A hipercolesterolemia familiar (HF) é uma doença autossômica dominante caracterizada por níveis elevados de LDL-c e doença arterial coronária (DAC) precoce. Existem evidências de maior prevalência de aterosclerose subclínica nesta população avaliada pelo escore de cálcio (CAC) e pela espessura íntima-média carotídea (EIMC). O objetivo do nosso estudo foi avaliar aterosclerose subclínica por meio da angiotomografia de coronárias em portadores de HF sem aterosclerose manifesta, correlacionando os achados com parâmetros clínicos, laboratoriais, rigidez aórtica e carotídea e com a EIMC. Incluímos 102 HFs, (45±13 anos, 36% homens, LDL-c 280±54mg/dL) e 35 controles (46±12 anos, 40% homens, LDL-c 103±18mg/dL). O grupo HF apresentava maior carga de placa aterosclerótica representado por: maior número de pacientes com placa (48% versus 14%, p=0,0005), maior número de pacientes com estenose luminal acima de 50% (19% versus 3%, p=0,015), maior número total de segmentos com placas (2,0±2,8 versus 0,4±1,3, p=0,0016), maior número de segmentos com placas calcificadas (0,8±1,54 versus 0,11±0,67, p= 0,0044) e maior escore de cálcio pelo método de Agatston (55±129, mediana:0 versus 38±140, mediana:0; p=0,0028). Houve correlação positiva no grupo HF do número total de segmentos com placa com: idade (r=0,41, p<0,0001), escore de risco de Framingham (r=0,25, p=0,012), colesterol total (r=0,36, p<0,0002), LDL-c (r=0,27, p=0,005), HDL-c (r=0,24, p=0,017), apolipoproteína B (r=0,3, p=0,0032) e escore de cálcio (r=0,93, p<0,0001). Além disso, houve correlação negativa com: variação sísto-diastólica carotídea (r=-0,23, p=0,028) e percentual de distensão carotídeo (r=-0,24, p=0,014). A análise multivariada de determinantes da presença de placa aterosclerótica, revelou que idade (OR=1,105, IC95%: 1,049-1,164, p<0,001) e colesterol total (OR=1,013, IC95%:1,001-1,025) foram as variáveis associadas com a presença da mesma. A única variável associada com presença de obstrução luminal acima de 50% foi o escore de cálcio coronário (OR=1,004; IC95%:1,001-1,008; p=0,014). Em relação a determinantes da composição de placa, na análise multivariada a presença de placa não calcificada esteve associada com o sexo masculino (OR:15,45; IC95%: 1,72-138,23, p=0,014), a placa mista com antecedente familiar de DAC precoce (OR=4,90; IC95%:1,32-18,21, p=0,018) e placa calcificada a menor chance com o sexo masculino (OR=0,21; IC95%: 0,05-0,84, p=0,027). Conclusões: Os pacientes portadores de HF apresentam maior carga de placa avaliada pela angiotomografia em comparação aos controles; idade e colesterol total associaram-se a presença de placas no grupo HF; o escore de cálcio associou-se a presença de estenose luminal acima de 50%. / Familial hypercholesterolemia (FH) is an autosomal dominant disease characterized by high LDL-c levels and premature coronary artery disease (CAD) onset. There is evidence of greater prevalence of subclinical atherosclerosis in this population evaluated by coronary calcium score (CCS) and carotid intima-media thickness (IMT). The aim of our study was to assess subclinical atherosclerosis by computed tomography coronary angiography (CTCA) in patients with FH without manifest atherosclerosis and correlate the findings with clinical and laboratory parameters, aortic and carotid stiffness and IMT. We included 102 FHs (45 ± 13 years, 36% men, LDL-c 280 ± 54mg/dL) and 35 controls (46 ± 12 years, 40% men, LDL-c 103 ± 18mg/dL). The FH group had a greater atherosclerosis plaque burden represented by: higher number of patients with coronary plaque (48% versus 14%, p = 0.0005) and with luminal stenosis greater than 50% (19% versus 3% p = 0.015), higher total number of segments with plaques (2.0 ± 2.8 versus 0.4 ± 1.3, p = 0.0016), higher number of segments with calcified plaques (0.8 ± 1.54 versus 0.11 ± 0.67, p = 0.0044) and higher CCS by the Agatston method (55 ± 129, median: 0 vs. 38 ± 140, median = 0, p = 0.0028). There were positive correlations of total number of segments with plaque in FH group with the following variables: age (r=0.41, p<0.0001), Framingham risk score (r =0.25, p=0.012), total cholesterol (r=0.36, p<0.0002), LDL-c (r=0.27, p=0.005), HDL-c (r=0.24, p=0.017), apolipoprotein B (r=0,3, p=0.0032) and CCS (r=0.93, p<0.0001). In addition there was a negative correlation with: carotid systo-diastolic variation (r=- 0.23, p=0.028) and percentage of carotid distension (r=- 0.24, p=0.014). After multivariate analysis, the determinants of plaque presence were age (OR=1.105, 95% CI=1.049-1.164, p<0.001) and total cholesterol (OR=1.013, 95% CI:1.001-1.025). The only variable associated with presence of luminal stenosis greater than 50% was CCS (OR = 1.004, 95% CI: 1.001-1.008, p=0.014). After multivariate analysis, the presence of non-calcified plaque was associated with male gender (OR: 15.45, 95% CI 1.72-138.23, p = 0.014), mixed plaque with family history of early CAD (OR = 4.90, 95%:1.32-18.21, p=0.018) and calcified plaque negatively with males (OR = 0.21, 95% CI: 0.05-0.84, p = 0.027). Conclusions: FH subjects have higher plaque burden assessed by CTCA compared to controls; age and total cholesterol were associated with the presence of coronary plaque in the FH subjects; CCS was associated with luminal stenosis greater than50%.

