Bernard Terlain

Obesity and osteoarthritis: more complex than predicted!

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 …

Cyclo-oxygenase isoenzymes. How recent findings affect thinking about nonsteroidal anti-inflammatory drugs.

SummaryThe discovery of at least 2 cyclo-oxygenase (COX) isoenzymes, referred to as COX-1 and COX-2, has updated our knowledge of nonsteroidal anti-inflammatory drugs (NSAIDs). This has lead investigators to reconsider what can be awaited from this class of drugs. The 2 COX isoenzymes share structural and enzymatic similarities, but are specifically regulated at the molecular level and may be distinguished apart in their functions, although some physiological overlap between them does occur.The major goal in developing selective COX inhibitors is to improve NSAID tolerability. Classic NSAIDs preferentially inhibit COX-1 in vitro, but it appears hazardous to judge their gastrointestinal (GI) safety profile from these data. New compounds with a high selectivity for COX-2, especially those that are non-acidic, may be better tolerated in the GI tract.While these compounds also might have a potential use in various diseases such as colorectal cancer and neurodegenerative diseases of the Alzheimer type, the possible appearance of adverse effects, perhaps renally-related, must be taken into consideration. Finally, well-designed large clinical trials are required to adequately estimate both the promising therapeutic advantages that may be offered by highly selective NSAIDs, and the potential drawbacks that may be inherent with prolonged COX-2 inhibition.

Interleukin-1β down-regulates the expression of glucuronosyltransferase I, a key enzyme priming glycosaminoglycan biosynthesis influence of glucosamine on interleukin-1β-mediated effects in rat chondrocytes

OBJECTIVE To assess the variations of galactose-beta-1,3-glucuronosyltransferase I (GlcAT-I) expression related to the decrease in proteoglycan synthesis mediated by interleukin-1beta (IL-1beta) in rat chondrocytes, and to evaluate the influence of glucosamine on the effects elicited by this proinflammatory cytokine. METHODS Rat articular chondrocytes in primary monolayer cultures or encapsulated into alginate beads were treated with recombinant IL-1beta in the absence or presence (1.0-4.5 gm/liter) of glucosamine. Variations of GlcAT-I and expression of stromelysin 1 (matrix metalloproteinase 3 [MMP-3]) messenger RNA (mRNA) were evaluated by quantitative multistandard reverse transcriptase-polymerase chain reaction. In vitro enzymatic activity of GlcAT-I was measured by thin-layer chromatography, with radiolabeled UDP-glucuronic acid and a digalactoside derivative as substrates. Proteoglycan synthesis was determined by ex vivo incorporation of Na2-35SO4. Nitric oxide synthase and cyclooxygenase activities were monitored by the evaluation of nitrite (NO2-) and prostaglandin E2 (PGE2) produced in the culture medium, respectively. RESULTS IL-1beta treatment resulted in a marked inhibition of GlcAT-I mRNA expression and in vitro catalytic activity, together with a decrease in proteoglycan synthesis. In addition, glucosamine was able to prevent, in a dose-dependent manner, the inhibitory effects of IL-1beta. In the same way, the amino sugar reduced NO2- and PGE2 production induced by IL-1beta. Finally, the up-regulation of stromelysin 1 (MMP-3) mRNA expression by IL-1beta was fully prevented by glucosamine. CONCLUSION The results of this study suggest that the deleterious effect of IL-1beta on the anabolism of proteoglycan could involve the repression of GlcAT-I, a key enzyme in the biosynthesis of glycosaminoglycan. Glucosamine was highly effective in preventing these IL-1beta-mediated suppressive effects. The amino sugar also prevented the production of inflammatory mediators induced by the cytokine. This action could account for a possible beneficial effect of glucosamine on osteoarthritic articular cartilage.

Glucosamine modulates IL-1-induced activation of rat chondrocytes at a receptor level, and by inhibiting the NF-κB pathway

We recently reported that glucosamine reversed the decrease in proteoglycan synthesis and in UDP‐glucuronosyltransferase I mRNA expression induced by interleukin‐1β (IL‐1β) [Arthritis Rheum. 44 (2001) 351–360]. In the present work, we show that glucosamine does not exert the same effects when chondrocytes were stimulated with reactive oxygen species (ROS). In order to better understand its mechanism of action, we determined if glucosamine could prevent the binding of IL‐1β to its cellular receptors or could interfere with its signaling pathway at a post‐receptor level. Addition of glucosamine to rat chondrocytes treated with IL‐1β or with ROS decreased the activation of the nuclear factor κB, but not the activator protein‐1. After treatment with IL‐1β, glucosamine increased the expression of mRNA encoding the type II IL‐1β receptor. These results emphasize the potential role of two regulating proteins of the IL‐1β signaling pathway that could account for the beneficial effect of glucosamine in osteoarthritis.

