L'arthrose (OA) est la maladie dégénérative la plus fréquente (ou) et la principale cause d'incapacité physique avec un coût socioéconomique important. Les manifestations cliniques de l'arthrose peuvent inclure des douleurs, des raideurs et des mouvements articulaires réduits. Pathologiquement, l'OA se caractérise par une dégénérescence progressive du cartilage articulaire, une augmentation de l'expression des médiateurs inflammatoires et cataboliques et le remodelage osseux sous-chondral. Il a été démontré que la protéine prostaglandine D2 (PGD2) est synthétisée par différents types cellulaires et possède des propriétés pro et anti-inflammatoires, selon le récepteur activé. Plusieurs stimuli pro-inflammatoires ont été discernés et étudiés, mais par contre, les voies anti-inflammatoires restent toujours un univers inexploré. Le récepteur DP1 de la PGD2, ainsi que l’enzyme de synthèse L-PGDS, jouent des rôles importants dans l'inflammation et le métabolisme du cartilage. Cependant, leurs rôles dans la pathogenèse de l'arthrose (OA) restent inconnus. Nous avons entrepris l’étude (de quoi) d’une part, pour explorer les rôles de la L- PGDS et de DP1 dans le développement d'OA, et d’autre part pour évaluer l'efficacité d'un agoniste sélectif de DP1 et d’un virus d'AAV2 / 5 codant pour L-PGDS dans le traitement de l'OA. En premier, nos travaux par histologie ont démontré que la dégradation du cartilage est plus prononcée chez les souris Knock-out L-PGDS et DP1, comparativement au souris sauvages Wild-Type (WT). Ensuite une augmentation de l’expression des médiateurs cataboliques (ADAMTS5 et MMP-13), chez les souris L-PGDS -/- et DP1 -/- par rapport au WT a été démontré. Après, la stimulation des explants de cartilage des souris L-PGDS -/- et de DP1 -/- avec l’IL-1a, ont montré une dégradation élevée en protéoglycanes. En outre ces souris ont développer aussi des modifications osseuses sous- chondral. Enfin, nos résultats suggèrent qu’à la suite d'injection intrapéritonéale de l’agoniste spécifique de DP1, le BW245C a atténué la gravité de la dégradation du cartilage induite par une déstabilisation du ménisque médiale (DMM) et des modifications osseuses chez les souris WT. Pareillement, l'injection intra-auriculaire d'AAV2 / 5 codant pour L- PGDS a atténué aussi la dégradation du cartilage induite par DMM et l'expression de ADAMTS-5 et MMP-13 chez des souris L-PGDS -/-. En conclusion, l’ensemble de nos résultats suggèrentque le récepteur DP1 et l’enzyme L-PGDS au niveau du cartilage articulaire arthrosique joue un rôle très important. . Elle pourrait constituer une voie thérapeutique potentielle dans le traitement de l’OA et aussi dans le traitement d’autres pathologies musculo-squelettiques. / Osteoarthritis (OA) is the most common degenerative disease (or) and the leading cause of physical disability with significant socioeconomic costs. Clinical manifestations of osteoarthritis can include pain, stiffness, and reduced joint movement. Pathologically, OA is characterized by progressive degeneration of articular cartilage, increased expression of inflammatory and catabolic mediators, and subchondral bone remodeling. It has been shown that prostaglandin D2 protein (PGD2) is synthesized by different cell types and has pro and anti-inflammatory properties, depending on the activated receptor. Several pro-inflammatory stimuli have been discerned and studied, but the anti- inflammatory pathways remain an unexplored universe. The DP1 receptor of PGD2, as well as the synthetic enzyme L-PGDS, play important roles in inflammation and cartilage metabolism. However, their roles in the pathogenesis of osteoarthritis (OA) remain unknown. We undertook the study (of what) on the one hand, to explore the roles of L-PGDS and DP1 in the development of OA, and