A hipercolesterolemia familiar (HF) é uma alteração de origem genética comum que pode se manifestar clinicamente desde o nascimento e provoca um aumento nos níveis plasmáticos de LDL-colesterol (LDL-c), xantomas e doença coronária prematura. Sua detecção e tratamento precoce reduzem a morbidade e mortalidade coronária. A identificação e rastreamento em cascata familiar usando níveis de LDL-c e detecção genética é a estratégia mais aconselhável e rentável para descoberta de novos casos. O tratamento crônico com estatinas reduz o risco cardiovascular da população em geral, contudo, estudos clínicos com estatinas revelam risco cardiovascular residual mesmo após correção das concentrações de LDL-c. Com o surgimento de novas drogas e mais recentemente um inibidor da enzima pró-proteína convertase subtilisina/kexina tipo 9 (PCSK9), este estudo enfatizou na investigação específica para aqueles acometidos com defeitos genéticos nessa enzima, por ser de frequência ainda mais rara e pouco estudada, necessitando de melhor investigação na população em estudo a fim de rastrear a ocorrência de mutações patológicas na PCSK9. O objetivo desse estudo foi identificar e caracterizar mutações e/ou deleções patológicas no gene PCSK9 em pacientes com Hipercolesterolemia Familiar provenientes do Hospital das Clínicas de Ribeirão Preto da FMRP/USP selecionados para o teste genético. Foi feito o rastreamento de mutações pelo método Hight Resolution Melting (HRM), de forma prática, rápida e eficiente, onde mutações detectadas foram seqüenciadas. Foram identificadas 7 mutações não patogênicas, caracterizando que a população estudada não apresenta Hipercolesterolemia Familiar associada a mutações no gene PCSK9, fato que não exclui o diagnóstico por outros defeitos genéticas associados a doença. / Familial hypercholesterolemia (FH) is an alteration of common genetic origin that can manifest clinically from birth and which causes an increase in the LDL-cholesterol plasma levels (LDL-c), xanthomas and premature coronary disease. Its early detection and treatment reduce morbidity and coronary mortality. The identification and tracking in familial cascade using levels of LDL-c and genetic detection is the most advisable and profitable strategy to find new cases. The chronic treatment with statins reduces the cardiovascular risk in the population in general. However, clinic studies on statins show a residual cardiovascular risk even after the correction of LDL-c concentrations. With the appearance of new drugs and, more recently, of a proprotein convertase subtilisin/kexin type 9 enzyme inhibitor (PCSK9), this study highlighted the specific investigation for those stricken by genetic defects in this enzyme, once it is even rarer and understudied and needs further investigation in the study\'s population aiming at tracking the occurrence of a pathological mutation in the PCSK9. This study aimed at identifying and characterizing mutations and/or pathological deletions in the PCSK9 gene in patients with Familial Hypercholesterolemia from the RPMS/USP Ribeirão Preto Clinical Hospital which were selected for the genetic test. We performed the mutation tracking by using the High Resolution Melting (HRM) method in a practical, fast and efficient way, where the mutations detected were sequenced. We identified 7 non-pathogenic mutations, showing that the population studied does not present Familial Hypercholesterolemia associated to mutations in the PCSK9 gene, which doesn\'t exclude the diagnosis by other genetic defects associated to the disease.

A total of 146 Chinese patients with various degrees of hyperlipidaemia and high cardiovascular risk, suitable for treatment with rosuvastatin 10 mg daily and in whom it was possible to obtain baseline lipid profiles measured on no lipid lowering drug, were enrolled in to the study. The drug compliance was assessed by personal interview and 9 patients were excluded from the efficacy analysis because they stated their compliance was less than 80%. From the remaining 137 subjects, 62 had a clinical diagnosis of familial hypercholesterolaemia. Data for dietary intake were available in 121 of the 137 subjects. The average reduction in LDL-cholesterol in these subjects was 48.8 +/- 12.8% and as anticipated there was a wide range between individuals. The percentage reductions in LDL-cholesterol were significantly greater in the female than in the male subjects (-51.35 +/-10.89% vs. -46.38 +/-13.96%; p = 0.025), but this was no longer significant after adjustment for body weight. In patients with familial hypercholesterolaemia the absolute reductions in total cholesterol and LDL-cholesterol were significantly greater (p&lt;0.001) than in those without familial hypercholesterolaemia, but the percentage reductions were not significantly different in the two groups. The increases in HDL-cholesterol and the decreases in triglycerides were significantly greater in the subjects with familial hypercholesterolaemia than in those without familial hypercholesterolaemia, both for the absolute changes and for the percentage changes. There were no significant effects on the percentage changes in lipids with rosuvastatin treatment due to age, measurements of body fatness, smoking or alcohol drinking status, or having hypertension or diabetes. / Polymorphisms in the CYP2D6 gene were analyzed and the subjects were divided into 4 groups as wild-type or extensive metabolisers, heterozygotes for CYP2D6*10 and wild-type, homozygotes for CYP2D6*10, and subjects with one allele for poor metaboliser status. The groups in this order would be expected to have decreasing activity of the CYP2D6 enzyme. There was a tendency for greater reduction in LDL-cholesterol in groups with lower CYP2D6 activity, most obvious in male subjects and this was significant in the patients with familial hypercholesterolaemia comparing the first 3 groups. The fourth group had a low number of subjects, which may have biased that result. In the subjects without familial hypercholesterolaemia, the % change in LDL-cholesterol was similar in all genotype groups, but the % reduction in triglycerides was numerically higher in the wild-type group than in groups with CYP2D6*10 alleles and the group with poor metaboliser status showed a lower % reduction. These differences were not significant and may be influenced by the baseline levels of triglycerides, which were not corrected for in this analysis. / The daily calorie intake and percentage of different macronutrient intake was obtained by using seven days food recall records. Dietary intake of most nutrients with higher in male than in female patients and was higher in the patients compared to gender-matched population data. Higher intake of most nutrients was associated with higher baseline triglyceride