While cellular levels of arginine greatly exceed the apparent Km for endothelial nitric oxide synthase (eNOS), nitric oxide (NO) production is limited by availability of arginine. Results from this work have provided a unique understanding of endothelial NO production, showing that arginine regeneration, that is the recycling of citrulline back to arginine by argininosuccinate synthase (AS) and argininosuccinate lyase (AL), defines the essential source of arginine for NO production. Using RNA interference analysis, selective reduction of AS expression was shown to directly correspond with a diminished capacity of endothelial cells to produce NO, despite saturating levels of arginine in the medium. In addition, the viability of AS siRNA-treated endothelial cells was compromised due to apoptotic cell death.AS expression was also investigated in response to two major vascular effectors. Tumor necrosis factor (TNF)-alpha; which is known to impair endothelial NO production, was shown to provoke a dose-dependent reduction of AS expression that corresponded to a decrease in NO production. Furthermore, TNF-alpha was shown to suppress AS expression through a NFkappaB mediated pathway, which involves three essential Sp1 elements in the proximal AS gene promoter. On the other hand, peroxisome proliferator-activated receptor gamma (PPARgamma) agonists, troglitazone and ciglitazone, which are known to elicit a vascular protective response against TNF-alpha effects, were shown to coordinately induce NO production and AS expression via a PPARgamma response element in the distal AS gene promoter. Importantly, these PPARgamma agonists were shown to restore AS expression and NO production following down-regulation by TNF-alpha, consistent with their vascular protective properties.

Les prostaglandines sont de petites molécules qui jouent d'importants rôles immunomodulateurs au niveau du système immunitaire. Elles contribuent notamment à l'établissement de l'inflammation et à la résolution de celle-ci. La prostaglandine J 2 (15d-PGJ2 ) est d'ailleurs l'une des prostaglandines les plus étudiées en vertu de ses activités anti-inflammatoires. En effet, il a été démontré que celle-ci peut inhiber plusieurs molécules dont plusieurs cytokines et le facteur de transcription NF-?B. D'un autre côté, l'effet de la 15d-PGJ 2 sur l'interleukine-13 (IL-13) est encore inconnu à ce jour. L'IL-13 est une cytokine produite principalement par les cellules T et qui module plusieurs types de cellules immunitaires, tant au niveau de leur différentiation que de leur activation. De plus, cette cytokine contribue à l'établissement de certaines pathologies telles que l'asthme et la colite ulcéreuse lorsqu'elle est surexprimée. L'objectif principal de ce mémoire est de vérifier l'effet de la 15d-PGJ 2 sur l'expression et la production de l'IL-13 par les cellules T. Les résultats obtenus dans ce projet démontrent que la I 5d-PGJ2 inhibe la production de l'IL-13 par les cellules T. En effet, autant la lignée cellulaire de lymphocytes T CD4+ Jurkat E6.1 que les cellules mononucléaires circulant dans le sang (de l'anglais PBMCs ) stimulés par des agents mimant l'activation des cellules T tels que le PMA et la ionomycine, puis traités à la 15d-PGJ 2 ont montré une inhibition dans l'expression et la production de l'IL-13. Les résultats ont également révélé un effet similaire lorsque les cellules étaient pré-traitées à I 5d-PGJ2 , puis stimulées avec PMA/ionomycine. Par ailleurs, plusieurs éléments sont essentiels pour activer la transcription de l'IL-13, dont le facteur de transcription NF-?B. D'un autre côté, il est connu que la 15d-PGJ2 inhibe la cascade de signalisation de NF-?B. Ainsi, nous avons voulu vérifier si NF-?B était impliqué dans le mécanisme d'inhibition de l'IL-13 par la 15d-PGJ2 . Pour ce faire, des extraits nucléaires ont été réalisés avec les Jurkat E6.1 et les PBMCs traitées à la 15dPGJ2 puis un essai de retard sur gel (de l'anglais EMSA, Electromobility shift assay ) a été fait avec une sonde NF-?B consensus ou une sonde d'un site de liaison putatif de NF-?B dans le promoteur IL-13. Les résultats ont révélé que le facteur de transcription NF-?B était effectivement activé lors de la transcription de l'IL-13 et inhibé lors d'un traitement à la 15d-PGJ 2 . Par la suite, puisqu'il a déjà été démontré que la 15d-PGJ2 active le récepteur nucléaire PPAR-?, nous avons voulu vérifier si le mécanisme d'inhibition de l'IL-13 dépendait ou non de ce dernier. Un antagoniste irréversible de PPAR-? a donc été utilisé dans les expériences, et l'essai a permis d'établir que le mécanisme d'inhibition de l'IL-13 parla 15d-PGJ 2 était indépendant de PPAR-?. Finalement, ces résultats dévoilent que la 15d-PGJ 2 inhibe l'expression et la production de l'IL-13, que cet effet serait indépendant de PPAR-?, et qu'il impliquerait probablement une inhibition du facteur de transcription NF-xB. Ces résultats pourraient avoir des implications cliniques où l'IL-13 joue un rôle d'importance, tels que l'asthme et la colite ulcéreuse. L'utilisation de cette prostaglandine en combinaison avec les traitements habituels, par exemple les corticostéroïdes, pourrait être envisagée.

