Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Obesity is a systemic low grade inflammation, that is associated to the increased production of proinflammatory molecules by adipose tissue. Besides, the adiposity elevated induces cellular apoptosis and release of a large amounts of free fatty acids from disrupted adipocyte. These molecules tend to accumulate in the liver and other body tissues like skeletal muscles causing a phenomenon known as lipotoxicity. Evidence suggests that in addition to fatty acids there are other molecules that could have a severe effect lipotoxicity. This is the case of the inflammatory cytokine interleukin 1 beta (IL-1β). The IL-1β is a major inflammatory cytokines in immunity system. Studies suggest that its continued production may act by reducing the glucose influx, and induces the free radicals production contributing to the maintenance of a state of oxidative stress. For this reason, some authors have suggested that IL-1β would be a molecule glicolipotóxic. In humans the expression of IL-1B is under strong genetic control. The single nucleotide polymorphism (SNP) of C to T at nucleotide position +3953 of the IL-1B gene appears to be functional because it has been associated with increased production of IL-1β in vivo. Therefore, the aim of this study was to analyze the association between the polymorphism +3953 IL-1B gene with obesity, the influence on glycemic control, lipid and oxidized LDL molecule (oxLDL). For this, two studies were performed. The first was a case-control study with 880 Caucasian individuals, classified to obesity (non-obese = 283, overweight = 334, obese = 263), age between 18-92 years, and genotyped to IL-1B +3953 polymorphism. It was measured the bioanthropometric variables (DBP, SBP, IBM, height, weight, waist circumference) and serum parameters of blood glucose, lipid profile. In the second study, we included 225 subjects genotyped previously and measured the oxLDL serum levels, besides bioanthropometric and lipid profile measures. In both studies the volunteers did not smoke and had no cardiovascular disease or chronic degenerative diseases that could affect results. The IL-1B polymorphism genotypes were determined by PCR-RFLP and biochemical parameters by spectrophotometry. The first study results showed that the C allele (CC and CT) had a higher frequency in the group of obese and overweight when compared to the non obese group. The odds ratio showed 1.340 (95% CI: 1.119-1.605) times more chance of the obese group being CC carriers compared to non-obese group independent of gender and age. The second study showed that TT genotype carriers presented lower levels of oxLDL than patients with other genotypes. However no significant influence was observed of this polymorphism in blood glucose levels. Multivariate analysis showed that this result is independent of sex, age, obesity and hypertension. Thus, these results support the hypothesis that IL-1β is a molecule indeed obesogenic and lipotoxicity as previously suggested from animal models studies. Key-word: obesity. Interleukin-1beta. Oxidized-LDL / A obesidade é um inflamação sistêmica de baixo grau associado a elevada liberação de moléculas pró-inflamatórias pelo tecido adiposo. O aumento da adiposidade resulta em necrose tecidual e liberação de ácidos graxos, a partir dos adipócitos, que tendem a se acumular em outros tecidos corporais causando um fenômeno conhecido como lipotoxicidade. A IL-1β é uma das principais citocinas pró-inflamatórias do sistema imune, e a sua produção continuada pode agir diminuindo o influxo da glicose para dentro da célula, e induzir a produção de radicais livres contribuindo para a manutenção de um estado de estresse oxidativo. Por este motivo, alguns autores sugeriram que a IL- 1β seria uma molécula glicolipotóxica. Nos seres humanos a IL-1β possui um polimorfismo +3953 do gene da IL-1B parece ser funcional pois tem sido associado a variações na produção da IL-1β in vivo. Portanto, o objetivo deste estudo foi o de analisar a associação entre o polimorfismo +3953 do gene da IL-1B com a obesidade, a influencia nos níveis glicêmicos, lipídicos e da molécula de LDL- oxidado (LDL-ox). Para isso, foram realizados dois estudos. O primeiro foi um estudo caso-controle com 880 indivíduos Caucasianos classificados para obesidade (não obesos= 283, sobrepeso=334, obesos= 263), com idade entre 18 e 92 anos, genotipados para IL-1B +3953 determinados por PCR-RFLP. As variáveis PAD, PAS, circunferência abdominal, IMC, altura, peso, bem como o perfil glicêmico e lipídico foram medidos. No segundo estudo foram selecionados 225 indivíduos, previamente genotipados para IL-1B +3953, e medido os níveis séricos de LDL-ox, além das medidas bioantropométricas e perfil glicêmico e lipídico. Em ambos os estudos os voluntários não fumavam e não apresentavam doenças cardiovasculares ou crônico-degenerativas que pudessem interferir nos resultados. Os resultados, do primeiro estudo, mostraram que o alelo C (CT CC) apresentou uma frequência maior no grupo dos obesos e sobrepeso quando comparado ao grupo dos não obesos. A chance dos indivíduos obesos de serem portadores do genótipo CC foi de 1,34 (IC 95%, 1,119- 1,605). Estes resultados foram independente do gênero e idade. O segundo estudo mostrou que indivíduos com o genótipo CC e CT apresentaram níveis mais elevados de LDL-ox do que os portadores do genótipo TT. A análise multivariada mostrou que este resultado é independente do gênero, idade e obesidade. Entretanto, em ambos os estudos, não foi observada influência deste polimorfismo nos níveis glicêmicos. Assim, podemos concluir que a IL-1β é uma molécula, com efeito obesogênico e lipotóxico, corroborando estudos prévios em modelos animais.