levels, but not LDL-cholesterol levels in all patients, and in lower HDL-cholesterol levels in the patients without familial hypercholesterolaemia. Higher intake of total calories was associated with less percentage reduction in LDL-cholesterol with rosuvastatin in the patients without familial hypercholesterolaemia and a similar non-significant tendency was seen with higher intake of total fat, saturated fat and cholesterol. / The study described in this thesis examined the role of the CYP2D6*10 polymorphism on the lipid response to rosuvastatin in addition to a number of phenotypic factors such as diet, gender, measures of obesity and other medical conditions. / These findings suggest that the CYP2D6 genotype may have some influence on the lipid response to rosuvastatin, but it appears to interact with other factors including, gender, diet and the presence of familial hypercholesterolaemia. (Abstract shortened by UMI.) / Lui, Siu Hung. / "February 2007." / Adviser: Brian Tomlinson. / Source: Dissertation Abstracts International, Volume: 69-01, Section: B, page: 0248. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 165-190). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

A hipercolesterolemia familiar (HF) é uma doença de herança genética autossômica dominante caracterizada pela elevação dos níveis séricos de colesterol total e das lipoproteínas de baixa densidade (LDL- c). Conhecida por estar estreitamente relacionada ao processo aterosclerótico, a HF pode determinar o desenvolvimento de lesões obstrutivas precoces em distintos leitos arteriais. Nesse contexto, a HF também tem sido proposta como um fator de risco para a doença arterial periférica (DAP). Avaliamos assim de forma sistemática por meio de um estudo transversal e observacional, a prevalência de DAP em uma população brasileira de portadores de HF. Estudamos também sua associação com diversos fatores de risco cardiovascular, incluindo sexo, idade, hipertensão arterial sistêmica, diabetes mellitus, tabagismo, perfil lipídico, níveis séricos de glicemia e creatinina. Avaliou-se também a associação da DAP com histórico prévio de doença cardiovascular (DCV) bem como sua associação com marcadores de aterosclerose subclínica representados pela angiotomografia coronariana e escore de cálcio coronário. Foram estudados 212 portadores de HF, sendo que em 86% foi comprovada presença de mutação do receptor da LDL e um grupo de comparação composto por 524 indivíduos normolipidêmicos. O rastreamento da DAP foi realizado por dois avaliadores treinados, pela medida do índice tornozelo-braquial (ITB) avaliado em repouso na posição supina, com Doppler vascular portátil. Houve maior prevalência de DAP definida por ITB <= 0,90 em portadores de HF comparados aos controles (17,5% vs. 2,3%, respectivamente; p < 0,001). As variáveis que se associaram independentemente com a alteração dos valores do ITB nos grupos estudados foram, a idade, antecedente prévio de doença cardiovascular e o indivíduo ser portador de HF (OR= 5,77 IC 95% 2,83-11,77, p < 0,001). Na população de HF as variáveis que se associaram independentemente à alteração dos valores de ITB foram a idade e a presença de histórico de tabagismo ativo ou passado. Houve uma associação univariada entre o histórico de doença cardiovascular e o diagnóstico de doença arterial periférica nesta população (OR= 3,20 IC 95% 1,53-6,67, p=0,001), porém tal associação não se manteve significativa quando ajustada por variáveis de confusão. Da mesma forma não se encontrou associação entre os valores alterados de ITB e a presença de placa coronariana e sua gravidade, bem como com o escore de cálcio coronário. Os dados sugerem dissociação entre o desenvolvimento da aterosclerose em diferentes leitos arteriais .Em conclusão, nossos resultados indicam que a DAP é mais frequente na HF do que em indivíduos normolipidêmicos e que outros fatores de risco potencializam o colesterol para sua presença. Não foi encontrada associação independente da alteração do ITB com manifestação de DCV prévia e com a aterosclerose coronária subclínica. Contudo, mais estudos são necessários para determinar o papel do uso do ITB como ferramenta para avaliação do risco de eventos cardiovasculares nessa população / Familial hypercholesterolemia (FH) is a genetic disease of autosomal dominant inheritance characterized by elevated serum levels of total and low density lipoprotein ( LDL - c ) cholesterol. FH is associated to atherosclerosis and can determine the early development of obstructive lesions in different arterial beds. In this context, FH has also been proposed as a risk factor for peripheral arterial disease (PAD). In a cross-sectional observational study the prevalence of PAD in a Brazilian population of patients with FH was determined . We also study its association with several cardiovascular risk factors, including gender, age , hypertension , diabetes mellitus , smoking , lipid profile , serum glucose and creatinine. The association of PAD with previous manifestations of cardiovascular disease (CVD) and with markers of subclinical coronary atherosclerosis detected by computed tomography coronary angiography and coronary calcium score was also evaluated. We studied 212 patients with FH, of which 86% had a confirmed diagnosis by the presence of LDL receptor mutations, and a comparison group consisting of 524 normolipidemic subjects . PAD diagnosis was made by 2 trained evaluators, by the ankle-brachial index ( ABI ) measured at rest in the supine position. There was a higher prevalence of PAD defined as ABI <= 0.90 in patients with HF compared with controls (17.5 % vs . 2.3% , p < 0.001 ) . The variables that were independently associated with altered ABI values in both groups were age, previous history of CVD and the diagnosis of FH (OR = 5.77 95% CI 2.83 to 11.77 , p < 0.001). In FH subjects variables independently associated with altered ABI values were age and the presence of current or past smoking history. There was a univariate association between CVD history and the diagnosis of PAD in this population (OR = 3.20 95% CI 1.53 to 6.67 , p = 0.001), but this association did not remain significant when adjusted for confounders . Likewise, no association was found between the values of altered ABI and the presence of coronary plaque and its severity, as well with the coronary calcium score. The data suggest that there is a dissociation of atherosclerosis development in different arterial beds. In conclusion, our results indicate that PAD is more common in FH than in normolipidemic subjects and that other risk factors potentiate cholesterol to determine its presence. No independent association was found between the alteration of ABI values with manifestations of prior CVD, as well as with the presence of subclinical coronary atherosclerosis. More studies are needed to determine the role of ABI use as a tool for assessing the risk of cardiovascular events in FH

