, IL-1) factors was investigated by real-time PCR to evaluate chondrocyte responsiveness to leptin. Furthermore, the effect of body mass index (BMI) on leptin signalling pathways was analyzed with an enzyme-linked immunosorbent assay for STATs activation. RESULTS: Leptin at 20 ng/ml was unable to modulate gene expression in chondrocytes, except for MMP-13 in obese OA patients. Higher leptin levels induced the expression of IGF-1, type 2 collagen, TIMP-2 and MMP-13. However, the activity of the adipokine was shown to be critically dependent on both the concentration and the BMI of the patients with a negative association between the activation of regulated genes and BMI for 100 ng/ml of adipokine, but a positive association between chondrocyte responsiveness and BMI for the highest leptin dose. In addition, the gene encoding MMP-13 was identified as a target of leptin for chondrocytes originated from obese patients while mRNA level of TIMP-2 was increased in leptin-treated chondrocytes collected from normal or overweight patients. The adipokine at 500 ng/ml triggered signal transduction through a STAT-dependent pathway while 100 ng/ml of leptin failed to activate STAT 3 but induced STAT 1alpha phosphorylation in chondrocytes obtained from obese patients. CONCLUSIONS: The current study clearly showed that characteristics of OA patients and more especially obesity may affect the responsiveness of cultured chondrocytes to leptin. In addition, the BMI-dependent effect of leptin for the expression of TIMP-2 and MMP-13 may explain why obesity is associated with an increased risk for OA.

[Leptin: a link between obesity and osteoarthritis?]. / La leptine: un lien entre obésité et arthrose?

In addition to aging, obesity is one of the most common underlying causes of osteoarthritis (OA). Mechanical loading, together with biochemical and systemic factors linked to altered lipid metabolism, are thought to contribute to the onset of OA. It has been suggested that OA is a systemic metabolic disease associated with lipid disorders affecting joint homeostasis. These gradual changes may be due to the local effect of adipokines, and especially leptin. Indeed, their relative levels in joints differ from that found in plasma. In particular, leptin levels are increased and adiponectin and resistin levels are reduced This hypothesis is supported by--leptin overexpression in OA cartilage and its correlation with the degree of cartilage destruction,--abundant leptin synthesis by osteophytes, and--the high leptin levels found in OA joints from female patients. This link between OA and adipokines provides new leads regarding the prevention of OA and the identification of new drug targets.

Evidence for a key role of leptin in osteoarthritis.

OBJECTIVE: To evaluate the contribution of leptin (an adipose tissue-derived hormone) to the pathophysiology of osteoarthritis (OA), by determining the level of leptin in both synovial fluid (SF) and cartilage specimens obtained from human joints. We also investigated the effect of leptin on cartilage, using intraarticular injections of leptin in rats. METHODS: Leptin levels in SF samples obtained from OA patients undergoing either knee replacement surgery or knee arthroscopy were measured by enzyme-linked immunosorbent assay. In addition, histologic sections of articular cartilage and osteophytes obtained during surgery for total knee replacement were graded using the Mankin score, and were immunostained using antibodies to leptin, transforming growth factor beta (TGFbeta), and insulin-like growth factor 1 (IGF-1). For experimental studies, various doses of leptin (10, 30, 100, and 300 microg) were injected into the knee joints of rats. Tibial plateaus were collected and processed for proteoglycan synthesis by radiolabeled sulfate incorporation, and for expression of leptin, its receptor (Ob-Rb), and growth factors by reverse transcriptase-polymerase chain reaction and immunohistochemical analysis. RESULTS: Leptin was observed in SF obtained from human OA-affected joints, and leptin concentrations correlated with the body mass index. Marked expression of the protein was observed in OA cartilage and in osteophytes, while in normal cartilage, few chondrocytes produced leptin. Furthermore, the pattern and level of leptin expression were related to the grade of cartilage destruction and paralleled those of growth factors (IGF-1 and TGFbeta1). Animal studies showed that leptin strongly stimulated anabolic functions of chondrocytes and induced the synthesis of IGF-1 and TGFbeta1 in cartilage at both the messenger RNA and the protein levels. CONCLUSION: These findings suggest a new peripheral function of leptin as a key regulator of chondrocyte metabolism, and indicate that leptin may play an important role in the pathophysiology of OA.