Contrairement aux HDL de sujets sains, les HDL de patients diabétiques ont perdu leur capacité à contrecarrer les effets inhibiteurs des LDL oxydées sur la vasorelaxation endothélium dépendante. Les mécanismes en cause ne sont pas connus. Or la glycation et l’oxydation sont deux phénomènes majeurs au cours du diabète. Nous avons donc étudié in vitro, le rôle de la glycation (associée ou non à une oxydation spontanée), et de l’oxydation d’HDL issues de sujets sains, sur leurs capacités à contrecarrer les effets inhibiteurs des LDL oxydées sur la vasorelaxation endothélium dépendante. Chaque condition a conduit au même constat: les HDL modifiées perdent leur pouvoir vasorelaxant en présence de LDL oxydées. En revanche, en l’absence de LDL oxydées, elles n’altèrent pas la vasorelaxation induite par l’acétylcholine. Ainsi les modifications structurelles des HDL (glycation, oxydation, ou les deux) induisent une perte de leur capacité à protéger l’endothélium du stress oxydatif, plutôt qu’un effet délétère direct sur l’endothélium. Un des mécanismes majeur impliqué dans ce phénomène est probablement l’absence de fixation de ces HDL modifiées à leur récepteur SR-BI. Elles ne pourraient plus alors s’opposer au niveau des cavéoles aux effets délétères des LDL oxydées, et ne favoriseraient plus la production de NO. Mais si aussi bien la glycation que l’oxydation des HDL entraînent ces effets néfastes, il semblerait qu’en condition physiopathologique (oxydation spontanée des HDL glyquées), l’oxydation ne majore pas cette perte de capacité des HDL à contrecarrer les effets inhibiteurs des LDL oxydées sur la vasorelaxation. / Contrary to HDL from normolipidaemic and normoglycaemic subjects, HDL from diabetic patients have lost their capacity to reverse the inhibition of vasorelaxation induced by oxidized LDL. Mechanisms involved are unknown. The glycation and oxidation of HDL are two major phenomena in diabetes mellitus. The aim of this work was to study in vitro the role of glycation (with or without spontaneous oxidation) and oxidation of HDL, on their capacity to counteract the inhibitory effect of oxidized LDL on endothelium-dependent vasorelaxation. Each state showed the same result, modified HDL lost their vasorelaxing power in stress conditions (with oxidized LDL). Nevertheless, modified HDL alone (without oxidized LDL) did not alter vasorelaxation induced by acetylcholine, after noradrenaline-induced vasoconstriction. Thus, modifications of HDL induce a loss of the ability to protect vessels from oxidative stress rather than have a direct deleterious effect on the vessel. One of the major mechanisms involved in this phenomenon is probably the loss of SR-BI binding of these modified HDL, that could lead to the inability of HDL to protect caveolae from deleterious effects induced by oxidized LDL and could not preserve NO production. However, though glycation, like oxidation of HDL, leads to these deleterious effects, it would seem that during physiopathological conditions, with the spontaneous oxidation of glycated HDL, oxidation does not aggravate the loss of the capacity of diabetic HDL to counteract the inhibitory effect of oxidized LDL on endothelium-dependent vasorelaxation.