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Estudos em animais experimentais evidenciaram associações significativas entre esquistossomose mansoni e hipercolesterolemia. Estudos in vitro e in vivo já demonstraram que o colesterol é essencial para Schistosoma mansoni, embora este não tenha capacidade de sintetizá-lo. A captação é realizada a partir do ambiente (cultivo ou hospedeiro) através do tegumento. O colesterol está envolvido nos mecanismos de evasão do helminto contra a resposta imunológica, além de poder participar na modulação da sinalização celular e reprodução, estimulando os órgãos reprodutores dos helmintos adultos como observado na fase aguda da infecção experimental. Este trabalho tem como objetivo avaliar se o mesmo fenômeno ocorre na fase crônica. Os helmintos foram recuperados de dez camundongos submetidos à dieta hiperlipídica ou padrão (controle) foram corados pelo carmin cloridrico e montados, individualmente, em lâmina histológica com bálsamo do Canadá. A preparação foi analisada por microscopia de campo claro nos seguintes caracteres: tegumento e o sistema reprodutor nos vermes machos (lobos testiculares, vesícula seminal, lobos testiculares supranumerários e canal ginecóforo) e, nas fêmeas (ovário, oótipo, útero, ovo, glândulas vitelínicas e espermateca). Posteriormente, algumas lâminas foram separadas para visualização pela microscopia confocal dos órgãos do sistema reprodutores acima descritos. Apesar de ter sido observado uma maior quantidade de espermatozoides, uma maior quantidade de oócitos sendo liberados no grupo da dieta, não houve diferença estatística significativa (p>0,05) entre os grupos analisados. Houve um aumento na oogênese como observado na fase aguda. Dessa forma, o colesterol pode estar relacionado com a estimulação na atividade dos órgãos reprodutores dos helmintos adultos na fase crônica da infecção. / Studies in experimental animals showed significant associations between with schistosomiasis and hypercholesterolemia. In vitro and in vivo studies have demonstrated that cholesterol is essential for Schistosoma mansoni, although this is not able to synthesize it. The capture is carried out from the environment (cultivation or host) through the tegument. The capture is carried out from the middle (cultivation or host) through the tegument. Cholesterol is involved in the helminth evasion mechanisms against the immune response, and can participate in the modulation of cell signaling and reproduction of worms by stimulating the reproductive organs of adult worms as observed in the acute phase of experimental infection. This study aims to evaluate whether the same phenomenon occurs in the chronic phase. Helminthes recovered from ten mice subjected to high fat diet or standard (control) were stained with hydrochloric carmine and mounted individually on histological slide with Canada balsam. The preparation was analyzed by bright field microscopy the following characteristics: oral sucker and ventral sucker, tubercles on tegument and the reproductive system in male worms (lobes testicular, seminal vesicles, supernumerary testicular lobes and gynaecophoric canal), and in females (ovary, ootype, uterus, egg, vitelline glands and spermatheca). Subsequently, some slides were separated for confocal microscopy for visualization of the organs of the reproductive system described above. Despite having been observed a higher amount of sperm, a larger number of oocytes are released in the diet group, there was no statistically significant difference (p> 0.05) between the groups. There was an increase in oogenesis as observed in the acute phase. Thus, cholesterol may be related to the stimulation of the activity of the reproductive organs of adult helminths in the chronic phase of infection.

Les maladies cardiovasculaires représentent l’une des principales causes de mortalitédans le monde et leur prévention constitue un enjeu majeur de santé publique. Les études épidémiologiques montrent que la consommation de fruits et légumes est associée à un moindre risque cardiovasculaire. Ces effets pourraient être expliqués par leur richesse en micronutriments et plus particulièrement en polyphénols. La consommation de flavanones, une classe de polyphénols présente spécifiquement et en grandes quantités dans les agrumes, a été inversement associée au risque d’accidents coronaires et d’accidents vasculaires cérébraux. Des données expérimentales obtenues in vivo et in vitro suggèrent que les flavanones (hespéridine ou naringine) possèdent des propriétés hypolipémiantes, hypotensives et anti-inflammatoires. Ces effets pourraient notamment être médiés par une modulation de l’expression de gènes par les flavanones. Cependant jusqu’à présent, l’action anti-athérogène des flavanones in vivo n’a été explorée qu’à des doses supra-nutritionnelles et les mécanismes responsables de ces effets sont encore largement méconnus. Par ailleurs, les données mécanistiques in vitro sont peu informatives, car obtenues avec des formes de flavanones non présentes dans l’organisme. Les objectifs de cette thèse ont donc été : (1) d’évaluer l’impact d’une consommation de flavanones (naringine et hespéridine) à dose nutritionnelle sur le développement de la lésion athéromateuse chez différents modèles murins d’hypercholestérolémie et d’appréhender les mécanismes moléculaires mis en jeu par une approche transcriptomique ; (2) de déterminer l’impact des métabolites circulants sur la fonction des cellules endothéliales et d’identifier les mécanismes moléculaires sous-jacents. In vivo, nous avons montré que seule la supplémentation en naringine (0,02%), la flavanone majoritaire du pamplemousse, réduisait la progression des lésions athéromateuses dans un modèle murin d’athérosclérose induite par l’alimentation (souris C57Bl/6J sous régime riche en graisse et en cholestérol). Cet effet était associé à une réduction des concentrations plasmatiques de non HDL-Chol et de biomarqueurs de la dysfonctionendothéliale, mais semblait indépendant d’une modulation du statut anti-oxydant. Une analyse transcriptomique pangénomique de l’aorte de ces animaux, a montré, que la supplémentation en naringine induisait une variation de l’expression de gènes impliqués dans des processus, tels que l’adhésion cellulaire ou l’organisation du cytosquelette, qui sont impliqués dans la migration trans-endothéliale des leucocytes. Ces résultats mettent en évidence de nouvelles cibles moléculaires d’action des flavanones qui ont été plus largement abordées in vitro. Les données obtenues in vitro montrent que l’exposition de cellules endothéliales(HUVEC) à des concentrations physiologiques de métabolites