Con il primo lavoro è stato identificato un tratto fenotipico di un ceppo di L.crispatus associato alla capacità di persistere e colonizzare il colon dell’ospite e di modificarene la composizione microbica, tale L.crispatus M247 è in grado di modificare, nell’epitelio del colon, il livello di espressione dei TLR2 dei TLR4 sia in vitro che in vivo. Con il secondo studio si identifica un meccanismo antinfiammatorio, prima sconosciuto, indotto da un ceppo probiotico che coinvolge l’attivazione di PPAR-γ e fornisce una nuova visuale sui meccanismi molecolari coinvolti nel dialogo tra epitelio intestinale e microbiota simbionte. / The colonic microbiota is a major modulator of the mucosal immune system; therefore, its manipulation through supplementation with probiotics may significantly affect the host’s immune responses. Since different probiotics seem to exert various effects in vivo, we tested the relevance of the autoaggregation phenotype on the intestinal persistence of lactobacilli and their ability to modulate the host’s innate immune responses. After 14 days of diet supplementation, the aggregating strain Lactobacillus crispatus M247 but not aggregation-deficient isogenic mutant MU5 was recovered from the feces and colonic mucosa of mice. This observation was confirmed by strain-specific PCR amplification and by Lactobacillus-specific denaturing gradient gel electrophoresis analysis. Indeed, L. crispatus M247 increased Toll-like receptor 2 (TLR2) mRNA levels, while it reduced TLR4 mRNA and protein levels in the colonic mucosa, whereas MU5 was ineffective. In colonic epithelial cells (CMT-93 cells) L. crispatus M247 but not MU5 induced time-dependent extracellular signal-regulated kinase-1 (ERK1) tyrosine phosphorylation and TLR modulation, which were abolished in the presence of PD98059 (an ERK1 inhibitor). To assess the functional relevance of probiotic-induced TLR modulation, we determined the consequences of L. crispatus preexposure on TLR4 (lipopolysaccharide [LPS]) and TLR2 [Pam3Cys-Ser-(Lys)4] ligand-mediated effects in intestinal epithelial cells. Preexposure to L. crispatus M247 blunted LPS-induced interleukin-6 (IL-6) release and inhibition of CMT-93 migration over a wound edge, whereas it enhanced TLR2-mediated IL-10 up-regulation. In summary, the aggregation phenotype is required for L. crispatus persistence in the colon and for modulation of TLR2/TLR4 expression through an ERK-dependent pathway. We speculate that the aggregation phenotype in L. crispatus M247 is required to temper epithelial cell responsiveness to bacterial endotoxins, which thus affects the evolution of intestinal inflammatory processes. Accumulating evidence indicates that the peroxisome proliferator activated receptor (PPAR)- is a major player in maintaining intestinal mucosa homeostasis, but whether PPAR- is directly involved in probiotic-mediated effects and the molecular events involved in its activation are not known. Methods: We investigated the role of PPAR- in the immunomodulatory effects of Lactobacillus crispatus M247 on intestinal epithelial cells (IEC) and the role of probiotic-derived H2O2 on PPAR- activity. Results: L crispatus M247 supplementation in mice significantly increased PPAR- levels and transcriptional activity in the colonic mucosa. L crispatus M247 induced PPAR- nuclear translocation and enhanced transcriptional activity in epithelial (CMT-93) cells, as demonstrated by the increased luciferase activity of a PPAR- –responsive element, PPAR- – responsive gene up-regulation, and reduced activity of an nuclear factor- B–responsive element. Pharmacologic PPAR- inhibition or silencing by small interfering RNA cancelled the L crispatus M247–mediated effects in CMT-93 cells. Because Lactobacillus strains producing little H2O2 failed to activate PPAR- , we investigated the role of L crispatus M247– derived H2O2 in PPAR- activation. L crispatus M247 induced a transient rise in intracellular H2O2 and PPAR- transcriptional activity was cancelled by antioxidant or H2O2 scavenger. Toll-like receptor (TLR)-2 was not required for PPAR- up-regulation mediated by L crispatus M247 in mice, although the protective effects of L crispatus M247 on dextran sodium sulfate-induced colitis were less pronounced in TLR-2 / mice. Conclusions: L crispatus M247 uses H2O2 as a signal transducing molecule to induce PPAR- activation in IEC, directly modulating epithelial cell responsiveness to inflammatory stimuli.