Während die zellulären Mechanismen der Pathogenese der Arteriosklerose intensiv untersucht worden sind, ist über die Mechanismen, die zu einer bevorzugten Lokalisation arteriosklerotischer Läsionen in bestimmten Gefäßarealen führen, weniger bekannt. Zur Untersuchung dieser Mechanismen wurden Endothelzellen aus menschlichen Koronararterien, einem Gefäßbereich, in dem häufig arteriosklerotische Läsionen beobachtete werden, isoliert und kultiviert. Endothelzellen der Mikrozirkulation menschlicher Herzen wurden unter gleichen Bedingungen kultiviert und die Reaktionen beider Zellarten verglichen. Inkubation der Zellen mit den in Bezug auf die Bildung arteriosklerotischer Plaques besonders pathogenen oxidierten LDL induzierte in makrovaskulären koronaren Endothelzellen eine stärkere Zunahme der PAI-1 Aktivität (182%, p / While the cellular mechanisms of atherosclerosis have been intensively studied, the mechanisms leading to preferential localization of atherosclerotic lesions are less well understood. To further define these mechanisms, endothelial cells from coronary arteries, i.e. vessels with frequent atherosclerotic lesions, were isolated and grown in vitro. In order to compare the reactions of both cell types, endothelial cells derived from microvessels of human hearts were isolated and cultured under identical conditions. Incubation of endothelial cells with oxidized LDL (75 µg/ml protein) induced a significant increase in PAI-1 activity (182 %, p

A aterosclerose é uma doença degenerativa crônica dos vasos, com conseqüências clínicas agudas que incluem o infarto do miocárdio e o acidente vascular cerebral, resultantes geralmente da ruptura da placa e trombose. É atualmente reconhecida como de característica inflamatória, iniciada e propagada no contexto da hipercolesterolemia. Um trabalho de nosso grupo utilizou técnicas de phage display para comparar placas ateroscleróticas e carótidas normais objetivando a busca de proteínas alteradas potencialmente envolvidas na patogênese da doença. Diversas semaforinas e plexinas (receptores de semaforinas) foram identificadas dentre elas a plexina B1, que possui alta afinidade por CD100, sugerindo assim uma concentração aumentada de CD100 na placa aterosclerótica. CD100 foi a primeira semaforina descrita no sistema imune e a única até hoje descrita como possuidora de duas formas de funcionalidades distintas, sendo uma de membrana (mCD100) e outra solúvel (sCD100). Neste trabalho demonstramos a expressão da semaforina CD100 em macrófagos e células espumosas em placas ateroscleróticas humanas, assim como seu padrão de expressão ao longo da diferenciação monócito-macrófago-célula espumosa, e sob estímulos distintos. Além disso, identificamos pela primeira vez o receptor que medeia suas atividades nessas células, a plexina B2. Adicionalmente, detectamos também pela primeira vez detectamos a expressão de CD100 em células endoteliais teciduais e cultivadas in vitro, o que sugere um papel significativo da semaforina em fenômenos vasculares. Com base nessas observações e nos resultados de experimentos de bloqueio de adesão constatamos que CD100 pode atuar na fase mais precoce da aterosclerose, como uma molécula de adesão envolvida na ligação entre monócitos e células endoteliais. Verificamos ainda que CD100 diminui a captação de LDLox em macrófagos e células espumosas. Poucos estudos relatam a presença ou possível atividade biológica de CD100 tanto na aterosclerose quanto em macrófagos. Devido às já estabelecidas ações no sistema imune, acreditamos que a expressão diferencial dessa semaforina desempenha um papel amplificador na patogênese da aterosclerose. Posteriormente, essa proteína poderá servir como alvo de inibição da progressão da doença e de suas complicações / Atherosclerosis is a chronic degenerative disease affecting vessels, with acute clinical consequences that include myocardium infarction or stroke, generally resulting from plaque rupture and thrombosis. It is now recognized as an inflammatory disease, initiated and developed in a hipercholesterolemic context. A work in our lab has used phage display techniques to compare atherosclerotic plaques and normal carotids, searching for altered proteins potentially involved in the pathogenesis of the disease. Many semaphorins and plexins (semaphorin receptors) have been identified, among which plexin B1, a high affinity receptor for CD100, suggesting an augmented level of CD100 in the atherosclerotic plaques. CD100 is the first semaphorin described in the immune system, and the only to possess two forms with distinct functionalities, being one associated to the membrane, mCD100, and another soluble form, sCD100. In the present work we have demonstrated CD100 expression in macrophages and foam cells of human atherosclerotic plaques, as well as its pattern of expression along monocyte-macrophage-foam cell differentiation and under distinct stimuli. Furthermore, we