No diabete melito, produtos de glicação avançada (AGE) reduzem o efluxo de colesterol celular o que agrava o desenvolvimento da aterosclerose. Neste estudo, investigou-se o papel da albumina modificada por glicação avançada (albumina-AGE) sobre a sensibilização de macrófagos à resposta inflamatória e o impacto da secreção de citocinas, quimocinas e moléculas de adesão sobre o efluxo de colesterol mediado por apolipoproteína A-I e subfrações de HDL. Além disso, determinou-se a capacidade da HDL em modular a resposta inflamatória em macrófagos tratados com albumina-AGE. Macrófagos de peritônio de camundongo foram tratados com ou sem sobrecarga de colesterol, na presença de 2 mg/mL de albumina-controle (albumina-C) ou albumina-AGE, por 72 h, seguindo-se de incubação, por 24 h, com calgranulina S100B (20 g/mL) ou lipopolissacarídeo (LPS; 1 g/mL). Em comparação com albumina-C, a albumina-AGE, isenta em endotoxinas, isoladamente não alterou a secreção de citocinas em macrófagos. No entanto, a albumina-AGE sensibilizou macrófagos não enriquecidos em colesterol a uma maior secreção de interleucina 6 (IL-6), fator de necrose tumoral alfa (TNF-), proteína quimoatraente de monócitos 1 (MCP-1), interlucina 1 beta (IL-1) e molécula de adesão celular vascular 1 (VCAM-1) após estimulação com S100B ou LPS, o que foi potencializado pela sobrecarga de colesterol celular. Em macrófagos não estimulados, o meio condicionado, advindo das incubações de células com albumina-AGE e S100B (meio enriquecido em citocinas), reduziu o efluxo de 14C-colesterol mediado por apoA-I, HDL2 e HDL3 em, respectivamente, 23%, 43% e 20%, em comparação com células incubadas com meio isolado do tratamento com albumina-C e S100B. De forma similar, o efluxo de 14C-colesterol mediado por apoA-I, HDL2 e HDL3 foi reduzido em macrófagos tratados com meio advindo de incubações com albumina-AGE e LPS, respectivamente, 37%, 47% e 8,5% em comparação ao tratamento com albumina-C e LPS. Em macrófagos tratados com albumina-C e S100B, a incubação prévia com HDL reduziu a secreção de IL-6, TNF-, MCP-1 e VCAM-1 em, respectivamente, 72%, 57%, 50% e 41% quando comparada à incubação na ausência de HDL. Em incubações com albumina-C, a secreção de IL-6, TNF-, MCP-1, IL-1 e VCAM-1 induzida por LPS foi respectivamente, 58%, 54%, 42%, 74% e 45% menor mediante incubação com HDL, em comparação a incubações similares, porém na ausência desta lipoproteína. Por outro lado, em macrófagos tratados com albumina-AGE e S100B, a HDL não foi capaz de reduzir a secreção de TNF-, IL-1 e VCAM-1 e aumentou a secreção de IL-6 (54%) e MCP-1 (20%). Nas células tratadas com albumina-AGE e LPS, a HDL também não reduziu a secreção de TNF-, MCP-1 e IL-1 e aumentou a secreção de IL-6 (16%) e VCAM-1 (20%). Redução na secreção de mediadores inflamatórios foi observada em macrófagos tratados com albumina-AGE apenas quando a HDL foi incubada juntamente com S100B ou LPS. Em conclusão, a albumina-AGE sensibiliza macrófagos à resposta inflamatória induzida por calgranulina S100B e LPS, prejudicando o transporte reverso de colesterol de macrófagos. Além disso, a albumina-AGE reduz as propriedades anti-inflamatórias da HDL, o que pode agravar a aterosclerose no diabete melito / In diabetes mellitus, advanced glycation end products (AGE) reduces the cholesterol efflux from cells, which aggravates the development of atherosclerosis. In this study, we investigated the role of advanced glycated albumin (AGE-albumin) on macrophage inflammatory response and the impact of cytokines, chemokines and adhesion molecules secretion on cholesterol efflux mediated by apolipoprotein A-I (apoA-I) and HDL subfractions. Furthermore, the HDL ability in modulating inflammatory response in macrophages treated with AGE-albumin was also determined. Mouse peritoneal macrophages previously enriched or not with cholesterol were treated in the presence of 2 mg/mL of control-albumin (C-albumin) or AGE-albumin, for 72 h, followed by incubation, for 24 h, with S100B calgranulin (20 g/mL) or lipopolysaccharide (LPS; 1 g/mL). In comparison to free endotoxin-C-albumin, AGE-albumin, by itself did not alter cytokine secretion by macrophages. However, AGE-albumin primed non-cholesterol enriched macrophages to a higher secretion of interleukin -6 (IL-6), tumor necrosis factor alpha (TNF-), monocyte chemotactic protein 1 (MCP-1), interleukin 1 beta (IL-1) and vascular cell adhesion molecule 1 (VCAM-1) after stimulation with S100B or LPS, which was potentiated by cell cholesterol overload. In non-stimulated macrophages, conditioned medium, derived from incubation with AGE-albumin and S100B (cytokine