Obesity constitutes a problem whose manifestations have consequences in almost every field of the medicine and nowadays there is a lack of pharmacological therapy alternatives for its long term treatment. Lipidic compounds as endocannabinoids and PPAR-α ligands are known to play an important role in the modulation of appetite and metabolism. Three series of fatty acid derivatives of catechol compounds were synthesized and their biological activity evaluated. Some of the synthesized compounds presented LDL antioxidant activity and/or food intake modulation in an animal model and their mechanism of action was also evaluated. The pharmacodynamics of the synthesized compounds could be explained by CB1 and PPAR-α interactions nevertheless it does not explain the activity of all compounds. / La obesidad es un problema cuyas manifestaciones tienen consecuencias en casi todos los campos de la medicina y actualmente existe una escasez de terapias farmacológicas para su tratamiento de uso continuo. Se sabe que algunos compuestos lipídicos como los endocanabinoides y ligandos del PPAR-α participan de manera importante en la modulación del apetito y en el metabolismo. Tres series de compuestos derivados de ácidos grasos con compuestos catecólicos fueron sintetizadas y sus actividades biológicas fueron evaluadas. Algunos de los compuestos presentó inhibición de la oxidación de la LDL y/o modulación de la ingesta en modelo animal y sus mecanismos de acción fueron también evaluados. La actividad de los compuestos pasa por interacciones con el receptor CB1 y el PPAR-α pero estas interacciones no explican la actividad de todos los compuestos

Abstract Peroxisome proliferator-activated receptors (PPARs) are transcription factors that belong to the family of nuclear hormone receptors and exist as three isoforms namely PPARα, PPARβ and PPARγ. PPARs function as key regulators of glucose and lipid metabolism and are potential targets for drugs used in the treatment of glucose and lipid metabolism dysregulation. PPARs also regulate the expression of genes involved in the process of cellular proliferation and differentiation. Since it was discovered that PPAR ligands cause liver tumourigenesis in rodents, PPARs and their modulators have been investigated widely in in vitro and in vivo studies of carcinogenesis of the liver, colon, prostate, lung and skin. PPARα and PPARγ are the most studied PPAR isoforms in relation to cancer, while the association of PPARβ with cancer is increasingly being investigated. Some studies suggest that PPARβ and its ligands may have anticancer activity, while other studies identify a role for PPARβ in tumour promotion and progression. Breast cancer is one of the most common types of cancer in women with the majority caused by non-hereditary mechanisms. The activation of PPARα in breast cancer cells is associated with an increase in proliferation, while PPARγ activation in breast cancer cells is related to differentiation and an inhibition of cell proliferation. The role of PPARβ and its modulators in breast cancer is uncertain, as there have been limited studies addressing the effects of PPARβ modulation in breast cancer cell lines. Environmental contaminants such as the phthalate plasticizers and alcohol are putative risk factors for breast cancer. The phthalates di(2-ethylhexyl)phthalate (DEHP) and di-n-butyl phthalate (DBP) are plasticizers that are used in a range of common household, medical and beauty products and as a consequence humans are exposed to significant levels of these compounds. DEHP and DBP are known teratogens in rodents and DEHP induces hepatocarcinogenesis in a process thought to be mediated via PPARα. DEHP and DBP are metabolized in vivo by esterases to the monoesters, mono-(2-ethylhexyl) phthalate (MEHP) and mono-n-butyl phthalate (MBP), and these compounds have been identified in human biological samples. MEHP and MBP modulate PPARs in various tissues and cell types, but their ability to modulate PPARs in human breast cancer cells is not known. Like phthalates, ethanol is another modulator of PPARs and alcohol consumption is associated positively with breast cancer development, but the molecular mechanisms involved are unknown and there are no studies that examine the effects of ethanol and its