have identified for the first time the receptor involved in CD100 activities in these cells, namely plexin B2. Aditionally, we have detected CD100 expression in tissue as well as in in vitro cultured endothelial cells, also for the first time. According to these informations and adhesion blockage experiments we have shown that CD100 may act in the earliest phase of the establishment of atherosclerosis, as an adhesion molecule involved in monocyte-endothelial cell association. We have also verified that CD100 diminishes the intake of oxLDL in macrophages and foam cells. Only a few studies describe the presence or possible biological activity of CD100 in atherosclerosis or macrophages. Since the molecule has been shown to participate in the immune system, we believe that the differential expression of this semaphorin plays an amplifying role in the pathogenesis of atherosclerosis. In the future, this protein could act as an inhibition target of the disease progression as well as its complications

A aterosclerose é uma doença degenerativa crônica dos vasos, com conseqüências clínicas agudas que incluem o infarto do miocárdio e o acidente vascular cerebral, resultantes geralmente da ruptura da placa e trombose. É atualmente reconhecida como de característica inflamatória, iniciada e propagada no contexto da hipercolesterolemia. Um trabalho de nosso grupo utilizou técnicas de phage display para comparar placas ateroscleróticas e carótidas normais objetivando a busca de proteínas alteradas potencialmente envolvidas na patogênese da doença. Diversas semaforinas e plexinas (receptores de semaforinas) foram identificadas dentre elas a plexina B1, que possui alta afinidade por CD100, sugerindo assim uma concentração aumentada de CD100 na placa aterosclerótica. CD100 foi a primeira semaforina descrita no sistema imune e a única até hoje descrita como possuidora de duas formas de funcionalidades distintas, sendo uma de membrana (mCD100) e outra solúvel (sCD100). Neste trabalho demonstramos a expressão da semaforina CD100 em macrófagos e células espumosas em placas ateroscleróticas humanas, assim como seu padrão de expressão ao longo da diferenciação monócito-macrófago-célula espumosa, e sob estímulos distintos. Além disso, identificamos pela primeira vez o receptor que medeia suas atividades nessas células, a plexina B2. Adicionalmente, detectamos também pela primeira vez detectamos a expressão de CD100 em células endoteliais teciduais e cultivadas in vitro, o que sugere um papel significativo da semaforina em fenômenos vasculares. Com base nessas observações e nos resultados de experimentos de bloqueio de adesão constatamos que CD100 pode atuar na fase mais precoce da aterosclerose, como uma molécula de adesão envolvida na ligação entre monócitos e células endoteliais. Verificamos ainda que CD100 diminui a captação de LDLox em macrófagos e células espumosas. Poucos estudos relatam a presença ou possível atividade biológica de CD100 tanto na aterosclerose quanto em macrófagos. Devido às já estabelecidas ações no sistema imune, acreditamos que a expressão diferencial dessa semaforina desempenha um papel amplificador na patogênese da aterosclerose. Posteriormente, essa proteína poderá servir como alvo de inibição da progressão da doença e de suas complicações / Atherosclerosis is a chronic degenerative disease affecting vessels, with acute clinical consequences that include myocardium infarction or stroke, generally resulting from plaque rupture and thrombosis. It is now recognized as an inflammatory disease, initiated and developed in a hipercholesterolemic context. A work in our lab has used phage display techniques to compare atherosclerotic plaques and normal carotids, searching for altered proteins potentially involved in the pathogenesis of the disease. Many semaphorins and plexins (semaphorin receptors) have been identified, among which plexin B1, a high affinity receptor for CD100, suggesting an augmented level of CD100 in the atherosclerotic plaques. CD100 is the first semaphorin described in the immune system, and the only to possess two forms with distinct functionalities, being one associated to the membrane, mCD100, and another soluble form, sCD100. In the present work we have demonstrated CD100 expression in macrophages and foam cells of human atherosclerotic plaques, as well as its pattern of expression along monocyte-macrophage-foam cell differentiation and under distinct stimuli. Furthermore, we have identified for the first time the receptor involved in CD100 activities in these cells, namely plexin B2. Aditionally, we have detected CD100 expression in tissue as well as in in vitro cultured endothelial cells, also for the first time. According to these informations and adhesion blockage experiments we have shown that CD100 may act in the earliest phase of the establishment of atherosclerosis, as an adhesion molecule involved in monocyte-endothelial cell association. We have also verified that CD100 diminishes the intake of oxLDL in macrophages and foam cells. Only a few studies describe the presence or possible biological activity of CD100 in atherosclerosis or macrophages. Since the molecule has been shown to participate in the immune system, we believe that the differential expression of this semaphorin plays an amplifying role in the pathogenesis of atherosclerosis. In the future, this protein could act as an inhibition target of the disease progression as well as its complications