15-Deoxy-Δ12,14-PGJ2, but not troglitazone, modulates IL-1β effects in human chondrocytes by inhibiting NF-κB and AP-1 activation pathways

The activation of peroxisome proliferator‐activated receptor γ (PPARγ) has been shown to inhibit the production and the effects of proinflammatory cytokines. Since interleukin‐1β (IL‐1β) directly mediates cartilage degradation in osteoarthritis, we investigated the capability of PPARγ ligands to modulate IL‐1β effects on human chondrocytes. RT‐PCR and Western blot analysis revealed that PPARγ expression was decreased by IL‐1β. 15‐Deoxy‐Δ12,14‐prostaglandin J2 (15d‐PGJ2), in contrast to troglitazone, was highly potent to counteract IL‐1β‐induced cyclooxygenase‐2 and inductible nitric oxide synthase expression, NO production and the decrease in proteoglycan synthesis. Western blot and gel‐shift analyses demonstrated that 15d‐PGJ2 inhibited NF‐κB activation, while troglitazone was ineffective. Although 15d‐PGJ2 attenuated activator protein‐1 binding on the DNA, it potentiated c‐jun migration in the nucleus. The absence or the low effect of troglitazone suggests that 15d‐PGJ2 action in human chondrocytes is mainly PPARγ‐independent.

Relations between functional, inflammatory, and degenerative parameters during adjuvant arthritis in rats

We assessed the time-course of adjuvant arthritis (AA) in Lewis rats, using biotelemetry to monitor the rats spontaneous locomotor activity and body temperature, and studied the evolution of the arthritic index, circulating concentrations of inflammation-promoting cytokines, cartilage proteoglycan synthesis, and the effect of indomethacin as a cyclooxygenase inhibitor to evaluate prostaglandin (PG) contribution in AA. The injection of complete Freunds adjuvant on day 0( D0) induced a marked, transient loss of locomotor activity ( D1- D4; initial phase) and then a second phase of hypomobility peaking on D15 and thereafter irreversible ( D16- D20; arthritic phase). Fever peaked first on D1 and again between D13 and D17. The primary hyperthermia was associated with increases in plasma interleukin-6 and tumor necrosis factor-α concentrations and seemed to be partly PG dependent. Proteoglycan synthesis inhibition in the patellar cartilage increased gradually, spreading from the injected paw to the contralateral paw. It was corrected on D20 by preventive and curative indomethacin treatments. Indomethacin also greatly relieved hypomobility during the systemic phase of AA ( D10- D15). The combination of information about cartilage metabolism, body temperature, locomotor activity, and cytokine in this study permits analysis of analgesic, antipyretic, anti-inflammatory, and chondroprotective properties of drugs in the various phases of AA. Thus, using a new methodology, we have discriminated the different phases of the disease and confirmed the symptomatic and systemic inhibitory effect of indomethacin on fever, activity, and cartilage metabolism.

Pefloxacin-Induced Achilles Tendon Toxicity in Rodents: Biochemical Changes in Proteoglycan Synthesis and Oxidative Damage to Collagen

ABSTRACT Despite a relatively low incidence of serious side effects, fluoroquinolones and the fluoroquinolone pefloxacin have been reported to occasionally promote tendinopathy that might result in the complication of spontaneous rupture of tendons. In the present study, we investigated in rodents the intrinsic deleterious effect of pefloxacin (400 mg/kg of body weight) on Achilles tendon proteoglycans and collagen. Proteoglycan synthesis was determined by measurement of in vivo and ex vivo radiosulfate incorporation in mice. Collagen oxidative modifications were measured by carbonyl derivative detection by Western blotting. An experimental model of tendinous ischemia (2 h) and reperfusion (3 days) was achieved in rats. Biphasic changes in proteoglycan synthesis were observed after a single administration of pefloxacin, consisting of an early inhibition followed by a repair-like phase. The depletion phase was accompanied by a marked decrease in the endogenous serum sulfate level and a concomitant increase in the level of sulfate excretion in urine. Studies of ex vivo proteoglycan synthesis confirmed the in vivo results that were obtained. The decrease in proteoglycan anabolism seemed to be a direct effect of pefloxacin on tissue metabolism rather than a consequence of the low concentration of sulfate. Pefloxacin treatment for several days induced oxidative damage of type I collagen, with the alterations being identical to those observed in the experimental tendinous ischemia and reperfusion model. Oxidative damage was prevented by coadministration of N-acetylcysteine (150 mg/kg) to the mice. These results provide the first experimental evidence of a pefloxacin-induced oxidative stress in the Achilles tendon that altered proteoglycan anabolism and oxidized collagen.