enriched-medium), reduced the 14C-cholesterol efflux mediated by apoA-I, HDL2 and HDL3 in, respectively, 23%, 43% and 20%, in comparison to cells incubated with conditioned medium isolated from treatment with C-albumin and S100B. Similarly, 14C-cholesterol efflux mediated by apoA-I, HDL2 and HDL3 was reduced in macrophages treated with medium derived from incubation with AGE-albumin and LPS, respectively, 37%, 47% and 8,5% in comparison to treatment with C-albumin and LPS. In macrophages treated with C-albumin and S100B, previous incubation with HDL reduced the secretion of IL-6, TNF-, MCP-1 and VCAM-1 in, respectively 72%, 57%, 50% and 41% in comparison to incubation in the absence of HDL. In incubations with C-albumin, the secretion of IL-6, TNF-, MCP-1, IL-1 and VCAM-1 induced by LPS was respectively, 58%, 54%, 42%, 74% and 45% lower in cells treated with HDL in comparison to similar incubations in the absence of this lipoprotein. On the other hand, in macrophages treated with AGE-albumin and S100B, HDL was unable to reduce the TNF-, IL-1 and VCAM-1 secretion and increased the secretion of IL-6 (54%) and MCP-1 (20%). In cells treated with AGE-albumin and LPS, HDL was unable to reduce the secretion of TNF-, MCP-1 and IL-1 and increased IL-6 (16%) and VCAM-1 (20%). Reduction in inflammatory mediators was observed in macrophages treated with AGE-albumin only when HDL was incubated simultaneously with S100B or LPS. In conclusion, AGE-albumin primes macrophages to an inflammatory response elicited by S100B calgranulin and LPS, impairing macrophage reverse cholesterol transport. Moreover, AGE-albumin impairs the HDL anti-inflammatory properties, which can aggravate the atherosclerosis in diabetes mellitus

Les maladies cardiovasculaires sont la principale cause de morbidité et de mortalité dans les pays industrialisés. Le récepteur CD36, exprimé à la surface des macrophages, joue un rôle déterminant dans l’internalisation des lipoprotéines oxydées menant à la formation des cellules spumeuses dans l’espace sous endothélial, première étape du développement des lésions athérosclérotiques. Nous avons montré précédemment que les sécrétines de l’hormone de croissance sont des ligands du récepteur CD36 qui possèdent un site de liaison qui chevauche celui des lipoprotéines oxydées. Cependant, aucune étude n’avait rapporté les effets potentiels des ligands sélectifs du CD36 sur la progression des lésions athérosclérotiques et le métabolisme lipidique au niveau des macrophages. Ainsi, ce projet de doctorat visait à évaluer le potentiel anti-athérosclérotique du EP 80317, un ligand sélectif du CD36, et élucider les mécanismes à l’origine de ses effets sur le métabolisme et le transport des lipides au niveau des macrophages. À cette fin, des souris déficientes en apolipoprotéine E (apoE-/-), nourries avec une diète riche en lipides et en cholestérol, ont été traitées quotidiennement pendant 12 semaines avec le EP 80317, montrant un puissant effet anti-athérosclérotique associé à une réduction de 51% des lésions aortiques et de 30% du taux plasmatique de cholestérol total. Cette même étude a permis de montrer une réduction de l’internalisation des lipoprotéines oxydées ainsi qu’une augmentation de l’expression des gènes/protéines impliqués dans l’efflux du cholestérol au niveau des macrophages, comme le peroxisome proliferator-activated receptor γ (PPARγ), liver x receptor α (LXRα) et les transporteurs ABCA1 et ABCG1, entraînant une réduction de la formation des cellules spumeuses. Ces observations nous ont conduits à élucider les mécanismes moléculaires engendrés par la liaison d’un ligand sélectif au récepteur CD36 dans les macrophages. Les études ont permis de montrer que les ligands du CD36 entraînent une augmentation de l’efflux du cholestérol vers les transporteurs ABCA1 et ABCG1 en augmentant l’expression protéique de la cyclooxygénase 2 (COX-2) consécutive à la phosphorylation de la MAP kinase ERK1/2. L’activation de COX-2 stimule la production intracellulaire de la prostaglandine 15d-PGJ2, cette dernière conduisant à l’activation du PPARγ. Finalement, une troisième étude nous a permis de mettre en évidence les effets du EP 80317 sur le transport inverse du cholestérol in vivo. L’injection de macrophages J774 radiomarqués avec du cholestérol tritié dans la cavité péritonéale de souris avec le EP 80317 nous a permis de montrer que le EP 80317 entraîne une réduction de la radioactivité retrouvée dans le foie tandis qu’il augmente celle retrouvée dans les fèces par comparaison aux souris contrôles, sans néanmoins modifier le profil plasmatique du radiotraceur entre les