metabolite acetaldehyde on PPARs in breast cancer cell lines. This thesis describes studies establishing and validating a breast cancer cell line that conditionally expresses human PPARβ under the control of a tetracycline regulator. Using this model, the ability of PPARβ over-expression and/or activation by the PPARβ specific ligand GW0742 to promote breast cancer cell proliferation was studied. Furthermore, putative PPARβ regulated genes were examined for alterations in expression in the presence of the PPARβ ligand. This work determined that over-expression of PPARβ and/or its activation by GW0742 does not promote proliferation in MCF-7 breast cancer cells. This thesis also investigated the effects of the phthalate monoesters MEHP and MBP on PPARs in MCF-7 breast cancer cells. It was found that MEHP activated both PPARα and PPARγ but was unable to activate PPARβ, whereas MBP could not activate any of the PPAR isoforms. MBP was an antagonist for both PPARγ and PPARβ. Using breast cancer cell lines, studies were conducted addressing the effects of an increasing concentration of ethanol (0-300 mM) on the transcription and transactivation of PPARα and PPARβ isoforms. Estrogen receptor positive MCF-7 breast cancer cells were more sensitive to the effects of ethanol than estrogen receptor negative MDA-MB-231 cells, with changes in PPARα mRNA more pronounced than PPARβ mRNA. Studies in MCF-7 cells conditionally expressing either PPARα or PPARβ in the presence of their respective specific ligands, GW7647 and GW0742, revealed that ethanol concentrations of 20 mM and 100 mM suppressed the maximal response to ligand-mediated activation for PPARα. Studies using the ethanol metabolism enzyme inhibitors 4-methylpyrazole and cyanamide, suggested that while ethanol was responsible for the modulation of PPARβ transactivation, the primary metabolite acetaldehyde was responsible for the effects on PPARα transactivation. Lastly, it was determined that ethanol and/or GW0742 did not increase the proliferation of MCF-7 Tet-off cells. The findings in this thesis suggest that given the different consequences of MEHP, MBP and ethanol on PPARs, PPAR expression and activation by ligands may have tissue specific consequences and that PPARβ may have a complex role in mammary gland tumourigenesis.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2009. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 54-62).

Submitted by Anderson Silva (avargas@icict.fiocruz.br) on 2012-10-26T17:48:32Z No. of bitstreams: 1 clarissa_c_menezes_ioc_bcm_0054_2011.pdf: 2530597 bytes, checksum: 62f04a75c90de18a85463693ba14fb9b (MD5) / Made available in DSpace on 2012-10-26T17:48:32Z (GMT). No. of bitstreams: 1 clarissa_c_menezes_ioc_bcm_0054_2011.pdf: 2530597 bytes, checksum: 62f04a75c90de18a85463693ba14fb9b (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. / A bactéria Pseudomonas aeruginosa (PA) é um dos principais agentes etiológicos de pneumonias nosocomiais, cujo tratamento é dificultado por sua resistência a antibióticos e pela secreção de fatores de virulência. Novas alternativas para o tratamento destas infecções incluem manipular o sistema de comunicação bacteriano conhecido como quorum sensing (QS), reduzindo a expressão destes fatores de virulência e prevenindo seus efeitos deletérios em células e sistemas eucariotos. Os efeitos do componente de QS de PA 3-oxo dodecanoil homoserina lactona (3-oxo C12 HSL) nos pulmões não são conhecidos; portanto, a primeira etapa de nosso trabalho consistiu na caracterização dos efeitos desta molécula no ambiente pulmonar. Para isso, camundongos swiss desafiados com 3-oxo C12 HSL por via intratraqueal tiveram amostras de sangue, lavado bronco-alveolar (BAL) e tecido pulmonar e coletadas seis horas após o procedimento. Nossos resultados demonstram que inflamação pulmonar causada por 3-oxo C12 HSL se caracteriza pela migração de células mononucleares e neutrófilos para o espaço alveolar, níveis elevados de atividade mieloperoxidase no tecido pulmonar e formação de edema. A resposta inflamatória causada por 3-oxo C12 HSL não alterou os níveis de TNF-α, MIF, IL- 10 e IL-12 no tempo analisado. Foram observados aumentos nos níveis de IL-6, CCL2/MCP-1, CXCL1/KC e LTB4 no BAL de animais desafiados, sendo o aumento deste eicosanóide