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Epidemiological studies have suggested that intake of fruits and vegetables act in the prevention of several diseases such as cancer and cardiovascular diseases. This can be explained by these bioactive compounds are present in their food nutritional matrix that may have different biological activities, including antioxidant, antitumor, genoprotective and in preventing the oxidation of LDL. However, much of the fruit remains to be studied. This is the case of Solanum sessiliflorum popularly known as mana-cubiu or cubíu fruit coming from the Amazon region of Brazil, considered the "Amazonian apple". This fruit is popularly used as a medicine to control the itching of the skin and reducing cholesterol levels, glucose and uric acid in the blood. Once S. sessiliflorum studies are still incomplete, the aim of this study was to chemically characterize the extract and perform tests to determine their prospecting potential antioxidant, antitumoral and effect in vitro on preventing the oxidation of LDL. For this, the study was conducted in two stages, the first stage was prepared hydroalcoholic extract of the peel and seed and pulp. After preparation of the extract, the chemical characterization was conducted by the same determination of total bioactive compounds (flavonoids, tannins and polyphenols), then the extract was chemically characterized by high performance liquid chromatography (HPLC). In the second step, we evaluated the antioxidant potential and in citoprotetor in and in PBMC (Peripheral Blood Mononuclear Cells) and human plasma exposed to hydrogen peroxide (H2O2). We also evaluated the antitumor activity in lines of breast (MCF7) and colorectal (HT29) cancer cell cancer, assessing cell proliferation and viability. After this, the effect has been evalueted on the oxidation of LDL (isolated from healthy individuals). The results obtained indicate that the extract of Solanum sessiliflorum presents in its composition total bioactive compounds such as flavonoids, tannins and polyphenols. These results corroborate the analysis performed by HPLC which indicated that the extracts present in its composition gallic acid, caffeic acid, quercetin, rutin, β carotene and catechin (the latter except in the pulp and seed extract). When examined the in vitro antioxidant effect was observed that extracts at different concentrations did not show any pro-oxidant potential. However extract the pulp and the seeds had antioxidant and cytoprotective activity. It was also observed that the extract of the pulp and the seeds had antigenotoxic and specific antitumor activity for colorectal cancer cells (in vitro tests performed with strain HT29 cells), and no antitumor activity was observed for breast cancer cells (MCF7), instead of the expected high concentrations in the extract increased the viability and proliferation. Another important finding was that both extracts, seeds and pulp and peel showed potential for inhibiting oxidation of LDL. In synthesis, the in vitro results obtained data indicate that S. sessiliflorum has antioxidant, antitumor and citoprotetor activity and present potential to inhibit the oxidation of LDL. These results should be proof of the presence of bioactive compounds in its composition as flavonoids, polyphenols and catechins. In literature there are several studies that report that eating foods rich in these types of compounds are related to the prevention of several chronic diseases, such as cancer and cardiovascular diseases. / Estudos epidemiológicos têm sugerido que a ingestão de frutas e verduras atuam na prevenção de diversas patologias, tais como o câncer e doenças cardiovasculares. Isto pode ser explicado por estes alimentos apresentarem compostos bioativos na sua matriz nutricional que podem ter diversas atividades biológicas, incluindo atividade antioxidante, antitumoral, genoprotetor e na prevenção da oxidação do LDL. Entretanto, uma grande parte dos frutos ainda precisa ser estudada. Este é o caso do Solanum sessiliflorum popularmente conhecido como mana-cubiu ou cubíu fruto oriundo da região amazônica do Brasil, considerado a maçã amazônica que é popularmente utilizado como medicamento no controle do prurido da pele e na redução dos níveis de colesterol, glicose e ácido úrico no sangue. Uma vez que estudos sobre o