circulants de la naringénine(dérivés glucuronidés) ou de l’hespérétine (dérivés glucuronidés et sulfatés) diminue l’adhésion des monocytes (U937) aux cellules endothéliales, une étape clé de la migrationtrans-endothéliale. En accord avec ces résultats, une analyse TaqMan Low Density Array a montré que l’exposition des cellules endothéliales aux métabolites circulants de flavanones affectait l’expression de gènes impliqués dans l’inflammation, le chimiotactisme, l’adhésion ou encore la migration trans-endothéliale des leucocytes. Pris dans leur ensemble, les résultats de ce travail de thèse montrent que les effets antiathérogènesdes flavanones ne découlent pas exclusivement d’un effet hypolipémiant, mais aussi d’une action directe sur des étapes clés du développement de l’athérosclérose au niveau de l’endothélium, en particulier les processus d’adhésion et migration trans-endothéliale. / Cardiovascular disease is a major cause of mortality worldwide and its prevention is a major public health issue. Epidemiological studies have shown that fruit and vegetables consumption is associated with a lower cardiovascular risk. These effects could be explainedby their richness in micronutrients, especially polyphenols. Consumption of flavanones, a class of polyphenols present specifically and in large amounts in citrus fruits, was inversely associated with risk of coronary events and stroke. Experimental data obtained in vivo and invitro suggest that the flavanones (hesperidin or naringin) have lipid-lowering, hypotensive and anti-inflammatory properties. These effects could be mediated via modulation of gene expression by these bioactives. However, the anti-atherogenic effect of flavanones in vivo has been only explored at supra-nutritional doses and the mechanisms responsible for these effects are largely unknown. Furthermore, in vitro mechanistic data are questionable as they have been obtained using native forms of flavanones which are not present in the body.The objectives of this thesis were: (1) to assess the impact of consumption of flavanones (naringin and hesperidin) at nutritional doses on the development of atherosclerotic lesions indifferent mouse models of hypercholesterolemia and decipher molecular mechanisms brought into play, using a transcriptomic approach, (2) to determine the impact of physiological concentrations of plasma flavanone metabolites on endothelial cell function and theunderlying molecular mechanisms. In vivo, we showed that only naringin supplementation (0.02%), the major flavanonein grapefruit, reduced the progression of atherosclerotic lesions in a mouse model of dietinduced atherosclerosis (C57BL/6J mice fed an enriched diet in fat and cholesterol). Thiseffect was associated with a reduction in plasma non-HDL-Chol and biomarkers ofendothelial dysfunction, but appeared independent of a modulation of antioxidant status. Agenome-wide transcriptome analysis of the aorta of these animals showed that naringin supplementation modulated expression of genes involved in processes such as cell adhesion and cytoskeleton organization; these latter being involved in leukocytes trans-endothelialmigration. These results reveal new molecular targets of action of flavanones that have beenfurther studied in vitro.The in vitro data showed that exposure of endothelial cells (HUVEC) to physiological concentrations of circulating metabolites of naringenin (glucuronides) or hesperetin(glucuronides and sulfate) decreased the adhesion of monocytes (U937) to endothelial cells, a key step in the trans-endothelial migration. Consistent with these results, a TaqMan Low Density Array analysis showed that exposure of endothelial cells to these flavanone metabolites affected the expression of genes involved in inflammation, chemotaxis, adhesionas well as leukocytes trans-endothelial migration. Overall, the results of this work show that the anti-atherogenic effect of flavanones is not exclusively derived from lipid-lowering effect, but also due to a direct action on the endothelium by modulating key processes of atherosclerosis development, particularly adhesion and trans-endothelial migration.

La present tesi avalua els efectes de certs factors ambientals sobre la paraoxonasa1 (PON1), enzim antioxidant, possiblement protector enfront les malalties cardiovasculars, a través de dos estudis d'intervenció i un de transversal. En primer lloc, el tractament amb simvastatina dels pacients amb hipercolesterolèmia familiar, que presentaven una activitat paraoxonasa baixa, s'associava a un increment de l'activitat fins a valors similars als d'individus normolipèmics, independentment dels polimorfismes PON1-55 o PON1-192. En segon lloc, l'entrenament físic s'associava a un augment de l'activitat paraoxonasa en els individus QQ i una disminució de la mateixa en els portadors de l'al·lel R pel polimorfisme PON1-192. L'increment de l'activitat paraoxonasa immediatament després de l'exercici físic agut era seguit per una disminució subseqüent de l'activitat. La recuperació dels nivells basals d'activitat paraoxonasa a les 24h de l'exercici físic agut es donava en els individus QQ independentment del seu estat d'entrenament, i en els individus portadors de l'al·lel R només quan estan entrenats. En tercer lloc, el consum elevat d'àcid oleic comportava un augment de la concentració de c-HDL i de l'activitat paraoxonasa en els homes portadors dels genotips QR i RR del polimorfisme PON1-192, respectivament.Paraules claus: paraoxonasa, PON1, genotips, simvastatina, hipercolesterolèmia familiar, interacció gen-dieta, lipoproteïna d'alta densitat (HDL), exercici físic agut, entrenament físic, estrès oxidatiu, àcid oleic, oli d'oliva, peròxids lipídics, malaltia cardiovascular. / The present thesis evaluates some environmental factor effects on paraoxonase1 (PON1), an possibly protective against cardiovascular disease antioxidant enzyme, through two intervention studies and a cross-sectional one. First, treatment with simvastatin of the familial hypercholesterolemic patients, which had low paraoxonase activity, was associated with an increase in the activity to values similar to the normolipemic ones, regardless of the PON1-55 or PON1-192 polymorphisms. Second, Regular exercise was associated with an increase in PON1 activity in QQ subjects and with a decrease in R carriers. Increased PON1 activity immediately after a bout of exercise was subsequently followed by a decrease of activity. The recovery of the basal PON1 activity levels at 24 h was found in QQ subjects regardless of their training status and in trained R carriers, but not in untrained R carriers. Third, high oleic acid intake was associated with increased HDL cholesterol and PON1 activity levels only in men who were QR and RR of the PON1-192 polymorphism, respectively.