deux groupes. De plus, l’expression des gènes impliqués dans le transport du cholestérol au niveau intestinal comme le LXRα, ABCA1, ABCG5 ainsi que ABCG8 ont été régulés à la hausse par le EP 80317 tandis que l’expression de NPC1L1, un transporteur impliqué dans l’absorption du cholestérol, a été régulé à la baisse. Toutefois, les gènes impliqués dans le métabolisme du cholestérol au niveau du foie ne sont pas modulés par le EP 80317. En conclusion, les travaux effectués dans le cadre de cette thèse nous ont permis de montrer que l’activation du récepteur CD36 par le EP 80317 pourrait s’avérer être une nouvelle approche thérapeutique pour le traitement de l’athérosclérose. Les effets anti-athérosclérotiques et hypocholestérolémiants des ligands synthétiques du récepteur CD36 sont en partie engendrés par 1) la régulation du métabolisme des lipides au niveau des macrophages en réponse à l’activation du PPARγ par son ligand endogène, le 15d-PGJ2 et 2) par une augmentation du transport inverse du cholestérol, particulièrement par une augmentation de l’efflux transintestinal. / Cardiovascular diseases are the major cause of mortality and morbidity in industrialized countries. CD36, a type B scavenger receptor expressed on macrophages, appears to play a major role in foam cell formation through scavenging oxidatively modified lipoproteins, thus leading to fatty streak lesion formation in the arterial wall. We have previously reported that growth hormone-releasing peptides (GHRP) are synthetic ligands that share the same binding site as oxidized low density lipoprotein (oxLDL) on the CD36 receptor. However, no study has reported the anti-atherosclerotic effects of CD36 ligands and their role in macrophage lipid metabolism. Thus, this project aimed to evaluate the anti-atherosclerotic effects of EP 80317, a CD36 selective ligand, and to elucidate the role of GHRP on macrophage lipid metabolism and transport. Apolipoprotein E deficient mice (apoE-/-) fed a high fat high cholesterol diet were treated for 12 weeks with EP 80317. Our study showed that EP 80317 exerted potent anti-atherosclerotic effects as shown by reduced lesion areas (up to 51%) and hypocholesterolemia. We further showed that a chronic treatment with EP 80317 reduced oxLDL uptake and increased the expression of genes/proteins involved in macrophage cholesterol efflux, such as peroxisome proliferator-activated receptor γ (PPARγ), liver x receptor α (LXRα) and ATP-binding cassette A1 (ABCA1) and ABCG1, thus reducing foam cell formation. Our second study aimed to elucidate the molecular mechanisms by which CD36 ligands lead to an increase in macrophage cholesterol efflux following PPARγ activation. [3H]-cholesterol-labeled murine macrophages incubated in the presence of EP 80317 showed a significant increase in cholesterol efflux to both ABCA1 and ABCG1 transporters of cholesterol. EP 80317-mediated macrophage cholesterol efflux through PPARγ involved an increase in intracellular 15d-PGJ2 levels that were elicited by extracellular signal-regulated kinase 1/2 (ERK1/2) stimulation, itself dependent on COX-2 activation. The third and last study of this thesis aimed to investigate the effect of CD36 selective ligand on reverse cholesterol transport in vivo. ApoE-/- mice treated or not with EP 80317 were injected intraperitoneally with [3H]-cholesterol-labeled murine J774 macrophage-like cells. The radioactivity recovered in the livers of EP 80317-treated mice was significantly lower than that found in vehicle-treated mice whereas feces radioactivity was higher. Yet, the radioactivity in plasma did not achieve statistical differences between the two groups. Furthermore, the expression of genes involved in intestinal cholesterol transport such as LXRα, ABCA1, ABCG5 and ABCG8 was upregulated in EP 80317-treated mice while the expression of NPC1L1, a transporter involved in cholesterol absorption, was downregulated compared to vehicle-treated mice. In contrast, genes involved in hepatic cholesterol metabolism were not modulated by EP 80317. In conclusion, the work conducted in this thesis supported that activation of CD36 signaling pathways by EP 80317 may constitute a novel therapeutic approach for the treatment of atherosclerosis. The anti-atherosclerotic and hypocholesterolemic effects of synthetic CD36 selective ligands might be explained, at least in part, by 1) the regulation of macrophage cholesterol metabolism as a result of an increase in PPARγ activation by its endogenous ligand, 15d-PGJ2 and 2) the stimulation of reverse cholesterol transport, in particular that of transintestinal cholesterol efflux.