S. sessiliflorum são ainda incipientes, o objetivo deste estudo foi caracterizar quimicamente o extrato de S. sessiliflorum e realizar ensaios de prospecção para determinar seu potencial efeito antioxidante, antitumoral e na prevenção da oxidação do colesterol LDL através de ensaios in vitro. Para isto, o estudo foi conduzido em duas etapas, na primeira etapa foram preparados dois extratos hidroalcóolicos 1) da casca e 2) da semente e polpa do S. sessiliflorum. Após preparo do extrato, foi conduzida a caracterização química do mesmo através da determinação de compostos bioativos totais (flavonoides, taninos e polifenóis), em seguida, o extrato foi caracterizado quimicamente por cromatografia líquida de alta eficiência (CLAE). Na segunda etapa, foi avaliado o potencial antioxidante e citoprotetor em plasma humano exposto ao peróxido de hidrogênio (H2O2) e também em PBMCs (Peripheral Blood Mononuclear Cells). Também foi verificada a atividade antitumoral em linhagens de célula de câncer de mama (MCF7) e de câncer de colorretal (HT29), avaliando a proliferação e a viabilidade celular. Após, foi avaliado o efeito na oxidação do LDL (isolado de indivíduos saudáveis). Os resultados obtidos indicam que o extrato de Solanum sessiliflorum apresenta em sua composição compostos bioativos totais como os flavonóides, taninos e polifenóis. Estes resultados corroboram com as análises realizadas por CLAE que indicaram que os extratos apresentam em sua composição ácido gálico, ácido caféico, quercetina, rutina, β-caroteno e catequina (este último exceto no extrato de polpa e semente). Quando analisado o efeito antioxidante in vitro foi observado que os extratos nas concentrações testadas não apresentaram potencial pró-oxidante. No entanto o extrato da polpa e da semente apresentou atividade antioxidante e citoprotetora. Também foi observado que o extrato da polpa e da semente apresentou atividade antigenotóxica e antitumoral específica para células de câncer de colorretal (teste in vitro realizado com linhagem de células HT29), e não foi observado atividade antitumoral em relação à linhagem de células de câncer de mama (MCF7), pelo contrário do esperado, em altas concentrações o extrato aumentou a viabilidade e a proliferação celular. Outro importante achado foi que ambos os extratos, semente e polpa e casca apresentaram potencial de inibição da oxidação do LDL. Em síntese, os dados obtidos nos experimentos in vitro indicam que o S. sessiliflorum apresenta atividade antioxidante, antitumoral, antigenotóxica e potencial de inibir a oxidação do LDL. Estes resultados devem-se a presença de compostos bioativos em sua composição como flavonóides, polifenóis e catequina. Na literatura há diversos estudos que relatam que a ingestão de alimentos ricos nestes tipos de compostos está relacionada à prevenção de diversas doenças crônicas, como o câncer e doenças cardiovasculares.

Abstract Atherosclerosis is a multifactorial disease with numerous genes and environmental factors affecting its intiation and progression. During the past years many candidate genes for atherosclerosis have been suggested, and it has also become evident that the immune system plays a part in atherogenesis. Early atherosclerotic changes can be effectively detected by measuring carotid artery intima-media thickness (IMT). In the present study the associations between IMT and polymorphisms of three candidate genes for atherosclerosis were studied: endothelial nitric oxide synthase (eNOS), apolipoprotein E (apoE) and paraoxonase-1 (PON1). To assess the role of immunological factors determining carotid atherosclerosis, CRP and circulating autoantibodies to oxidised LDL were studied in relation to IMT. The study population consisted of 519 hypertensive and 526 control subjects from a middle-aged population in Oulu, Finland. The results showed that the investigated polymorphisms of eNOS and PON1 genes were not associated with IMT, suggesting that these polymorphisms are not major risk factors for atherosclerosis in the general Caucasian population. A significant interaction between the apoE polymorphism and smoking in relation to IMT was observed among men, indicating that carriers of the ε4 allele may be particularly prone to the atherogenic effects of smoking. This interaction was especially clear in hypertensive subjects. CRP was strongly associated with IMT before adjusting for confounding factors. After the adjustment, this association diasppeared. The finding suggests that instead of early atherosclerosis CRP may be related to the later phases of the disease. This may partly explain the strong correlation between CRP and future cardiovascular events. IgM type of autoantibodies binding to oxidised LDL were inversely associated with IMT, and this finding remained after adjusting for previously known risk factors for atherosclerosis, implying a possible protective role for these antibodies in atherogenesis.