La proprotéine convertase subtilisine/kexine-9 (PCSK9) a été identifiée comme le troisième locus impliqué dans l’hypercholestérolémie autosome dominante (ADH). Les deux autres gènes impliqués dans l’ADH encodent le récepteur des lipoprotéines de faible densité (LDLR) et l’apolipoprotéine B. La PCSK9 est une convertase qui favorise la dégradation du LDLR dans les hépatocytes et augmente le niveau plasmatique de cholestérol des LDL (LDL-C). Les mutations « gain de fonction » de la PCSK9 sont associées à un phénotype d’hypercholestérolémie familiale, tandis que les variantes « perte de fonction » sont associées à un LDL-C réduit et à un risque coronarien plus faible. Pour élucider le rôle physiologique de la PCSK9, nous avons étudié sa régulation génique. En utilisant le RT-PCR quantitatif dans des hépatocytes humains, nous avons analysé la régulation de PCSK9 sous différentes conditions modulant l’expression des gènes impliqués dans le métabolisme du cholestérol. Nous avons démontré que l’expression de la PCSK9 était induite par les statines de manière dose-dépendante et que cette induction était abolie par le mévalonate. De plus, le promoteur de PCSK9 contenait deux motifs conservés pour la régulation par le cholestérol : le sterol regulatory element (SRE) et un site Sp1. La PCSK9 circule dans le plasma sous des formes mature et clivée par la furine. Grâce à notre anticorps polyclonal, nous avons mis au point un test ELISA mesurant la PCSK9 plasmatique totale. Une étude transversale a évalué les concentrations plasmatiques de PCSK9 chez des sujets sains et hypercholestérolémiques, traités ou non par des statines ou une combinaison statine/ezetimibe. Chez 254 sujets sains, la valeur moyenne de PCSK9 (écart-type) était de 89,5 (31,9) µg/L. La concentration plasmatique de la PCSK9 corrélait avec celle de cholestérol total, du LDL-C, des triglycérides (TG), de la glycémie à jeun, l’âge et l’indice de masse corporelle. Le séquençage de PCSK9 chez des sujets aux extrêmes de la distribution des concentrations de PCSK9 de notre cohorte a révélé la présence d’une nouvelle variation « perte de fonction » : R434W. Chez 200 patients hypercholestérolémiques, la concentration de PCSK9 était plus élevée que chez les sujets sains (P<0,04). Elle a augmenté avec une dose croissante de statine (P<0,001), et a augmenté encore plus suite à l’ajout d’ezetimibe (P<0,001). Chez les patients traités, ceux présentant une hypercholestérolémie familiale (HF; due à une mutation du LDLR) avaient des concentrations plus élevées de PCSK9 que les non-HF (P<0,005), et la réduction de LDL-C corrélait positivement avec la concentration de PCSK9 atteinte de la même manière dans les deux sous-catégories (P<0,02 et P<0,005, respectivement). Par ailleurs, une incubation des cellules HepG2 (hépatocytes) et Caco-2 (entérocytes) avec de l’ezetimibe a provoqué une augmentation de l’ARNm de PCSK9 et de NPC1L1 de 1,5 à 2 fois (P<0,05), mais aucune variation significative de PCSK9 sécrétée n’a été observée, suggérant que ces lignées cellulaires ne sont pas un modèle idéal. Nous avons également mesuré le niveau de PCSK9 chez 1 739 Canadiens-français âgés de 9, 13 et 16 ans. La valeur moyenne (écart-type) de PCSK9 dans cette cohorte était de 84,7 (24,7) µg/L, légèrement plus basse que dans la cohorte d’adultes (89,5 (31,9) µg/L). Chez les garçons, la PCSK9 circulante diminuait avec l’âge, tandis que c’était l’inverse chez les filles. Il y avait des associations positives et significatives entre la PCSK9 et la glycémie à jeun, l’insulinémie, le HOMA-IR, et les paramètres lipidiques (TC, LDL-C, TG, HDL-C, apoAI et apoB). Dans l’analyse multivariée, une hausse de 10% de l’insulinémie à jeun était associée à une augmentation de 1 à 2% de PCSK9. La régulation de PCSK9 est typique de celle d’un gène impliqué dans le métabolisme des lipoprotéines et est probablement la cible du facteur de transcription «sterol regulatory element-binding protein » (SREBP-2). La concentration plasmatique de la PCSK9 est associée avec l’âge, le sexe, et de multiples marqueurs métaboliques chez les enfants et les adultes. La détection de la PCSK9 circulante chez les sujets HF et non-HF signifie que ce test ELISA spécifique à PCSK9 pourrait servir à suivre la réponse à la thérapie chez un grand éventail de sujets. PCSK9 semble être une cible thérapeutique prometteuse dans le traitement de l’hypercholestérolémie et de la maladie cardiovasculaire. / Proprotein convertase subtilisin/kexin type 9 (PCSK9) has been identified as the third locus implicated in autosomal dominant hypercholesterolemia (ADH). The two other known genes implicated in ADH encode the low-density lipoprotein receptor (LDLR) and apolipoprotein B. PCSK9 is a protein convertase that post-translationally promotes the degradation of the LDLR in hepatocytes and increases plasma LDL cholesterol concentration (LDL-C). Heterozygote “gain-of-function” mutations of PCSK9 are associated with the familial hypercholesterolemia phenotype, whereas “loss-of-function” variants are associated with reduced LDL-C concentrations and lower coronary risk. As an approach toward the elucidation of the physiological role(s) of PCSK9, we studied its transcriptional regulation. Using quantitative RT-PCR, we assessed PCSK9 regulation under conditions known to regulate genes involved in cholesterol metabolism in HepG2 cells and in human primary hepatocytes. We found that PCSK9 expression was strongly induced by statins in a dose-dependent manner and that this induction was efficiently reversed by mevalonate. The