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.

Aims In heart failure with preserved ejection fraction (HFpEF), the reduction of nitric oxide (NO)-bioavailability and consequently endothelial dysfunction leads to LV stiffness and diastolic dysfunction of the heart. Besides shear stress, high-density lipoprotein (HDL) stimulates endothelial cells to increased production of NO via phosphorylation of endothelial nitric oxide synthase (eNOS). For patients with heart failure with reduced ejection fraction, earlier studies demonstrated a positive impact of exercise training (ET) on HDL-mediated eNOS activation. The study aims to investigate the influence of ET on HDL-mediated phosphorylation of eNOS in HFpEF patients. Methods and results The present study is a substudy of the OptimEx-Clin trial. The patients were randomized to three groups: (i) HIIT (high-intensity interval training; (ii) MCT (moderate-intensity continuous training); and (iii) CG (control group). Supervised training at study centres was offered for the first 3 months. From months 4–12, training sessions were continued at home with the same exercise protocol as performed during the in-hospital phase. Blood was collected at baseline, after 3, and 12 months, and HDL was isolated by ultracentrifugation. Human aortic endothelial cells were incubated with isolated HDL, and HDL-induced eNOS phosphorylation at Ser1177 and Thr495 was assessed. Subsequently, the antioxidative function of HDL was evaluated by measuring the activity of HDL-associated paraoxonase-1 (Pon1) and the concentration of thiobarbituric acid-reactive substances (TBARS). After 3 months of supervised ET, HIIT resulted in increased HDL-mediated eNOS-Ser1177 phosphorylation. This effect diminished after 12 months of ET. No effect of HIIT was observed on HDL-mediated eNOS-Thr495 phosphorylation. MCT had no effect on HDL-mediated eNOS phosphorylation at Ser1177 and Thr495. HIIT also increased Pon1 activity after 12 months of ET and reduced the concentration of TBARS in the serum after 3 and 12 months of ET. A negative correlation was observed between TBARS concentration and HDL-associated Pon1 activity in the HIIT group (r = −0.61, P < 0.05), and a trend was evident for the correlation between the change in HDL-mediated eNOS-Ser1177 phosphorylation and the change in peak V̇O2 after 3 months in the HIIT group (r = 0.635, P = 0.07). Conclusions The present study documented that HIIT but not MCT exerts beneficial effects on HDL-mediated eNOS phosphorylation and HDL-associated Pon1 activity in HFpEF patients. These beneficial effects of HIIT were reduced as soon as the patients switched to home-based ET.

Résumé : L’apolipoprotéine E (apoE) joue un rôle important dans le transport des acides gras (AG) via les lipoprotéines. Cependant, il existe possiblement une perturbation dans l’homéostasie des AG au niveau des lipoprotéines chez les porteurs du génotype de l’apolipoprotéine E epsilon 4 (E4+). L’objectif de cette étude est de déterminer le profil en AG dans les lipoprotéines de hautes et de faibles densités (HDL et LDL) chez les E4+ et les non-porteurs (E4-), pendant une supplémentation en AG oméga-3 (n-3) de 28 jours. Matériels et Méthodes: 80 participants (34 hommes et 46 femmes) en santé, âgés entre 20-35 ans, ont consommé 1,6 g/jour d’AG n-3 sur une période de 28 jours. Des prélèvements sanguins à jeun ont été récoltés chaque semaine. Les lipoprotéines ont été séparées par ultracentrifugation sur gradient discontinu de sucrose. Les lipides totaux des particules de HDL et de LDL ont été analysés par chromatographie en phase gazeuse. Les génotypes de l’APOE (E4+ ou E4-) ont été déterminés par la méthode de polymorphisme de longueur des fragments de restriction (RFLP) et les données ont été analysées par logiciel SAS à l’aide d’une procédure MIXED. Résultats: Les caractéristiques anthropométriques et habitudes de vies ne variaient pas significativement entre les E4+ et E4-. Le ratio d’AG n-6/n–3 était environ 17% plus élevé chez les E4+ dans les LDL (P = 0.043) pendant la supplémentation. Ceci peut être attribuable au niveau plus élevé d’AG n-6, sans changement dans le niveau d’AG n-3 chez les E4+. Une interaction génotype × temps a été trouvée pour l’acide linoléique (LA) dans les HDL ainsi qu’un effet génotype pour les AG n-6 totaux dans les HDL et LDL (P ≤ 0.05). De plus, l’acide palmitique (PA) et palmitoléïque (PAL) est plus bas chez les E4+ comparativement aux E4-. Conclusion: Le débalancement de la distribution des AG dans les HDL et LDL chez les E4+ peut être causé par une altération de la spécificité de la β-oxydation des AG chez les E4+. Plus d’investigation doit être faite à cet égard afin de confirmer ces hypothèses.