on the other hand to evaluate the efficacy of a selective agonist DP1 and an AAV2 / 5 virus encoding L-PGDS in the treatment of OA. First, our histology work demonstrated that cartilage degradation is more pronounced in L- PGDS Knock-out and DP1 mice compared to Wild-Type (WT) wild-type mice. Then an increase in the expression of catabolic mediators (ADAMTS5 and MMP-13), in L-PGDS - / - mice and DP1 - / - compared to WT was demonstrated. Subsequently, stimulation of cartilage explants with IL-α from L-PGDS - / - and DP1 - / - mice showed high degradation in proteoglycans. In addition, these mice also develop subchondral bone changes. Finally, our results suggest that following intraperitoneal injection of the DP1-specific agonist, BW245C attenuated the severity of cartilage degradation induced by medial meniscus destabilization (DMM) and bone changes in mice. WT. Similarly, the intra-atrial injection of AAV2 / 5 encoding L-PGDS also attenuated DMM-induced cartilage degradation and the expression of ADAMTS-5 and MMP-13 in L-PGDS - / - mice. In conclusion, all our results suggest that the recepteur DP1 and the L-PGDS enzyme in osteorthritis cartilage plays a very important role. It may be a potential therapeutic avenue in the treatment of OA and also in the treatment of other musculoskeletal conditions.

Submitted by Anderson Silva (avargas@icict.fiocruz.br) on 2012-11-30T11:59:08Z No. of bitstreams: 1 fabio_p_m_santos_ioc_bcm_0037_2011.pdf: 10305796 bytes, checksum: 74de5b830c8354cd532f74bd40993c6a (MD5) / Made available in DSpace on 2012-11-30T11:59:08Z (GMT). No. of bitstreams: 1 fabio_p_m_santos_ioc_bcm_0037_2011.pdf: 10305796 bytes, checksum: 74de5b830c8354cd532f74bd40993c6a (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. / Durante a resposta alérgica, dentre os vários mediadores inflamatórios de natureza lipídica, a prostaglandina D2 (PGD2) é considerada um mediador-chave. Em adição aos seus conhecidos efeitos quimiotáticos para eosinófilos, recentemente, foi descrito que a PGD2 é também capaz de promover a ativação dos eosinófilos, induzindo a biogênese de corpúsculos lipídicos e a síntese de leucotrieno C4 (LTC4) nessas organelas recém-formadas. Esses efeitos são atribuídos a ação da PGD2 sobre seus 2 receptores – DP1 e DP2 – os quais encontram-se expressos de maneira constitutiva na membrana dos eosinófilos. Então, o objetivo principal do estudo foi identificar o receptor específico da PGD2 envolvido no mecanismo de síntese de LTC4 por eosinófilos estimulados com PGD2. In vivo, num modelo murino de pleurisia alérgica e induzida por PGD2, a utilização dos antagonistas seletivos do receptor DP1 (BW A868c) ou do receptor DP2 (CAY10471) inibiu a síntese de LTC4 nessas respostas inflamatórias. No entanto, somente BWA868C foi capaz de inibir a biogênese de corpúsculos lipídicos nos eosinófilos recrutados para o sítio inflamatório; enquanto que o tratamento com o CAY10471, diminuiu o número de eosinófilos infiltrantes na cavidade pleural, mas não inibiu a biogênese de corpúsculos lipídicos nessas poucas células recrutadas. In vitro, eosinófilos humanos purificados estimulados com PGD2 tiveram a síntese de LTC4 inibida tanto pelo pré-tratamento com BWA868c, quanto pelo prétratamento com CAY10471. Além disso, a ativação do receptor DP1, com seu agonista seletivo (BW245c) e a ativação do receptor DP2 com o agonista seletivo do receptor DP2 (DK-PGD2) corroborou a observação de que no processo de síntese de LTC4 nos eosinófilos, ambos os receptores são necessáior, pois somente quando ambos os receptores foram ativados simultaneamente foi observada síntese de LTC4 nos corpúsculos lipídicos recém-formados (Eicosacell). Além disso, caracterizamos que uma das vias de sinalização intracelular envolvida na formação de corpúsculos