acompanhado por uma indução de corpúsculos lipídicos. Evidências in vitro relatam o envolvimento do receptor nuclear PPARγ nos efeitos pró-inflamatórios atribuídos a 3-oxo C12 HSL; portanto, decidimos estudar os efeitos do tratamento com o agonista de PPARγ rosiglitazona no modelo de inflamação pulmonar causado por este componente de QS. Nós observamos que o tratamento com rosiglitazona (0,5 mg/kg) uma hora após o estímulo diminuiu a formação de corpúsculos lipídicos e de edema pulmonar, causando também uma redução na migração de células mononucleares e neutrófilos e uma menor atividade mieloperoxidase no pulmão dos animais tratados. A redução da migração de células mononucleares parece estar associada à uma redução dos níveis de CCL2/MCP-1, enquanto o decréscimo nos neutrófilos parece envolver a modulação de CXCL1/KC. A instilação com 3-oxo C12 HSL diminuiu a expressão da enzima paraoxonase (PON) no tecido pulmonar, fenômeno que não foi revertido pelo tratamento com rosiglitazona nesta dose. Estudos demonstram que o PPARγ está envolvido na expressão de PON e que a superexpressão desta enzima é capaz de proteger animais da mortalidade por PA em função de sua atividade lactonase; por este motivo, decidimos testar uma dose maior de rosiglitazona (5 mg/kg) para alcançar mais um benefício nesta proposta terapêutica. O tratamento com rosiglitazona nesta dose foi capaz de reverter a redução da expressão de paraoxonase causada por 3-oxo C12 HSL, diminuindo a formação de edema, a migração de neutrófilos e os níveis de atividade mieloperoxidase no pulmão de animais desafiados. O número de células mononucleares recuperado no BAL de animais estimulados e tratados com a droga não foi reduzido, bem como os níveis de CCL2/MCP-1. De fato, a droga por si só causou um aumento de CCL2/MCP-1 que parece ter contribuído para a indução de corpúsculos lipídicos observada. Devido ao envolvimento de macrófagos e da quimiocina CCL2/MCP-1 no processo de eliminação bacteriana, estudos em modelos de pneumonia por PA precisam ser conduzidos para melhor avaliar esta dose de rosiglitazona e validar esta droga como uma estratégia terapêutica no combate à esta bactéria. / Pseudomonas aeruginosa (PA) is a major pathogen involved in nosocomial pneumonia. These infections are difficult to manage due bacterial antibiotic resistance and secretion of virulence factors; therefore, current approaches to treat PA infections now focus on manipulating the bacteria communication system known as quorum sensing in order to reduce the production of those virulence factors and mitigate their deleterious effects to the host. Since there are no reports about the effects of the PA main quorum sensing component 3-oxo dodecanoyl homoserine lactone (3-oxo C12 HSL) in the lung the first step of our work consisted in investigating the host pulmonary response to this molecule in a mouse model. In order to do so, swiss mice submitted to intratracheal instillation with 3- oxo C12 HSL had blood, BAL and lung tissue samples collected six hours after the procedure. Our results show that the inflammatory response elicited by 3-oxo C12 HSL is characterized by the migration of both mononuclear cells and neutrophils to the alveolar space, together with high levels of myeloperoxidase activity in lung tissue and development of pulmonary edema. We found that the levels of TNF-α, MIF, IL-10 and IL-12 were not altered, while there was a significant increase in IL-6 CCL2/MCP-1 and CXCL1/KC in BAL. The increase in LTB4 levels in the BAL of defied animal was accompanied by an induction of lipid bodies. In vitro evidences report the involvement of the nuclear receptor PPARγ in the inflammatory events ascribed to 3-oxo C12 HSL; therefore, we decided to study the effects of PPARγ agonist rosiglitazone in the pulmonary inflammation caused by this quorum sensing component. We observed that rosiglitazone administration (0,5 mg/kg) one hour after stimulation was able to reduce the formation of lipid bodies and pulmonary edema, besides decreasing the migration of mononuclear cells, neutrophils and myeloperoxidase activity in the lung of defied animals. This reduction in the migration of mononuclear cells seems to be due to a decrease in the levels of CCL2/MCP-1, while the impairment in neutrophil migration