Dysregulation of lipid homeostasis is one of the mechanisms leading to osteoarthritis Osteoarthritis is usually considered to be a joint disorder the central pathological feature of which is cartilage destruction. However, this concept has evolved, and today osteoarthritis is generally regarded as a disease that may affect the whole joint (bone, muscles, ligaments and synovium). Although the aetiology of osteoarthritis is not established, the main risk factors are well known and commonly include mechanical, biochemical and genetic factors. Of these risk factors, obesity is beyond doubt considered a prominent one. The overload effect on joint cartilage may explain part of the increased risk of osteoarthritis, at least for osteoarthritis of the knee, in overweight people. A recent discovery in the discipline of cartilage biology is the presence of mechanoreceptors at the surface of chondrocytes, which are sensitive to pressure and link extracellular environment to intracellular signalling cascades. Three types of mechanoreceptors have been described on chondrocytes: the stretch-activated channels, the α-5β1 integrin and CD44. Compression and stretch stimulate integrins and stretch-activated channels leading to the activation of signalling pathways (mitogen-activated protein kinase, NF-κB), as well as the release of second messengers (calcium, Inositol triphosphate and Adenosine monophosphate cyclic). 1 After mechanoreceptor activation, cytokines, growth factors and metalloproteinases may be expressed, and mediators such as prostaglandins or nitric oxide may be produced.2 As experimental studies have shown that under specific conditions overload may trigger both inhibition of matrix synthesis and cartilage degradation, we can speculate that obesity may induce cartilage damage through activation of these mechanoreceptors. In the same manner, the mechanoreceptors expressed on osteoblasts3,4 may also be involved in the impaired response of chondrocytes to the obesity-induced overload. Even if it is usually accepted that mechanical loading contributes to joint cartilage destruction in overweight patients, recent advances …

Objective: To analyze the distribution of leptin, adiponectin and resistin between paired serum and synovial fluid (SF) samples of patients with osteoarthritis (OA) and to determine the potential sources of these adipokines in the joint. The active free form of leptin was also examined by evaluating the level of the soluble leptin receptor (sOb-R).Methods: Levels of adipokines and sOb-R were measured by a sandwich enzyme-linked immunosorbent assay in serum and SF collected from OA patients. The levels of adipokines were also determined in conditioned media from cultured joint tissues (synovium, infrapatellar fat pad, meniscus, osteophyte, cartilage and bone).Results: The adipokines exhibited different patterns of distribution between the joint and the circulating compartment. Serum levels of resistin and adiponectin exceeded those in the paired SF. Conversely, leptin SF concentrations were similar or higher than those measured in serum counterparts. Leptin and adiponectin in SF may derive from each joint tissue examined, whereas resistin was not detected in conditioned media of cultured explants. Synovium and infrapatellar fat pad were the major sources of adipokines, but osteophytes released also large amounts of leptin. The sOb-R deficiency found in SF further increased the difference in the bioactive leptin levels between serum and SF. A gender-specific difference was observed with women exhibiting the highest level of free leptin in the joint.Conclusion: These data demonstrated that adipokines serum levels are not predictive values for SF determination. The joint cavity is a special space where each adipokine undergoes specific regulatory pathways, strengthening the hypothesis that adipokines may have local effects in the joint and may account for the high prevalence of OA in women. (C) 2006 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

O steoarthritis is usually considered to be a joint disorder the central pathological feature of which is cartilage destruction. However, this concept has evolved, and today osteoarthritis is generally regarded as a disease that may affect the whole joint (bone, muscles, ligaments and synovium). Although the aetiology of osteoarthritis is not established, the main risk factors are well known and commonly include mechanical, biochemical and genetic factors. Of these risk factors, obesity is beyond doubt considered a prominent one.