This study investigated whether oxLDL and/or angiotensin (Ang) II signaling pathways mediate traffic-generated air pollution- exposure induced alterations in blood-brain barrier (BBB) integrity and permeability in a healthy wild type (C57Bl/6) mouse model; additionally, whether these outcomes are exacerbated by a high fat-diet investigated. An environmentally relevant concentration of a mixture of vehicle engine exhaust (MVE) was used. To investigate the hypotheses, 12 wk old male C57Bl/6 mice on either a high fat (HF) or low fat (LF) diet were randomly assigned to inhalational exposure of either filtered-air (FA) or 30 µg PM/m3 diesel exhaust + 70 µg PM/m3 gasoline exhaust (MVE) for 6 hr/day for 30 days. Additionally, we examined mechanisms involved in MVE-mediated alterations BBB integrity using a novel BBB co-culture in vitro model, consisting of mouse primary cerebral vascular endothelial cells on an apical transwell and astrocytes in the basal compartment, which was treated with plasma from the mice on our exposure study. Our in vivo exposure study results showed that MVE inhalation resulted in increased circulating plasma oxLDL and Ang II, compared to FA controls. Additionally, we observed increased cerebral microvascular expression of oxLDL receptors, LOX-1 and CD-36, and Ang II receptor subtype 1 (AT1) in MVE-exposed C57Bl/6 mice, which was further exacerbated with consumption of an HF diet. Increased signaling of both Ang II and oxLDL was associated with decreased BBB integrity, as evidenced by the concurrent reduction in expression of tight junction (TJ) protein claudin-5 and increased permeability of sodium fluorescein (Na-F) from the blood into the cerebral parenchyma. Our results suggest that possible mechanisms involved in oxLDL and/or Ang II-mediated alterations in BBB integrity include oxidative stress and upregulated expression and activity of matrix metalloproteinase (MMP)-9, which is associated with degradation of TJ proteins in the BBB. Our in vitro BBB co-culture results confirm our in vivo findings, as we observe increased BBB permeability (TEER) and decreased integrity (decreased expression of TJ proteins) in the endothelial (apical) layer when treated with plasma from MVE-exposed mice, which was further exacerbated when treated with plasma from MVE-exposed mice on an HF diet. Pre-treatment of the endothelial cells with the AT1 receptor antagonist, Losartan, prior to applying plasma, resulted in attenuation of the alterations observed in endothelial integrity in the BBB co-culture treated with plasma from either MVE+LF or MVE+HF animals. These results suggest Ang II – AT1 signaling mediate, at least in part, the alterations in the BBB integrity observed after exposure to MVE. Moreover, we observed that treatment of the endothelial (apical) layer with plasma from MVE-exposed animals resulted in increased production of inflammatory mediators interleukin-6 (IL-6) and transforming growth factor-β in the astrocyte media (basal compartment). Additionally, these same astrocytes also displayed increased production of angiotensin-converting enzyme (ACE) and also AT1 receptor mRNA expression, while showing decreased expression of the aryl hydrocarbon receptor (AhR) and glutathione peroxidase (GPx). Collectively, these results suggest that exposure to the ubiquitous environmental air pollutant, vehicle engine emissions, results in increased oxLDL and Ang II signaling in the cerebral microvasculature, which is associated with decreased vessel integrity and increased oxidative stress and inflammatory signaling in the CNS. The observed detrimental outcomes are even further exacerbated when coupled with the consumption of an HF diet.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-06-14T19:33:16Z No. of bitstreams: 1 alicebelleigolirezende.pdf: 4554252 bytes, checksum: 45624650ab925d7640e01b2b01e2a245 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-06-29T12:16:33Z (GMT) No. of bitstreams: 1 alicebelleigolirezende.pdf: 4554252 bytes, checksum: 45624650ab925d7640e01b2b01e2a245 (MD5) / Made available in DSpace on 2017-06-29T12:16:33Z (GMT). No. of bitstreams: 1 alicebelleigolirezende.pdf: 4554252 bytes, checksum: 45624650ab925d7640e01b2b01e2a245 (MD5) Previous issue date: 2011-08-30 / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / A aterosclerose é uma doença imune-inflamatória associada ao acúmulo de lípides na camada íntima das artérias. O baço desempenha uma importante função imunológica, mas a sua participação no desenvolvimento da aterosclerose permanece controversa. Considerando que a incidência de esplenectomias totais ainda é alta e que a doença aterosclerótica é prevalente, é de grande importância que se verifique se o tecido esplênico influencia no processo de aterosclerose. Neste trabalho, o impacto da esplenectomia no desenvolvimento da aterosclerose foi avaliado em camundongos deficientes em apoproteína E (apoE). Os animais foram divididos em dois grupos: grupo controle (CT), composto de camundongos deficientes em apoE submetidos a um procedimento cirúrgico simulado; e grupo esplenectomia total (ET), camundongos deficientes em apoE submetidos à esplenectomia total. Trinta dias após a cirurgia, os animais foram submetidos a uma dieta aterogênica enriquecida com 0,15% de colesterol. Após oito semanas, os animais foram eutanasiados e o sangue, o coração e a aorta foram submetidos à análise. Os níveis séricos de colesterol total e de anticorpos anti-LDL oxidada foram