seems to involve the modulation of CXCL1/KC. 3-oxo C12 HSL instillation also decreased paraoxonase (PON) expression in lung tissue, which was not reverted by rosiglitazone treatment at this dose. Since it was reported that PPARγ is able to regulate PON expression and that the overexpression of this enzyme seems to protect animals from PA mortality due its lactonase activity we searched to modulate this enzyme with a higher dose of rosiglitazone (5 mg/kg) in order to achieve this extra benefit in our proposal. We observed that this dose was able to revert the decrease in PON expression caused by 3-oxo C12 HSL, reducing the pulmonary edema, neutrophil migration and myeloperoxidase activity in defied animals. There was no reduction in the number of mononuclear cells recovered from the BAL of stimulated animals, which was also observed for CCL2/MCP-1 levels. In fact, the drug itself at his dose caused an increase in CCL2/MCP-1 that possibly accounted for the induction of lipid bodies. Because macrophages and CCL2/MCP-1 have a crucial role in the clearance of bacteria, this dose of rosiglitazone has to be tested in a PA pneumonia model so the effects observed in our investigation can be validate rosiglitazone as an therapeutic approach to the treatment of these infections.

Submitted by Priscila Nascimento (pnascimento@icict.fiocruz.br) on 2013-04-03T17:21:22Z No. of bitstreams: 1 Claudia_Valeria_de_Araujo.pdf: 3901306 bytes, checksum: 6449c70c8037ebc0049d5989078927c0 (MD5) / Made available in DSpace on 2013-04-03T17:21:22Z (GMT). No. of bitstreams: 1 Claudia_Valeria_de_Araujo.pdf: 3901306 bytes, checksum: 6449c70c8037ebc0049d5989078927c0 (MD5) Previous issue date: 2012 / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. / A regulação deficiente da resposta inflamatória do indivíduo aos produtos microbianos é crucial para a mortalidade de paciente com SIRS (síndrome da resposta inflamatória sistêmica) e sepse. Vários mediadores inflamatórios são liberados e a regulação da expressão destes mediadores é crítica para a defesa do hospedeiro, mas também pode resultar em dano tecidual, disfunção orgânica múltipla e morte. Estratégias anti-inflamatórias são investigadas no tratamento da sepse. Estudos têm focado em fatores transcricionais que possuem um interesse terapêutico, como receptor nuclear PPAR (receptor ativado por proliferador de peroxissomo do tipo gama). Nosso objetivo principal foi caracterizar o papel do PPARγ na sepse experimental. Foram feitas análises de sobrevida e parâmetros inflamatórios, como eliminação bacteriana, produção de mediadores inflamatórios e migração celular, além da avaliação da microcirculação cerebral 24 horas após a ligadura e punção cecal (CLP) em animais tratados com agonista de PPARγ (Rosiglitazona) 15 min após a indução de sepse. Observamos um aumento da sobrevida de camundongos submetidos à CLP e tratados com Rosiglitazona. Em animais submetidos à sepse grave, a Rosiglitazona foi igualmente eficaz em aumentar a sobrevida dos animais e induzir uma melhora no quadro clínico. Nos animais tratados com Rosiglitazona houve um aumento nos níveis plsmáticos de mediadores anti-inflamatórios como a IL-10 e CCL2 e decréscimo de mediadores pró-inflamatórios como TNF-α, IL-6 e de corpúsculos lipídicos, sem alteração da glicemia. Houve uma diminuição plasmática da quimiocina CXCL1 nos animais tratados com Rosiglitazona quando comparados ao grupo controle. Além disso, observamos um aumento na migração de neutrófilos peritoneaisl de animais submetidos à CLP e o pós-tratamento com Rosiglitazona foi capaz de reverter este efeito. Observamos que a Rosiglitazona diminuiu o número de unidades formadoras de colônias (UFC) do lavado peritoneal, que se correlacionou com o aumento no metabolismo oxidativo de células fagocíticas com a diminuição da taxa de mortalidade. Em um estudo in vitro com E.coli incubadas com ligantes de PPARγ observamos que não houve alteração do crescimento bacteriano, mostrando que a Rosiglitazona não parece ter um efeito direto sobre o patógeno. Em outro experimento in vitro com neutrófilos humanos incubados com LPS ou E. coli,a Rosiglitazona aumentou a eliminação bacteriana por estas células e levou a um aumento na formação de redes extracelulares de neutrófilos (NETs) por estas células. O