AIDA-I (adhesin involved in diffuse adherence) est une importante adhésine autotransporteur exprimée par certaines souches de Escherichia. coli impliquée dans la colonisation des porcelets sevrés causant la diarrhée post-sevrage et la maladie de l’œdème. Une précédente étude de notre laboratoire a identifié l’apolipoprotéine AI (ApoAI) du sérum porcin, la protéine structurale des lipoprotéines à haute densité, comme récepteur cellulaire putatif de AIDA-I. L’interaction entre ces deux protéines doit être caractérisée. Ici, nous montrons par ELISA que AIDA-I purifiée est capable d’interagir avec l’ApoAI humaine, mais également avec les apolipoprotéines B et E2. L’ApoAI est rencontrée sous deux formes, soit libre ou associée aux lipides. Nous montrons que la forme libre n’interagit pas avec les bactéries AIDA-I+ mais s’associe spécifiquement à l’ApoAI membranaire de cellules épithéliales HEp-2. Afin d’étudier le rôle de l’ApoAI dans l’adhésion des bactéries, nous avons infecté des cellules HEp-2 en présence d’anticorps dirigés contre l’ApoAI, mais l’adhésion des bactéries AIDA I+ n’a jamais été réduite. De plus, l’induction de l’expression de l’ApoAI par fénofibrate et GW7647 chez les cellules Caco 2 polarisée et Hep G2, n’a pas permis l’augmentation de l’adhésion cellulaire des E. coli exprimant AIDA-I. Notre étude suggère davantage que l’interaction entre AIDA-I et ApoAI n’intervient pas dans les mécanismes d’adhésion cellulaire. / The adhesin involved in diffuse adherence (AIDA-I) is an important autotransporter adhesin expressed by some strains of Escherichia coli and is involved in the intestinal colonisation of weaned piglets, causing the postweaning diarrhea and the edema disease. A previous study from our laboratory identified the apolipoprotein AI (ApoAI) from porcine serum, the structural protein of high density lipoproteins, as a putative receptor of AIDA-I. The interaction between these two proteins must be characterized. Here, we show that purified AIDA-I, using an ELISA assay, is able to bind the human ApoAI and the apolipoprotein B and E2. The ApoAI is found under two forms, either free or bound to lipid. We show that the free form of ApoAI does not interact with AIDA-I+ bacteria but specifically interact with membrane bound ApoAI on Hep-2 epithelial cells. To study the role of ApoAI in the adhesion of bacteria, we infected Hep-2 cells preincubated with antibodies to ApoAI. The adhesion of AIDA-I+ bacteria to the cells couldn’t be reduced. Additionally, the induction of ApoAI synthesis using fenofibrate and GW7647 on polarized Caco-2 or Hep G2 cells did not increase the adhesion of AIDA-I+ bacteria. Our study suggests that the interaction between AIDA-I and ApoAI is not involved in the cellular adhesion of the bacteria.

No diabete melito, produtos de glicação avançada (AGE) reduzem o efluxo de colesterol celular o que agrava o desenvolvimento da aterosclerose. Neste estudo, investigou-se o papel da albumina modificada por glicação avançada (albumina-AGE) sobre a sensibilização de macrófagos à resposta inflamatória e o impacto da secreção de citocinas, quimocinas e moléculas de adesão sobre o efluxo de colesterol mediado por apolipoproteína A-I e subfrações de HDL. Além disso, determinou-se a capacidade da HDL em modular a resposta inflamatória em macrófagos tratados com albumina-AGE. Macrófagos de peritônio de camundongo foram tratados com ou sem sobrecarga de colesterol, na presença de 2 mg/mL de albumina-controle (albumina-C) ou albumina-AGE, por 72 h, seguindo-se de incubação, por 24 h, com calgranulina S100B (20 g/mL) ou lipopolissacarídeo (LPS; 1 g/mL). Em comparação com albumina-C, a albumina-AGE, isenta em endotoxinas, isoladamente não alterou a secreção de citocinas em macrófagos. No entanto, a albumina-AGE sensibilizou macrófagos não enriquecidos em colesterol a uma maior secreção de interleucina 6 (IL-6), fator de necrose tumoral alfa (TNF-), proteína quimoatraente de monócitos 1 (MCP-1), interlucina 1 beta (IL-1) e molécula de adesão celular vascular 1 (VCAM-1) após estimulação com S100B ou LPS, o que foi potencializado pela sobrecarga de colesterol celular. Em macrófagos não