OBJECTIVE To investigate the potency of selective agonists of peroxisome proliferators-activated receptors' (PPAR) isotypes (alpha, beta/delta or gamma) to modulate the stimulating effect of transforming growth factor-beta1 (TGF-beta1) on proteoglycans' (PGs) synthesis in chondrocytes. METHOD Rat chondrocytes embedded in alginate beads and cultured under low serum conditions were exposed to TGF-beta1 (10 ng/ml), alone or in combination with the following agonists: Wy14643 for PPARalpha, GW501516 for PPARbeta/delta, rosiglitazone (ROSI) for PPARgamma, in the presence or absence of PPAR antagonists (GW6471 for PPARalpha, GW9662 for PPARgamma). PGs' synthesis was evaluated by radiolabelled sulphate incorporation and glycosaminoglycans' (GAGs) content by Alcian blue staining of beads and colorimetric 1.9 dimethyl-methylene blue assay after beads' solubilization. Phosphorylation of Extracellular Signal-related Kinase1/2 (ERK1/2), Smad2/3 and p38-MAPK was assessed by Western Blot and production of prostaglandin E2 (PGE2) by Enzyme immuno-assay (EIA). Levels of mRNA for PPAR target genes [acyl-CoA oxidase (ACO) for PPARalpha; mitochondrial carnitin palmitoyl transferase-1 (CPT-1) for PPARbeta/delta and adiponectin for PPARgamma], aggrecan, TGF-beta1 and genes controlling GAGs' side chains' synthesis were quantified by real time polymerase chain reaction and normalized over RP29 housekeeping gene. RESULTS ACO was selectively up-regulated by 100 microM of Wy14643, CPT-1 by 100 nM of GW501516 and adiponectin by 10 microM of ROSI without cell toxicity. TGF-beta1 increased PGs' synthesis by four-fold, GAGs' content and deposition by 3.5-fold and six-fold, respectively, while inducing aggrecan expression around 10-fold without modifying mRNA levels of GAGs' controlling enzymes. PPAR agonists inhibited the stimulating effect of TGF-beta1 by 24-44% on PGs' synthesis and over 75% on aggrecan, GAGs' content and deposition with the following rank order of potency: ROSI>GW501516> or =Wy14643. TGF-beta1-induced phosphorylation of Smad2/3 and ERK1/2 was reduced by ROSI over GW501516 but not by Wy14643 whereas stimulated PGE2 production was inhibited by Wy14643 over GW501516 but not by ROSI. The effect of PPAR agonists on PPAR target genes and TGF-beta1-induced aggrecan expression was reversed selectively by PPAR antagonists. CONCLUSION In chondrocytes' beads, PPAR agonists reduced the stimulating effect of TGF-beta1 on PGs by inhibiting TGF-beta1-induced aggrecan expression in an isotype-selective manner. Thus, PPAR agonists could be deleterious in situation of cartilage repair although being protective in situation of cartilage degradation.

Osteoarthritis is characterized by a gradual degradation of extracellular matrix, resulting from an excess of chondrocyte cell death, mainly due to an increase in apoptotis. Recent studies have revealed the essential role of HSP70 in protecting cells from stressful stimuli. Therefore, overexpressing HSP70 in chondrocytes could represent a good strategy to prevent extracellular matrix destruction. To this end, we have developed a vector carrying HSP70/GFP, and transduced chondrocytes were thus more resistant to cell death induced by mono-iodoacetate (MIA). To overcome the barrier-effect of matrix, we investigated the efficacy of plasmid delivery by electroporation (EP) in rat patellar cartilage. Two days after EP, 50% of patellar chondrocytes were HSP/GFP+. After 3 months, long-term expression of transgene was only depicted in the deep layer (20-30% positive cells). HSP70 overexpression inhibited the natural endochondral ossification in the deep layer, thus leading to a lesser decrease in chondrocyte distribution. Moreover, overexpression of HSP70, after a preventive EP transfer in rat patella, was sufficient to decrease the severity of osteoarthritis-induced lesions, as demonstrated histologically and biochemically. In conclusion, intracellular overexpression of HSP70, through EP delivery, could protect chondrocytes from cellular injuries and thus might be a novel chondroprotective modality in rat OA.