PCSK9 promoter contains two typical conserved motifs for cholesterol regulation: a sterol regulatory element (SRE) and an Sp1 site. PCSK9 circulates in plasma as mature and furin-cleaved forms. A polyclonal antibody against human PCSK9 was used to develop an ELISA that measures total plasma PCSK9 rather than only the mature form. A cross-sectional study evaluated plasma levels in normal and hypercholesterolemic subjects treated or untreated with statins or statin plus ezetimibe. In 254 healthy subjects, the mean plasma PCSK9 (SD) concentration was 89 (32) µg/L. PCSK9 levels correlated positively with plasma cholesterol, LDL-C, triglycerides, fasting glucose, age and body mass index. Sequencing PCSK9 from subjects at the extremes of PCSK9 plasma distribution revealed a new loss-of-function R434W variant. In 200 hypercholesterolemic patients, circulating PCSK9 was higher than in controls (P<0.04), increased with increasing statin dose (P<0.001), and further increased when ezetimibe was added (P<0.001). In treated patients (n = 139), those with familial hypercholesterolemia (FH; due to LDLR gene mutations) had higher PCSK9 values than non-FH (P<0,005), and LDL-C reduction correlated positively with achieved plasma PCSK9 levels to a similar extent in both subsets (P<0.02 and P<0.005, respectively). However, incubation with ezetimibe of HepG2 (hepatocytes) and Caco-2 (enterocytes) cells caused an increase in PCSK9 and NPC1L1 mRNA of 1.5 to 2-fold (P<0.05), but no significant rise in PCSK9 protein secretion, suggesting that these transformed cells are not an ideal model. We also studied PCSK9 levels in 1,739 French Canadian youth ages 9, 13, and 16 years old. The mean (SD) plasma PCSK9 concentration, measured by ELISA, was 84.7 (24.7) µg/L in the cohort, slightly lower than in the adult cohort (89.5 (31.9) µg/L. In boys, plasma PCSK9 decreased with age, whereas the inverse was true for girls. There were significant positive associations between PCSK9 and fasting glucose, insulin, and HOMA-IR (homeostasis model assessment of insulin resistance). In multivariable analysis, a 10% higher fasting insulin was associated with a 1%-2% higher PCSK9 in both sexes. There were also positive associations between PCSK9 and total cholesterol, LDL-C, and triglycerides, as well as with HDL-C and apolipoproteins A1 and B. PCSK9 regulation is typical of that of the genes implicated in lipoprotein metabolism. In vivo, PCSK9 is probably a target of the transcription factor “sterol response element-binding protein” (SREBP)-2. The PCSK9 plasmatic concentration is associated with age, sex, and multiple metabolic markers in youth and adult samples. The detection of circulating PCSK9 in both FH and non-FH subjects means that this PCSK9 ELISA test could be used to monitor response to therapy in a wide range of patients. PCSK9 seems to be a promising drug target in the treatment of hypercholesterolemia and coronary heart disease.

Les maladies cardiovasculaires (MCV) sont les principales causes de mortalité et de morbidité à travers le monde. En Amérique du Nord, on estime à 90 millions le nombre d’individus ayant une ou plusieurs MCV, à près de 1 million le nombre de décès reliés par année et à 525 milliards de dollars les coûts directs et indirects en 2010. En collaboration avec l’équipe du Dre. Boileau, notre laboratoire a récemment identifié, le troisième locus impliqué dans l’hypercholestérolémie familiale. Une étude publiée dans le New Engl J Med a révélé que l’absence de la convertase PCSK9 réduit de 88% le risque de MCV, corrélé à une forte réduction du taux de cholestérol plasmatique (LDL-C). Il fut démontré que PCSK9 lie directement le récepteur aux lipoprotéines de faible densité (LDLR) et, par un mécanisme méconnu, favorise sa dégradation dans les endosomes/lysosomes provoquant ainsi une accumulation des particules LDL-C dans le plasma. Dans cet ouvrage, nous nous sommes intéressés à trois aspects bien distincts : [1] Quels sont les cibles de PCSK9 ? [2] Quelle voie du trafic cellulaire est impliquée dans la dégradation du LDLR par PCSK9 ? [3] Comment peut-on inhiber la fonction de PCSK9 ? [1] Nous avons démontré que PCSK9 induit la dégradation du LDLR de même que les récepteurs ApoER2 et VLDLR. Ces deux membres de la famille du LDLR (fortes homologies) sont impliqués notamment dans le métabolisme des lipides et de la mise en place de structures neuronales. De plus, nous avons remarqué que la présence de ces récepteurs favorise l’attachement cellulaire de PCSK9 et ce, indépendamment de la présence du LDLR. Cette étude a ouvert pour la première fois le spectre d’action de PCSK9 sur d’autres protéines membranaires. [2] PCSK9 étant une protéine de la voie sécrétoire, nous avons ensuite évalué l’apport des différentes voies du trafic cellulaire, soit extra- ou intracellulaire, impliquées dans la dégradation du LDLR. À l’aide de milieux conditionnées dérivés d’hépatocytes primaires, nous avons d’abord démontré que le niveau extracellulaire de PCSK9 endogène n’a pas une grande influence sur la dégradation intracellulaire du LDLR, lorsqu’incubés sur des hépatocytes provenant de souris déficientes en PCSK9 (Pcsk9-/-). Par analyses de tri cellulaire (FACS), nous avons ensuite remarqué que la surexpression de PCSK9 diminue localement les niveaux de LDLR avec peu d’effet sur les cellules voisines. Lorsque nous avons bloqué l’endocytose du LDLR dans les cellules HepG2 (lignée de cellules hépatiques pour l’étude endogène de PCSK9), nous n’avons dénoté aucun changement des niveaux protéiques du récepteur. Par