estimulados, o meio condicionado, advindo das incubações de células com albumina-AGE e S100B (meio enriquecido em citocinas), reduziu o efluxo de 14C-colesterol mediado por apoA-I, HDL2 e HDL3 em, respectivamente, 23%, 43% e 20%, em comparação com células incubadas com meio isolado do tratamento com albumina-C e S100B. De forma similar, o efluxo de 14C-colesterol mediado por apoA-I, HDL2 e HDL3 foi reduzido em macrófagos tratados com meio advindo de incubações com albumina-AGE e LPS, respectivamente, 37%, 47% e 8,5% em comparação ao tratamento com albumina-C e LPS. Em macrófagos tratados com albumina-C e S100B, a incubação prévia com HDL reduziu a secreção de IL-6, TNF-, MCP-1 e VCAM-1 em, respectivamente, 72%, 57%, 50% e 41% quando comparada à incubação na ausência de HDL. Em incubações com albumina-C, a secreção de IL-6, TNF-, MCP-1, IL-1 e VCAM-1 induzida por LPS foi respectivamente, 58%, 54%, 42%, 74% e 45% menor mediante incubação com HDL, em comparação a incubações similares, porém na ausência desta lipoproteína. Por outro lado, em macrófagos tratados com albumina-AGE e S100B, a HDL não foi capaz de reduzir a secreção de TNF-, IL-1 e VCAM-1 e aumentou a secreção de IL-6 (54%) e MCP-1 (20%). Nas células tratadas com albumina-AGE e LPS, a HDL também não reduziu a secreção de TNF-, MCP-1 e IL-1 e aumentou a secreção de IL-6 (16%) e VCAM-1 (20%). Redução na secreção de mediadores inflamatórios foi observada em macrófagos tratados com albumina-AGE apenas quando a HDL foi incubada juntamente com S100B ou LPS. Em conclusão, a albumina-AGE sensibiliza macrófagos à resposta inflamatória induzida por calgranulina S100B e LPS, prejudicando o transporte reverso de colesterol de macrófagos. Além disso, a albumina-AGE reduz as propriedades anti-inflamatórias da HDL, o que pode agravar a aterosclerose no diabete melito / In diabetes mellitus, advanced glycation end products (AGE) reduces the cholesterol efflux from cells, which aggravates the development of atherosclerosis. In this study, we investigated the role of advanced glycated albumin (AGE-albumin) on macrophage inflammatory response and the impact of cytokines, chemokines and adhesion molecules secretion on cholesterol efflux mediated by apolipoprotein A-I (apoA-I) and HDL subfractions. Furthermore, the HDL ability in modulating inflammatory response in macrophages treated with AGE-albumin was also determined. Mouse peritoneal macrophages previously enriched or not with cholesterol were treated in the presence of 2 mg/mL of control-albumin (C-albumin) or AGE-albumin, for 72 h, followed by incubation, for 24 h, with S100B calgranulin (20 g/mL) or lipopolysaccharide (LPS; 1 g/mL). In comparison to free endotoxin-C-albumin, AGE-albumin, by itself did not alter cytokine secretion by macrophages. However, AGE-albumin primed non-cholesterol enriched macrophages to a higher secretion of interleukin -6 (IL-6), tumor necrosis factor alpha (TNF-), monocyte chemotactic protein 1 (MCP-1), interleukin 1 beta (IL-1) and vascular cell adhesion molecule 1 (VCAM-1) after stimulation with S100B or LPS, which was potentiated by cell cholesterol overload. In non-stimulated macrophages, conditioned medium, derived from incubation with AGE-albumin and S100B (cytokine enriched-medium), reduced the 14C-cholesterol efflux mediated by apoA-I, HDL2 and HDL3 in, respectively, 23%, 43% and 20%, in comparison to cells incubated with conditioned medium isolated from treatment with C-albumin and S100B. Similarly, 14C-cholesterol efflux mediated by apoA-I, HDL2 and HDL3 was reduced in macrophages treated with medium derived from incubation with AGE-albumin and LPS, respectively, 37%, 47% and 8,5% in comparison to treatment with C-albumin and LPS. In macrophages treated with C-albumin and S100B, previous incubation with HDL reduced the secretion of IL-6, TNF-, MCP-1 and VCAM-1 in, respectively 72%, 57%, 50% and 41% in comparison to incubation in the absence of HDL. In incubations with C-albumin, the secretion of IL-6, TNF-, MCP-1, IL-1 and VCAM-1 induced by LPS was respectively, 58%, 54%, 42%, 74% and 45% lower in cells treated with HDL in comparison to similar incubations in the absence