The contribution of leptin, as a possible link between osteoarthritis (OA) and obesity, was studied in cartilage and synovial fluid samples obtained from osteoarthritic patients. Its effect on cartilage was evaluated in rats after intraarticular injections of leptin. Leptin levels were measured in the synovial fluid samples by enzyme linked immunosorbent assay; leptin concentrations were correlated with the body mass index. Leptin was strongly expressed in osteophytes and OA cartilage, while, in normal cartilage, few chondrocytes produced leptin. The level of leptin expression was related to the grade of cartilage destruction and was in good relation with those of growth factors as IGF1 and TGFb. Studies in rats showed that intraarticular leptin injection stimulated anabolic functions of chondrocytes and induced the synthesis of leptin, IGF1 and TGFB in cartilage at both the chondrocytes and induced the synthesis of leptin, IGF1 and TGFB in cartilage at both the mRNA and protein levels. In conclusion, leptin may be a link between osteoarthritis and obesity, and may play a key role in cartilage metabolism. Leptin may contribute to the pathophysiology of OA.

La contribution de la leptine comme lien possible entre l’arthrose et l’obésité a été étudiée sur des échantillons de cartilages et de liquides synoviaux provenant de patients arthrosiques. Son effet sur le cartilage a été évalué sur des rats soumis à des injections intra-articulaires de leptine recombinante.

AbstractMicrosomal prostaglandin E synthase (mPGES)-1 is a newly identified inducible enzyme of the arachidonic acid cascade with a key function in prostaglandin (PG)E2 synthesis. We investigated the kinetics of inducible cyclo-oxygenase (COX)-2 and mPGES-1 expression with respect to the production of 6-keto-PGF1α and PGE2 in rat chondrocytes stimulated with 10 ng/ml IL-1β, and compared their modulation by peroxisome-proliferator-activated receptor (PPAR)γ agonists. Real-time PCR analysis showed that IL-1β induced COX-2 expression maximally (37-fold) at 12 hours and mPGES-1 expression maximally (68-fold) at 24 hours. Levels of 6-keto-PGF1α and PGE2 peaked 24 hours after stimulation with IL-1β; the induction of PGE2 was greater (11-fold versus 70-fold, respectively). The cyclopentenone 15-deoxy-Δ12,14prostaglandin J2 (15d-PGJ2) decreased prostaglandin synthesis in a dose-dependent manner (0.1 to 10 μM), with more potency on PGE2 level than on 6-keto-PGF1α level (-90% versus -66% at 10 μM). A high dose of 15d-PGJ2 partly decreased COX-2 expression but decreased mPGES-1 expression almost completely at both the mRNA and protein levels. Rosiglitazone was poorly effective on these parameters even at 10 μM. Inhibitory effects of 10 μM 15d-PGJ2 were neither reduced by PPARγ blockade with GW-9662 nor enhanced by PPARγ overexpression, supporting a PPARγ-independent mechanism. EMSA and TransAM® analyses demonstrated that mutated IκBα almost completely suppressed the stimulating effect of IL-1β on mPGES-1 expression and PGE2 production, whereas 15d-PGJ2 inhibited NF-κB transactivation. These data demonstrate the following in IL-1-stimulated rat chondrocytes: first, mPGES-1 is rate limiting for PGE2 synthesis; second, activation of the prostaglandin cascade requires NF-κB activation; third, 15d-PGJ2 strongly inhibits the synthesis of prostaglandins, in contrast with rosiglitazone; fourth, inhibition by 15d-PGJ2 occurs independently of PPARγ through inhibition of the NF-κB pathway; fifth, mPGES-1 is the main target of 15d-PGJ2.