lipídicos é depende da ativação de proteína quinase A (PKA). Em um outro grupo de ensaios, investigamos a PGD2 como potencial alvo terapêutico em doenças alérgicas. Recentemente, foi descrito que o extrato aquoso de C. sympodialis e a warafteína (alcalóide isolado) têm propriedades antialérgicas, visto que não somente reduzem a eosinofilia, mas também, a biogênese de corpúsculos lipídicos, assim como a produção de leucotrienos cisteinados. Dessa forma, aqui demonstramos que os pré-tratamentos tanto com o extrato quanto com o alcalóide isolado, foram capazes de inibir a produção de PGD2 ocorrida durante a resposta alérgica. In vitro, embora a warafteína não tenha inibido a biogênese de corpúsculos lipídicos em eosinófilos induzida por PGD2, observamos que é capaz de bloquear a liberação de PGD2 por mastócitos ativados – mas, não a produção de PGE2 por macrófagos ativados com A23187 – demonstrando que o mecanismo de ação dos seus efeitos antiinflamatórios não parecem envolver antagonismo de receptores em eosinófilos, e sim inibição da síntese da PGD2 em sítios alérgicos. / During allergic response, among several lipid mediators produced, prostaglandin D2 (PGD2) has emerged as key mediator. In addition to its known eosinophilotatics effects, recently PGD2 was described to be able to promote eosinophil activation, inducing lipid bodies biogenesis and LTC4 synthesis within these newly formed organelles. These effects are attributed to the action of PGD2 on its 2 receptors – DP1 e DP2 – which are expressed constituvely on eosinophil cell membranes. So, the main objective of this study was to identify the PGD2 specific receptor involved in LTC4 synthesis mechanism by stimulated eosinophils with PGD2. In vivo, in a murine allergic model of pleurisy and in a pleurisy induced by PGD2, the use of selective DP1 receptor (BWA868c) and DP2 receptor (CAY10471) antagonists showed us that both treatments inhibited LTC4 synthesis during these inflammatory responses. However, only BWA868C treatment was able to inhibit lipid bodies biogenesis within recruited eosinophils to the inflammatory sites, while CAY10471, decreased the number of infiltrated eosinophils in the pleural cavity, but did not inhibit lipid bodies biogenesis within these low number of recruited cells. In vitro, pre-treatment with BWA868c or CAY10471 inhibited LTC4 synthesis by human eosinophils stimulated with PGD2. Moreover, the activation of DP1 receptor with its selective agonist (BW245c) and DP2 activation with DP2 selective agonist (DK-PGD2) reinforced the observation that during LTC4 synthesis within eosinophils, activation of both receptors are necessary, because only simultaneous activation of DP1 and DP2, induced LTC4 synthesis within eosinophilic lipid bodies (Eicosacell). Moreover, we observed that the pathway of cellular signaling involved on lipid bodies biogenesis induced by DP1 activation is dependent on protein kinase A (PKA). In another set of experiments, we investigated PGD2 as a therapeutical target of allergic diseases. Recently, it was described that aqueous extract of C.sympodialis and warafteine (isolated alkaloid) have antiallergic properties, because of its effects on the reduction of eosinophils recruitment, lipid bodies biogenesis and cysteinyl leukotrienes synthesis. Here, we demonstrated that pre-treatments with extract and its alkaloid were able to inhibit PGD2 production during allergic response. In vitro, warafteine did not inhibit eosinophil lipid bodies biogenesis induced by PGD2, but it was capable to inhibit PGD2 release by activated mast cells – otherwise fail to blockade PGE2 production by A23187- activated macrophages – suggesting that the action mechanism of its antiinflammatory effects could occur through PGD2 synthesis inhibition in allergic sites.