contre, nous avons pu démontrer que PCSK9 favorise la dégradation du LDLR par l’intermédiaire d’une voie intracellulaire. En effet l’interruption du trafic vésiculaire entre le réseau trans-Golgien (RTG) et les endosomes (interférence à l’ARN contre les chaînes légères de clathrine ; siCLCs) prévient la dégradation du LDLR de manière PCSK9-dépendante. [3] Par immunobuvardage d’affinité, nous avons identifié que la protéine Annexine A2 (AnxA2) interagit spécifiquement avec le domaine C-terminal de PCSK9, important pour son action sur le LDLR. Plus spécifiquement, nous avons cartographié le domaine R1 (acides aminés 34 à 108) comme étant responsable de l’interaction PCSK9AnxA2 qui, jusqu’à présent, n’avait aucune fonction propre. Finalement, nous avons démontré que l’ajout d’AnxA2 prévient la dégradation du LDLR induite par PCSK9. En somme, nos travaux ont pu identifier que d’autres membres de la famille du LDLR, soit ApoER2 et VLDLR, sont sensibles à la présence de PCSK9. De plus, nous avons mis en évidence que l’intégrité du trafic intracellulaire est critique à l’action de PCSK9 sur le LDLR et ce, de manière endogène. Finalement, nous avons identifié l’Annexine A2 comme unique inhibiteur naturel pouvant interférer avec la dégradation du LDLR par PCSK9. Il est indéniable que PCSK9 soit une cible de premier choix pour contrer l’hypercholestérolémie afin de prévenir le développement de MCV. Cet ouvrage apporte donc des apports considérables dans notre compréhension des voies cellulaires impliquées, des cibles affectées et ouvre directement la porte à une approche thérapeutique à fort potentiel. / Cardiovascular disease (CVD) is the primary cause of death and morbidity worldwide, claiming about 900 000 lives yearly in North America alone. A high level of circulating LDL-cholesterol is a major risk factor positively correlated with premature development of complex CVD mainly due to a rapid buildup of lipid deposition in the arteries. In collaboration with Dre Boileau, we recently discovered that the convertase PCSK9 is the third locus of familial hypercholesterolemia. A study published in the New Eng J Med revealed that the absence of PCSK9 reduces the risk of CVD by ~88%, resulting from a strong reduction of cholesterol in the bloodstream (LDL-C). It has been shown that PCSK9 directly binds the low-density lipoprotein receptor (LDLR) and by an unknown mechanism, reroutes it towards degradation in late endosomes/lysosomes, resulting in the accumulation of LDL-C particles in plasma. In this thesis, we addressed three different aspects of PCSK9 biology: [1] What are the targets of PCSK9? [2] Which cellular trafficking components are involved in PCSK9-induced LDLR degradation? [3] How can we inhibit the function of PCSK9? [1] We first demonstrated that PCSK9 induces the degradation of the LDLR and two of its closest family members. These include the very-low-density-lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2) implicated in neuronal development and lipid metabolism. In addition, we demonstrated that these receptors enhance the cellular association of PCSK9 independently of the presence of the LDLR. This study represents the first evidence that PCSK9 could target other proteins for degradation, reinforcing its role as a key regulator of some members of the LDLR family. [2] Since PCSK9 is a secreted protein, we decided to investigate the contributions of both the intra- and extracellular trafficking pathways in LDLR degradation. Using conditioned media derived from mice primary hepatocytes, we showed that endogenously secreted PCSK9 was not able to influence LDLR levels of PCSK9-deficient primary hepatocytes (Pcsk9-/-). By flow cytometry (FACS), we observed that overexpression of the gain-of-function PCSK9-D374Y, but not wild type PCSK9, decreases cell surface LDLR on adjacent cells suggesting that its spectrum of action is local. We also noticed that blockade of endocytosis in HepG2 cells (commonly used to study endogenous LDLR degradation by PCSK9) does not affect total LDLR protein levels. In contrast, disruption of the intracellular trafficking between the trans-Golgi network (TGN) and endosomes (siRNAs against clathrin light chains; CLCs) prevented LDLR degradation in a PCSK9-specific manner. [3] By Far Western blotting, we identified that Annexin A2 (AnxA2) specifically interacts with the C-terminal domain of PCSK9, which is crucial for its function in LDLR degradation. Moreover, we determined that the R1 domain (amino acids 34 to 108) is responsible for the PCSK9AnxA2 interaction, which confers a new function for this protein. Finally, we showed that addition of AnxA2 prevents PCSK9-induced LDLR degradation. In summary, this work allowed us to identify that PCSK9 induces the degradation of the LDLR and its closest family members, ApoER2 and VLDLR. We also highlighted that the integrity of the intracellular trafficking pathway is crucial for endogenous PCSK9-induced LDLR degradation. Furthermore, we discovered that AnxA2 is a unique, natural inhibitor capable of interfering with the action of PCSK9 in LDLR degradation. It is undeniable that PCSK9 is a genetically validated target to reduce circulating LDL-cholesterol and prevent CVD. This thesis brings forth important contributions in our understanding of the cellular pathways involved and opens the door for novel therapeutic approaches.

Thesis (M.S.P.H.)--University of South Florida, 2009. / Title from PDF of title page. Document formatted into pages; contains 34 pages. Includes bibliographical references.