La maladie cardiovasculaire (MCV) représente encore aujourd’hui l’une des principales causes de décès et d’hospitalisation au Canada. Parmi les facteurs de risque de la MCV, la dyslipidémie est l’un des plus importants. En effet, des concentrations plasmatiques élevées de cholestérol transporté par les lipoprotéines de faibles densités (c-LDL), de triglycérides, ainsi que des concentrations faibles de cholestérol transporté par les lipoprotéines de hautes densités (c-HDL) ont été à maintes reprises identifiées comme des facteurs de risque indépendants de la MCV. Plusieurs facteurs héréditaires et environnementaux ont été associés aux concentrations de lipides plasmatiques. Toutefois, les facteurs héréditaires permettraient d’expliquer la plus grande proportion de la variabilité interindividuelle du bilan lipidique, particulièrement pour le c-HDL. Malgré l’étude de plusieurs millions de modifications génétiques, les principales causes moléculaires responsables de la forte héritabilité des concentrations de c-HDL demeurent pour l’instant encore inconnues. Afin d’expliquer l’héritabilité manquante des concentrations de lipides plasmatiques, plusieurs hypothèses ont été avancées. La présente thèse de doctorat se concentre sur celle suggérant que l’étude de la méthylation de l’ADN, une modification épigénétique considérée comme un facteur héréditaire non traditionnel, permettrait d’identifier de nouveaux fondements moléculaires associés aux dyslipidémies. Dans un premier temps, nous avons analysé la méthylation de l’ADN de plusieurs gènes du métabolisme lipoprotéique chez des sujets atteints d’hypercholestérolémie familiale (HF), un modèle humain de la MCV. Grâce à cette approche, nous avons pu démontrer que la méthylation de l’ADN de plusieurs gènes candidats (ABCA1, ABCG1, CETP, LIPC, LPL et PLTP) était associée à la variabilité du bilan lipidique, ainsi qu’avec les antécédents de maladies coronariennes athérosclérotiques. Dans un deuxième temps, une étude de la méthylation à l’échelle du génome d’un sous-groupe de patients HF nous a permis d’identifier de nouveaux loci associés à la concentration de c-HDL. Nous avons notamment observé que la méthylation de l’ADN du promoteur des gènes TNNT1 et ADRB3 était non seulement associée aux concentrations de c-HDL, mais également avec d’autres facteurs de risque de la MCV. L’ensemble des résultats présentés dans cette thèse démontre que la méthylation de l’ADN contribue à la variabilité du bilan lipidique à jeun de patients atteints d’HF et que l’étude des modifications épigénétiques pourrait aider à expliquer l’héritabilité manquante des concentrations de c-LDL, de c-HDL et de triglycérides plasmatiques.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