of this lipoprotein. On the other hand, in macrophages treated with AGE-albumin and S100B, HDL was unable to reduce the TNF-, IL-1 and VCAM-1 secretion and increased the secretion of IL-6 (54%) and MCP-1 (20%). In cells treated with AGE-albumin and LPS, HDL was unable to reduce the secretion of TNF-, MCP-1 and IL-1 and increased IL-6 (16%) and VCAM-1 (20%). Reduction in inflammatory mediators was observed in macrophages treated with AGE-albumin only when HDL was incubated simultaneously with S100B or LPS. In conclusion, AGE-albumin primes macrophages to an inflammatory response elicited by S100B calgranulin and LPS, impairing macrophage reverse cholesterol transport. Moreover, AGE-albumin impairs the HDL anti-inflammatory properties, which can aggravate the atherosclerosis in diabetes mellitus

A eficácia das estatinas em reduzir o risco de eventos coronarianos não é completamente explicada por seus efeitos em diminuir colesterol de lipoproteína de baixa densidade (LDL-C). Um dos seus efeitos adicionais pode ser decorrente da modificação na concentração de lipoproteína de alta densidade (HDL), reconhecida como ateroprotetora, principalmente por seu papel no transporte reverso do colesterol (TRC). Os transportadores de membrana do tipo ATP-binding cassette, ABCA1 e ABCG1, e o scavenger receptor BI (SRBI) são proteínas importantes envolvidas no TRC e seus genes são regulados por vários fatores de transcrição, entre eles os liver-x-receptors (LXRs). Com a finalidade de avaliarmos os efeitos dos hipolipemiantes sobre expressão dos transportadores ABC e do receptor SRBI, a expressão de RNAm do ABCA1, ABCG1, SCARB1, NR1H3 (LXR&#945;) e NR1H2 (LRX&#946;) foi avaliada por PCR em tempo real em células das linhagens HepG2 (origem hepática) e Caco-2 (origem intestinal) tratadas com atorvastatina ou sinvastatina (10 µM) e/ou ezetimiba (até 5 µM) por até 24 horas. Além disso, a expressão desses genes também foi avaliada em células mononucleares do sangue periférico (CMSP) de 50 pacientes normolipidêmicos (NL) e 71 hipercolesterolêmicos (HC) tratados com atorvastatina (10mg/dia/4semanas, n=48) ou sinvastatina e/ou ezetimiba (10mg/dia/4 ou 8 semanas, n=23). A possível associação entre os polimorfismos ABCA1 C-14T e R219K e a expressão de RNAm em CMSP também foi avaliada por PCR-RFLP. O SCARB1 foi o gene mais expresso nas células HepG2 e Caco-2, seguido por NR1H2, NR1H3, ABCG1 e ABCA1 em HepG2 ou por ABCA1 e ABCG1 em Caco-2. O tratamento com estatinas (1 ou 10 µM) ou ezetimiba (5 µM), por 12 ou 24 horas, aumentou a expressão de RNAm do ABCG1, mas não de ABCA1 e SCARB1, em células HepG2. Ainda nesta linhagem, o aumento na transcrição dos genes NR1H2 e NR1H3 foi observado somente com a maior concentração de atorvastatina (10 µM) e, ao contrário, o tratamento com ezetimiba causou redução na transcrição de NR1H2, sem alteração de NR1H3. Em células Caco-2, o tratamento com atorvastatina ou sinvastatina por 12 ou 24 horas reduziu a quantidade do transcrito ABCA1 e não alterou a expressão do SCARB1 e do ABCG1, embora, para este último, tenha havido uma tendência à diminuição da expressão após tratamento com sinvastatina (p=0,07). Após tratamento com ezetimiba isolada (até 5 µM) nenhuma alteração de expressão de RNAm foi observada em células Caco-2; no entanto, após 24 horas de tratamento com sinvastatina e ezetimiba, foi reduzida a taxa de transcrição de ABCA1 e ABCG1, mas não de SCARB1. Ao contrário das linhagens celulares, em CMSP os genes NR1H2 e ABCG1 foram os mais expressos, seguidos pelos genes SCARB1 e ABCA1 e, finalmente, pelo NR1H3. Indivíduos HC tiveram maior expressão basal de NR1H2 e NR1H3, mas não de outros genes, quando comparados aos NL (p<0,05). Além disso, nos indivíduos HC, a expressão basal de ABCA1 foi maior em portadores do alelo -14T do polimorfismo ABCA1 -14C>T quando comparados aos portadores do genótipo -14CC (p=0,034). O tratamento com estatinas, com ezetimiba ou com a terapia combinada diminuiu a transcrição de ABCA1 e ABCG1. Para o SCARB1, NR1H2 e NR1H3, nenhuma alteração de expressão de RNAm em CMSP foi detectada após os tratamentos in vivo. Após todas as fases de tratamento, ABCA1 e ABCG1 e