Gene transfer with HSP 70 in rat chondrocytes confers cytoprotection in vitro and during experimental osteoarthritis

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.—Grossin, L., Cournil‐Henrionnet, C., Pinzano, A., Gaborit, N., Dumas, D., Etienne, S., Stoltz, J. F., Terlain, B., Netter, P., Mir, L. M., Gillet, P. Gene transfer with HSP 70 in rat chondrocytes confers cytoprotection in vitro and during experimental osteoarthritis. FASEB J. 20, 65–75 (2006)

Evidence for neurogenic transmission inducing degenerative cartilage damage distant from local inflammation.

OBJECTIVE To investigate involvement of the nervous system in ipsilateral and contralateral joint inflammation. METHODS Freunds complete adjuvant (CFA; 1 mg or 1 microg) was injected unilaterally and the messages (a) from the hind paw to the ipsilateral and contralateral knees and (b) from one knee to the contralateral knee were analyzed. The degenerative impact of the local injury on distant cartilage was assessed using patellar proteoglycan synthesis as an indicator. Neurogenic mechanisms were blocked either by spinal cord compression or by injection of neurokinin 1 (NK-1) antagonist, or they were mimicked by intraarticular injection of substance P. The data were compared with those gathered in a model of systemic inflammation, characterized by fever and serum interleukin-6 production. RESULTS After unilateral subcutaneous injection of CFA, proteoglycan anabolism decreased bilaterally. Spinal cord compression and administration of the NK-1 antagonist inhibited the response in the contralateral limb. Following 1 mg CFA subcutaneous injection, the ipsilateral response implicated both neurogenic and systemic mechanisms, whereas the nervous system alone was implicated after 1 microg subcutaneous CFA injection. The 1 microg CFA intraarticular injection induced a degenerative contralateral signal, which was abolished by spinal cord compression and by pretreatment with the NK-1 antagonist. Intraarticular injection of 1 microg CFA also induced an ipsilateral increase of anabolism, which was enhanced by spinal cord compression. Similar results were obtained after intraarticular injections of substance P. These effects were not reproduced with turpentine treatment, a systemic model, in which spinal cord compression had no effect. CONCLUSION A unilateral inflammation can induce, by neurogenic mechanisms, distal bilateral degeneration of articular cartilage, implicating involvement of neuropeptides.

Effects of the Inhibition of Cyclo‐Oxygenase 1 or 2 or 5‐Lipoxygenase on the Activation of the Hypothalamic‐Pituitary‐Adrenal Axis Induced by Interleukin‐1β in the Male Rat

The limited entry of interleukin‐1β (IL‐1β) into the central nervous system has led to the hypothesis that IL‐1β acts, through IL‐1β receptors located notably on endothelial cells, on the release of prostaglandins which in turn stimulate the hypothalamic‐pituitary‐adrenal (HPA) axis. We used cyclo‐oxygenase‐1 (COX‐1) and cyclo‐oxygenase‐2 (COX‐2) and 5‐lipoxygenase (5‐LOX) inhibitors, before the injection of IL‐1β, to explore the role of arachidonic acid metabolic pathways on HPA axis activation. Adult male rats were i.m injected 20 min before i.p injection of IL‐1β, with (i): a COX‐1/COX‐2 inhibitor (ketoprofen); (ii) a COX‐2 selective inhibitor (NS 398); or (iii) a 5‐LOX inhibitor (BW A4C). Following this, rats were killed 90 min after i.p. IL‐1β injection and analysis for plasma adrenocorticotropic hormone (ACTH) and corticosterone concentrations and determination of anterior pituitary pro‐opio melanocortin (POMC) gene transcription was conducted. Administration of the COX‐1/COX‐2 inhibitor led to a complete blockage of ACTH and corticosterone secretion and POMC gene transcription. The COX‐2 inhibitor led to a complete blockade of ACTH secretion and POMC gene transcription but had no effect on corticosterone secretion. The 5‐LOX inhibitor had no significant effect on any parameter. These results demonstrate the crucial role of eicosanoid pathways in mediating the stimulation of the HPA axis induced by IL‐1β. Moreover, we found a clear dissociation of the effect of the blockage of COXs upon ACTH and corticosterone secretion, suggesting that IL‐1β may act at the brain as well as at the adrenal cortex to stimulate the secretion of corticosterone.