The aim of this work was to determine whether Hsp70 overexpression via proteasome inhibitor MG132 was able to protect chondrocytes towards mono-iodoacetate (MIA) cytotoxicity both in vitro and in vivo. In vitro, overexpression of Hsp70 via MG132 was significantly able to protect chondrocytes from MIA toxicity (MTT/LDH analyses). Hsp70 essentially mediated this chondroprotective effect as demonstrated by antisense strategy. In vivo, chondrocytic overexpression of Hsp70, after a preventive intra-articular injection of MG132 in rat knee, was sufficient to decrease the severity of OA-induced MIA lesions, as demonstrated histologically and biochemically. In conclusion, intracellular overexpression of Hsp70, through proteasome inhibition, could be an interesting tool in protecting chondrocytes from cellular injuries, either necrotic or apoptotic in nature, and thus might be a novel chondroprotective modality in rat experimental OA.

Objectives: To characterize the molecular events underlying cartilage injury in the early phase of mono-iodoacetate-induced osteoarthritis (OA) in rats.Methods: Experimental osteoarthritis was induced by intra-articular injection of 0.03 mg mono-iodoacetate (MIA) in Wistar rats. Animals were killed 2, 5, 10, 15 and 20 days post-injection. Synovial tissue and standardized biopsies from different areas of knee cartilage were examined. Proteoglycan synthesis (S-35 incorporation) and gelatinase activities (zymography), semi-quantitative RT-PCR and immunohistochemistry for IL1beta, iNOS, COX2 and PPARgamma, were performed on these samples.Results: Changes in proteoglycan synthesis and gelatinase activities were time and site-dependent. Proteoglycan synthesis inhibition was maximal by day 2 while the highest gelatinase activities were observed at day 5. Central part of patella and posterior plateaus and condyles, i.e. the weight-bearing cartilage areas, were the most affected.1L1beta and iNOS transcripts were induced early in cartilage at time of maximal proteoglycan synthesis inhibition, especially in weight-bearing areas. COX-2 was slightly up-regulated whereas PPARgamma gene expression remained unchanged. Gene expression profile in synovium paralleled that of cartilage, except for PPARgamma which was up-regulated at day 15 and 20.Immunostaining for IL1beta and iNOS showed that proteins were located in diseased cartilage areas at early stage of the experimental OA (day 2). At later time-points (day 20), IL1beta and iNOS were expressed in perilesional areas whereas immunostaining became below control level for COX-2 and PPARgamma.Conclusions: Time-dependent degradation of cartilage after injection of low dose of MIA (0.03mg) into rat knee joint can be related to early loss of proteoglycan anabolism, increased gelatinase activities and expression of IL1beta and downstream inflammatory genes. Increased susceptibility to MIA in weight-bearing areas of cartilage further indicate that MIA-induced experimental OA is a relevant model to study not only metabolical but also biomechanical aspects of human OA. (C) 2003 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Peroxisome proliferator‐activated receptor γ (PPARγ) ligands have been shown to inhibit the effects of proinflammatory cytokines such as interleukin‐1β (IL‐1β). This cytokine plays a key role in articular pathophysiologies by inducing the production of inflammatory mediators such as nitric oxide (NO) and prostaglandin E2 (PGE2). We previously demonstrated that 15d‐PGJ2 was more potent than troglitazone to counteract IL‐1β effects on chondrocytes. Here, we studied the action of 15d‐PGJ2 on intracellular targets in nuclear factor‐κB (NF‐κB) signalling pathway in IL‐1β treated rat chondrocytes. We found that 15d‐PGJ2 decreased inhibitor κBα (IκBα) degradation but not its phosphorylation by specifically inhibiting IκB kinase β (IKKβ), but not IKKα, enzymatic activity. We further evaluated the involvement of PPARγ in the anti‐inflammatory action of its ligands. In chondrocytes overexpressing functional PPARγ protein, 15d‐PGJ2 pre‐treatment inhibited inducible NO synthase and COX‐2 mRNA expression, nitrite and PGE2 production, p65 translocation and NF‐κB activation. Troglitazone or rosiglitazone pre‐treatment had no effect. 15d‐PGJ2 exhibited the same effect in chondrocytes overexpressing mutated PPARγ protein. These results suggest that 15d‐PGJ2 exerts its anti‐inflammatory effect in rat chondrocytes by a PPARγ‐independent mechanism, which can be conferred to a partial inhibition of IκBα degradation.

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.