Interleukin 17, a nitric oxide-producing cytokine with a peroxynitrite-independent inhibitory effect on proteoglycan synthesis.

OBJECTIVE: To compare the potency of 2 cytokines, interleukin 17 (IL-17) and IL-1beta, on rat cartilage proteoglycan synthesis with special attention to nitric oxide (NO) and peroxynitrite formation. METHODS: Chondrocytes in alginate beads were stimulated with human recombinant (rh) IL-17 (0.03 to 300.0 ng/ml) and/or rhIL-1beta (0.25 to 25.0 ng/ml) in the presence or not of L-NMMA or CuDips. Alternatively, rats were injected with either IL-17 (10.0 micro g) or IL-1beta (1.0 micro g) into each knee joint. NO concentrations were determined by a spectrofluorimetric assay, proteoglycan synthesis by 35SO4-2 incorporation, peroxynitrite generation by immunostaining for 3-nitrotyrosine, and IL-1beta mRNA expression by reverse transcription-polymerase chain reaction. RESULTS: IL-17 inhibited proteoglycan synthesis and increased NO production, both in vitro and in vivo, without inducing expression of IL-1beta mRNA in cartilage. Additive effects were observed when IL-17 was combined with low concentrations of IL-1. Surprisingly, a similar NO synthesis between IL-1 and IL-17 led to a less suppressive effect of IL-17 on cartilage anabolism than with IL-1. Both in vitro and in vivo, peroxynitrite formation was extensive with IL-1beta, but negligible or nonexistent with IL-17. L-NMMA and CuDips completely corrected the suppressive effect of IL-1beta on proteoglycan synthesis, unlike with IL-17. CONCLUSION: These data showed that NO is weakly involved in the IL-17 mediated inhibition of proteoglycan synthesis in rat. NO overload may not be predictive of any inhibitory effect on cartilage anabolism, but instead superoxide is a key regulator of NO contribution to chondrocyte dysfunction. Since IL-17 is a NO-producing cytokine with additive effects when combined with IL-1, it may play a pivotal role in cartilage destruction during rheumatoid arthritis, for which infiltrating cells produce high levels of superoxide and proinflammatory cytokines.

Nitric oxide (NO) and cartilage metabolism: NO effects are modulated by superoxide in response to IL-1.

Nitric oxide (NO) is thought to mediate most effects of interleukin-1 (IL-1) on cartilage. In vitro evidence includes the decreased synthesis of extracellular matrix components, the abnormal cell renewal, the decreased production of IL-1 receptor antagonist, the induction of apoptosis and the enhanced sensitivity of chondrocytes to oxidative stress. Studies in NOS2(-/-) mice or administration of NO synthase inhibitors in animal models of joint disorders have confirmed its potent pathophysiological role in cartilage. Using L-NMMA (1 mM), as a NO synthase inhibitor, and CuDips (10 microM), as a SOD mimetic, we provide evidence that the inhibitory potency of IL-1beta on proteoglycan synthesis and its stimulating effect on COX-2 activity depend both on NO and O2-* production. Peroxynitrite formation is further demonstrated by the occurrence of 3-nitrotyrosines in chondrocytes stimulated in vitro with 2.5 ng/ml IL-1 and in femoral condyles of rats injected locally with 1 microg IL-1. Preliminary data suggest that such contribution of reactive oxygen species is not shared in common by IL-17, another NO-producing cytokine. We conclude that superoxide is a key modulator of NO-mediated effects in chondrocyte stimulated with IL-1 and that a combined therapy with NO synthase inhibitors and antioxidants may be promising for a full cartilage protection.