também NR1H2 e NR1H3 foram significativamente correlacionados entre si, mas nenhuma correlação com perfil lipídico sérico foi relevante. Coletivamente, esses resultados dão indícios de que os hipolipemiantes analisados (estatinas e ezetimiba) têm um importante papel na regulação da expressão de genes envolvidos no transporte reverso do colesterol e sugerem a existência de regulação tecido-específica para os dois transportadores ABC. Além disso, o efeito das estatinas ou da ezetimiba sobre a expressão do ABCA1, do ABCG1 ou do SCARB1 não sofreu influencia de alterações diretas da transcrição dos LXRs. / The efficacy of statins in reducing the risk of coronary events is not completely explained by their effects in decreasing cholesterol low-density lipoprotein (LDL-C). One of their additional effects may result from the change in concentration of high-density lipoprotein (HDL), recognized as atheroprotective, mainly for the role in reverse cholesterol transport (RCT). The membrane transporters, as ATP-binding cassette, ABCA1 and ABCG1, and scavenger receptor BI (SRBI) are important proteins involved in the RCT and their genes are regulated by various transcription factors, including the liver-X-receptors (LXRs) . In order to evaluate the effects of lipid lowering on expression of ABC transporters and SRBI receptor, the mRNA expression of ABCA1, ABCG1, SCARB1, NR1H3 (LXR&#945;) and NR1H2 (LRX&#946;) was assessed by real time PCR in HepG2 (hepatic origin) and Caco-2 (intestinal origin) cells treated with atorvastatin or simvastatin (10 µM) and/or ezetimibe (up to 5 µM) for 24 hours. Furthermore, the expression of these genes was evaluated in peripheral blood mononuclear cells (PBMC) of 50 normolipidemic (NL) and 71 hypercholesterolemic (HC) patients treated with atorvastatin (10mg/d/4 weeks, n = 48) or simvastatin and/or ezetimibe (10mg/d/4 or 8 weeks, n = 23). The possible association between ABCA1 C-14T and R219K polymorphisms and mRNA expression in PBMC was also evaluated by PCR-RFLP. SCARB1 was the most expressed in HepG2 and Caco-2 cells, followed by NR1H2, NR1H3, ABCG1 and ABCA1 in HepG2 or by ABCG1 and ABCA1 in Caco-2. The treatment with statins (1 or 10 µM) or ezetimibe (5 µM) for 12 or 24 hours, increased mRNA expression of ABCG1 but not ABCA1 and SCARB1 in HepG2 cells. Moreover, in HepG2 cells, atorvastatin also upregulated NR1H2 and NR1H3 only at 10.0 &#181;M, meanwhile ezetimibe downregulated NR1H2 but did not change NR1H3 expression. In Caco-2 cells, atorvastatin or simvastatin treatment for 12 or 24 hours reduced the amount of ABCA1 transcript and did not alter the ABCG1 and SCARB1 expressions, despite the tendency to decrease ABCG1 mRNA expression after simvastatin treatment (p = 0.07). After treatment with ezetimibe alone (up to 5 &#181;M) no change in mRNA expression was observed in Caco-2 cells; however, after 24 hours- simvastatin and ezetimibe treatments decreased the transcription of ABCA1 and ABCG1, but not of SCARB1. Unlike cell lines, in PBMC, NR1H2 and ABCG1 were the most expressed, followed by SCARB1 and ABCA1 and finally by the NR1H3. HC patients showed higher NR1H2 and NR1H3 basal expressions, but not of other genes, compared to NL (p <0.05). Moreover, in HC individuals, the ABCA1 basal expression was higher in individuals carrying -14T allele of -14C> T polymorphism when compared with -14CC carriers (p = 0.034). Treatment with statins, ezetimibe, or combined therapy downregulated ABCA1 and ABCG1 expression. For SCARB1, NR1H2 and NR1H3, no change in mRNA expression in PBMC was detected after treatments. After all phases of treatment, ABCA1 and ABCG1 as well as NR1H2 and NR1H3 were significantly correlated, but no correlation with serum lipid profile was relevant. Collectively, these results provide evidences that the lipid lowering (statins and ezetimibe) have an important role in mRNA expression regulation of genes involved in reverse cholesterol transport and suggest the existence of tissue-specific regulation for the ABC transporters. Furthermore, the effect of statins or ezetimibe on ABCA1, ABCG1 or SCARB1 expression was not directly influenced by changes of LXR transcription.