antagonista do PPARγ, o GW9662 foi capaz de reverter este efeito. A Rosiglitazona também foi capaz de aumentar a liberação da proteína histona em polimorfonucleares (PMNs), um importante marcador na formação de NETs e o GW9662 reverteu este efeito. Nos experimentos de microcirculação cerebral, a Rosiglitazona diminuiu o rolamento, a aderência dos leucócitos no endotélio vascular, assim como a rarefação capilar, aumentando a perfusão tecidual cerebral. Estes efeitos foram independentes de alterações na pressão arterial média e frequência cardíaca. Nossos estudos indicam que a Rosiglitazona atuou em diversos parâmetros da fisiopatologia da sepse, modulando a resposta inflamatória, aumentando a eliminação bacteriana, melhorando o quadro clínico e diminuindo a mortalidade em camundongos sépticos. É de extrema importância o entendimento dos mecanismos moleculares envolvidos na sepse, para que possa servir de potencial manobra ou intervenção terapêutica no futuro. / The defective regulation of the inflammatory response to microbial products is crucial in mortality rate of patients with SIRS (systemic inflammatory response syndrome) and sepsis. Several inflammatory mediators are released and the regulation in expression of these mediators is critical for host defense, but can also result in tissue damage, multiple organ failure, and death. Antiinflammatory strategies are investigated for sepsis treatment. Studies have been focusing on transcription factors with a therapeutic interest, such as the nuclear receptor PPAR γ (Peroxisome proliferator-activated receptors γ). Our main objective was to characterize the role of PPAR γ in experimental sepsis. Survival analyzes were performed, as well as the inflammatory parameters, such as bacterial clearance, inflammatory mediators productions and cellular migration. We assessed the brain microcirculation 24 hours after CLP in animals treated with Rosiglitazone. Our results have shown an increase in survival rate with Rosiglitazone treatment. In a model of severe sepsis Rosiglitazone was equally effective in increasing the survival rate accompanied by an improvement in clinical status. The Rosiglitazone treatment increased the inflammatory mediators levels, such as IL-10 and CCL2 with a decrease of pro-inflammatory mediators such as TNF-α, IL-6, as well as a decrease in lipid bodies formation. A reduction in chemokine CXCL1 was also observed in animals treated with Rosiglitazone compared to control groups. An increase of neutrophils migration was seen in the peritoneal cavity 24 hours after CLP and the Rosiglitazone post-treatment was effective into reversing this parameter. A reduced number of colony forming units (CFU) of peritoneal fluid was also observed in rosiglitazone treatment which was directly correlated with an increase in oxidative stress and survival rate. However, in our experiments we did not observed any alteration of animals blood glucose levels. In an in vitro study with E. coli incubated only with PPARγ ligands, no changes on bacterial growth was seen, demonstrating that Rosiglitazone by itself does not have an effect on the pathogen. In another experiment with human neutrophils incubated with LPS or E. coli in the presence of Rosiglitazone, we observed an increase in the extracellular bacterial clearance mediated by netosis. The PPAR γ antagonist, GW9662, was able to reverse this effect. Rosiglitazone also enhanced the release of histone protein in PMNs, an important marker of NETs formation, and this effect was abolished by GW9662. During the assessement of cerebral microcirculation, Rosiglitazone decreased leukocyte rolling and adhesion to the vascular endothelium, as well as the capillary rarefaction, resulting with an improvement of brain perfusion. It was supposed that these effects were independent of haemodynamic changes. Finally, our studies suggested that Rosiglitazone acts on several parameters in the pathophysiology of sepsis by modulating the inflammatory response, increasing the bacterial clearance, improving clinical score, and reducing mortality rate in septic mice. In addition, it is extremely important in the understanding of molecular mechanisms involved in sepsis syndrome, thus it might serve as a potential therapeutic intervention or maneuver in the future.

Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / Este trabalho teve como objetivo investigar os efeitos da telmisartana (agonista PPAR-gama parcial), losartana (puro bloqueador do receptor AT1 da angiotensina II) e rosiglitazona (agonista PPAR-gama) em modelo experimental de síndrome metabólica. Os alvos do estudo foram a pressão arterial, metabolismo de carboidratos, resistência insulínica, inflamação, tecido adiposo e fígado. Camundongos C57BL/6 (a partir de 3 meses de idade) foram alimentados com dieta padrão (SC, n = 10) ou dieta hiperlipídica rica em sal (HFHS, n = 40) por 12 semanas. Após esse tempo, os animais do grupo HFHS foram subdivididos em 4 grupos (n = 10): HFHS (sem tratamento), ROSI (HFHS tratado com rosiglitazona), TELM (HFHS tratado com telmisartana) e LOS (HFHS tratado com losartana) por 5 semanas. O grupo HFHS apresentou um significante ganho de peso e aumento da pressão arterial sistólica, hiperinsulinemia com resistência insulínica, hiperleptinemia, hipertrofia de adipócitos bem como um quadro de esteatose hepática e níveis aumentados da citocina inflamatória interleucina-6 (IL-6). Os animais tratados com telmisartana chegou ao final do experimento com massa corporal similar ao grupo SC, com reversão do quadro de resistência insulínica, com pressão arterial normal, adipócitos de tamanho normal e sem apresentar esteatose hepática. Além disso, o tratamento com telmisartana aumentou a expressão de PPARγ e adiponectina no tecido adiposo epididimal. A expressão da proteína desacopladora-1 (UCP-1) no tecido adiposo branco (TAB) também foi aumentada. O tratamento com losartana diminuiu a pressão arterial para valores normais, porém com menores efeitos nos parâmetros metabólicos dos animais. O presente modelo experimental de ganho de peso e hipertensão induzidos por dieta mimetiza a síndrome metabólica humana. Neste modelo, a telmisartana aumentou a expressão de UCP-1 no TAB, preveniu o ganho de peso e melhorou a sensibilidade à insulina e a esteatose hepática dos camundongos C57BL/6, provavelmente devido à ativação PPAR-gama. / The study aimed to investigate the effects of telmisartan (a partial PPAR gamma agonist), losartan (a pure angiotensin II receptor blocker) and rosiglitazone (PPAR gamma agonist) in a mice model of metabolic syndrome (MetS). The targets of this study were blood pressure (BP), carbohydrate metabolism, insulin resistance, inflammation, white adipose tissue (WAT) and liver. Male C57BL/6 mice were studied over 17 weeks after being separated into two major groups according to diet: standard chow (SC, 10% fat, n = 10) or high-fat high-salt chow (HFHS, 60% fat, 7% salt, n = 40). In the last 5 weeks of the experiment, the HFHS group was divided into four groups (n = 10): untreated HFHS, ROSI (HFHS plus rosiglitazone), TELM (HFHS plus telmisartan), and LOS (HFHS plus losartan). The HFHS group had significantly greater body mass and BP, in addition to hyperinsulinemia with insulin resistance, hyperleptinemia, adipocyte hypertrophy and hepatic steatosis as well as increased inflammatory cytokine levels. Animals treated with telmisartan had body weights similar to the SC group, in addition to reversed insulin resistance, reduced hypertension, reduced adipocyte hypertrophy, ameliorates hepatic steatosis and decreased IL-6. Telmisartan increased PPARγ and adiponectin expression in white adipose tissue. Interestingly, the expression of UCP-1 in white adipose tissue was also increased by treatment with telmisartan. Losartan decreased BP but had smaller effects on metabolic parameters. The present model of diet-induced weight gain and hypertension in mice mimics human features of MetS. In this model, telmisartan enhances UCP-1 expression in WAT, prevented weight gain and ameliorates insulin sensitivity and hepatic steatosis in C57Bl/6 mice, probably due to PPAR gamma activation.