avaliados. As áreas de lesão aterosclerótica na raiz da aorta foram quantificadas por morfometria em cortes histológicos corados com hematoxilina-eosina. As lesões ateroscleróticas nas porções torácica e abdominal das aortas foram determinadas pela porcentagem da superfície luminal corada com Sudan IV em relação à área total do vaso. Os resultados mostraram que os níveis séricos de colesterol total não foram alterados pela esplenectomia e que os títulos de anticorpos IgG anti-LDL oxidada foram semelhantes entre os grupos. No entanto, foi observada uma maior porcentagem de lesões ateroscleróticas nas porções torácica e abdominal das aortas de animais esplenectomizados. Além disso, camundongos esplenectomizados apresentaram lesões ateroscleróticas na raiz da aorta significativamente maiores do que os animais controles. Estes dados indicam, em conjunto, que a esplenectomia está associada ao aumento das lesões ateroscleróticas em camundongos deficientes em apoE submetidos à dieta aterogênica, sugerindo um papel ateroprotetor do baço no modelo estudado. / Atherosclerosis is an inflammatory immune disease associated with lipid accumulation in the intima layer of arteries. The spleen plays an important immune function, but its influence in development of atherosclerosis remains unclear. Evaluation of the role of the spleen in atherosclerosis is justified due to the high frequency of total splenectomies and the high prevalence of atherosclerotic disease. In this work, the effect of splenectomy on the development of atherosclerosis in apolipoprotein E (apoE) deficient mice was investigated. ApoE deficient mice were divided into a sham-operated control group (CT) and a splenectomized group (SP). Thirty days after surgery, animals were fed a high fat western diet (with 0.15% cholesterol). After eight weeks, mice were euthanized and their blood, heart, and aorta were subjected to analysis. Total serum cholesterol and anti-oxidized LDL antibodies were measured. Atherosclerotic lesion areas in the aortic root were stained with hematoxylin-eosin and quantified by morfometry. The atherosclerotic lesions in the thoracic and abdominal portions of aorta were determined by assessing the percentage of the luminal surface area stained by Sudan IV. Levels of total serum cholesterol did not vary significantly after splenectomy. Anti-oxidized LDL IgG antibodies were similar between groups. However, compared to the control group, atherosclerotic area in the thoracic and abdominal portions of aorta were significantly increased in splenectomized mice. These data were confirmed by the larger lesions in the aortic root in splenectomized mice. These data indicate that splenectomy increases atherosclerotic lesions in apoE deficient mice fed an atherogenic diet, suggesting a atheroprotector role of the spleen.

Cardiovascular disease is the leading cause of mortality in the western world. The pro-inflammatory and pro-proliferative etiology of vascular proliferative diseases is well characterized, while much less is known about the mechanisms of anti-inflammatory and anti-proliferative processes. Interleukin-19 (IL-19) is a newly described member of the IL-10 family of anti-inflammatory interleukins, and our group was the first to discover IL-19 expression in activated, synthetic, but not quiescent, contractile human vascular smooth muscle cells (hVSMC). We also found that IL-19 is anti-inflammatory and anti-proliferative for hVSMC. IL-19 is able to reduce the abundance of COX-2, IL-1β, IL-8, and Cyclin D1 transcripts which contain AU-rich elements (ARE) in their 3'-untranslated regions (3'-UTR). IL-19 is able to reduce the abundance of HuR, a stabilizing RNA-binding protein, which we feel provides a mechanism for these effects. The overall goal of this study is to elucidate IL-19's anti-inflammatory and anti-proliferative mechanism(s) in hVSMC in the context of vascular proliferative diseases. This goal has directed our overall hypothesis: IL-19's anti-proliferative and anti-inflammatory effects in hVSMC are mediated, at least in part, by modulation of HuR abundance and translocation, resulting in decreased stability of mRNA transcripts. HuR functions through a translocation mechanism, and IL-19 is able to reduce HuR cytoplasmic abundance. IL-19 also reduces HuR phosphorylation, which is a pre-requisite for HuR translocation, possibly through a PKCα-dependent mechanism. The stability of ARE-containing transcripts is reduced with IL-19 treatment, and reducing HuR expression by siRNA has the same inhibitory effect. VSMC are important mediators in the initiation of atherosclerosis. Oxidized low-density lipoprotein (ox-LDL) is able to induce IL-19 expression in these cells. VSMC are known to express scavenger receptors that take up ox-LDL. IL-19 is able to reduce the uptake of ox-LDL and the abundance of ox-LDL induced LOX-1 and CX-CL16 scavenger receptors. Interestingly, these scavenger receptors also have ARE in their 3'-UTR. IL-19 is able to reduce ox-LDL induced HuR cytoplasmic abundance. HuR knockdown by siRNA reduces the uptake of ox-LDL by hVSMC. These data suggest that IL-19 reduced scavenger receptor abundance may be due to decreased total and cytoplasmic HuR abundance. IL-19 reduces the abundance of ox-LDL induced COX-2 expression. Taken together, these results demonstrate that IL-19 down-regulates vital steps in vascular proliferative disease processes through an HuR-dependent mechanism. / Molecular and Cellular Physiology