La capacité des particules HDL à exercer des effets anti-athérogènes passe notamment par leur capacité à assurer le transport inverse du cholestérol (RCT). L'objectif principal de mon programme de recherche est l'étude de l'étape initiale du transport inverse du cholestérol que représente l'efflux de cholestérol du macrophage, dans le contexte des maladies métaboliques et du risque cardiovasculaire et de mortalité. J'ai étudié la relation entre l'efflux, et les conséquences sur le développement de l'athérosclérose dans un contexte métabolique particulier ; le syndrome métabolique (SM). J'ai démontré que les critères individuels du SM sont intimement liés à l'efflux et que ces deux notions sont associées de façon indépendante aux paramètres cliniques de l'athérosclérose. J'ai aussi évalué la pertinence de l'efflux de cholestérol comme biomarqueur de la mortalité. Cette étude identifie l'efflux comme prédicteur de la mortalité toutes causes confondues, indépendamment des taux de HDL-cholestérol et des facteurs de risques cardiovasculaires traditionnels, dans une population de patients traités par angioplastie coronaire primaire, suite à un infarctus du myocarde avec élévation du segment ST. Enfin, je me suis intéressée à à une situation métabolique particulière ; l'état postprandial. Mes travaux montrent que la réponse postprandiale hypertriglycéridémique physiologique observée chez des individus sans désordre métabolique ne s'accompagne pas d'altération majeure de l'efficacité du RCT ou de l'inflammation systémique. Mes travaux confirment le rôle déterminant de l'efflux dans la prévention du développement de l'athérosclérose et de la mortalité cardiovasculaire. / The contribution of high-density lipoprotein to cardiovascular benefit is closely linked to its anti-atherogenic role in the cellular cholesterol efflux. The main purpose of my project was to evaluate the efficiency of the first step of reverse cholesterol transport (RCT), which is the efflux capacity, on metabolic disorder context, on cardiovascular risk and on mortality. My research has focused on three independent and complementary parts. I have first evaluated the relationship between efflux and its consequences on atherosclerosis development in a metabolic syndrome (MetS) population. I have shown that individual criteria of MetS are closely related synergistically to cholesterol efflux capacity. In addition, established metabolic syndrome and cholesterol efflux capacity were independently associated with clinical features of atherosclerosis. In a second study I identified cholesterol efflux capacity as a predictor of all-cause mortality in consecutive ST-segment elevation myocardial infarction patients treated by primary angioplasty, independent of HDL-C, traditional cardiovascular risks or cardiac risk factors. Finally I have evaluated the consequences of postprandial hypertriglyceridemia on the functionality of key steps of RCT and associated anti-inflammatory components. My work has shown that the physiological postprandial hypertriglyceridemia response is not accompanied by a major alteration in the efficiency of RCT or systemic inflammation, on individual without metabolic syndrome. In conclusion, I have confirmed the crucial role of the first step of reverse cholesterol transport in preventing the development of atherosclerosis and cardiovascular mortality.

No description available.

Generation of foam cells, an essential step for reverse cholesterol transport (RCT) studies, uses the technique of receptor dependent macrophage loading with radiolabeled acetylated Low Density Lipoprotein (Ac-LDL). In this study, we used the ability of a biologically relevant detergent molecule, Lysophosphatidylcholine (Lyso PtdCho), to form mixed micelles with cholesterol or cholesteryl ester (CE) to generate macrophage foam cells. Fluorescent or radiolabelled cholesterol / Lyso PtdCho mixed micelles were prepared and incubated with RAW 264.7 or mouse peritoneal macrophages. Results showed that such micelles were quite stable at 4°C and retained the solubilized cholesterol during one month storage. Macrophages incubated with cholesterol or CE (unlabeled, fluorescently labeled or radiolabeled) / Lyso PtdCho mixed micelles accumulated CE as documented by microscopy, lipid staining, labeled oleate incorporation, and by thin layer chromatography (TLC). Such foam cells unloaded cholesterol when incubated with high density lipoprotein (HDL) and not with oxidized HDL (Ox-HDL). We propose that stable cholesterol or CE / Lyso PtdCho micelles would offer advantages over existing methods. Oxidative stress is associated with heart failure (HF). Previously our research group observed that the patients with low left-ventricular ejection fraction showed accumulation of high level of oxidized LDL (Ox-LDL) when compared with the heart failure patients with normal range of ejection fraction (EF). HDL is known to be atheroprotective and one of its important antioxidative functions is to protect LDL from oxidative modifications. However, HDL itself undergoes oxidation and Ox-HDL becomes functionally poor. It is expected to have a diminished ability to promote reverse cholesterol transport. Therefore, it was hypothesized that the quality of HDL present in the patients with EF would more compromised than those present in the patients with normal EF. Functionality of HDL was evaluated by measuring its cholesterol efflux capacity from foam cells generated in vitro. Functionality of HDL, which is strongly related to the oxidative modifications of HDL was further estimated by measuring paraoxonase 1 (PON1) enzyme activity associated with HDL. Higher the PON1 activity and RCT ability, better is the functionality of HDL.