La obesidad infantil es un importante problema de salud pública mundial, por su prevalencia y disminución de calidad de vida, transformándose en un factor de riesgo importante de morbimortalidad por enfermedad cardiovascular en la edad adulta. En el Perú, una de las principales causas de mortalidad cardiovascular en adultos y la creciente incidencia en niños y adolescentes se encuentra asociada al Síndrome Metabólico (SM), relacionada a factores de riesgo como obesidad, resistencia insulínica, hipertensión y dislipidemias. Por esta razón nos propusimos determinar la presencia de SM en niños y adolescentes escolares de 8 a 17 años de edad. Se estudió una población de 100 alumnos, 55 del género femenino y 45 del masculino, de dos (2) Instituciones Educativas de la zona Salamanca-Valdiviezo-Olimpo del distrito de Ate-Lima. La metodología del presente estudio estimó realizar pruebas antropométricas (peso, talla, circunferencia de la cintura, IMC), medición de la presión arterial y pruebas bioquímicas para determinar valores séricos de colesterol, HDL, LDL, triglicéridos y glucosa. Para la determinación del Síndrome Metabólico se utilizó la tabla de Must y col. en referencia al Índice de Masa Corporal (IMC) con los siguientes criterios diagnósticos: Sobrepeso, de p85 a p95 (percentil), Obeso, > p95 (percentil); además se utilizó las recomendaciones de Cook. y col. que establece: déficit de Colesterol HDL : < = 40 mg/dL, Colesterol LDL elevados : > = 110 mg/dL, Triglicéridos elevados : > = 110 mg/dL; Glucosa elevada (en ayunas) : > = 110 mg/dL y Presión Arterial elevada (Hipertensión) : > = p90 (percentil). La presencia de tres o más factores en la población estudiada indicaría la existencia de Síndrome Metabólico. De acuerdo a los resultados obtenidos, el 6 % de la población estudiada presentó síndrome Metabólico, encontrándose una mayor frecuencia entre los alumnos de 12 a 17 años de edad (83,3 %) y de 8 a 11 años (16,7 %), todos ellos del género masculino quienes presentaron como factor principal la obesidad (34 %), Colesterol HDL (31 %), Triglicéridos (11 %) y Presión Arterial (9 %), en ningún caso se presentó hiperglicemia.

Emulsões artificiais sem proteína simulando quilomicrons e remanescentes de quilomícron foram preparadas por sonicalcação de trioleína, lecitina, colesteril oleato e colesterol em solução aquosa. A seguir foram ultracentrifugadas em gradiente descontínuo de densidade. As emulsões, marcadas com 3H-trioleína e 14C-colesteril oleato foram injetadas via intra-arterial em ratos controle e em ratos tratados com Triton WR1339, sulfato de protamina e heparina, medindo-se a seguir a remoção plasmática do colesteril-ester e dos triglicérides, durante dez minutos. O Triton WR 1339 e a protamina inibiram a lipólise dos quilomícrons artificiais, diminuindo a remoção destas partículas do plasma. O Triton WR 1339 mostrou ser mais efetivo que a protamina nestes efeitos. Por outro lado, a heparina promoveu uma lipólise rápida e brusca nos quilomícrons artificiais, assim como uma aceleração na remoção destas partículas do plasma. Em contraste flagrante com esses resultados, o metabolismo dos remanescentes de quilomícron não foi consideravelmente afetado pelo tratamento com Triton e heparina. Estas experiências indicam que as emulsões artificiais reproduzem o comportamento metabólico dos quilomícrons e remanescentes de quilomícron naturais, em condições em que a atividade da lipase lipoproteica esteja alterada. / Protein-free emulsions models of chylomicrons and chylomicron remnants were prepared by sonicating triolein, lecithin., cholesteryl oleate and cholesterol in aqueous saline media, followed by ultracentrifugation in density gradient solution. The 3H-triolein and 14C-cholesteryl oleate labeled emulsions were injected into the carotid artery of control rats and rats treated with Triton WR 1339, protamine sulphate and heparin. Plasma removal of both labels was measured during ten minutes in two minutes intervals. Triton WR 1339 and protamine sulphate strongly inhibited lipolysis of chylomicron-like emulsions leading to delayed removal of the particles from blood. Triton WR 1339 de appeared to be more effective than protamine to elicit these effects. On the other hand, heparin produced instantaneous lipolysis of the chylomicron-like particles markedly enhancing its removal from plasma. Contrarily, chylomicron remnant-like emulsions were not considerably affected either by Triton WR 1339 or by heparin treatment. The above described results obtained with artificial emulsions support current concepts on the metabolic behavior of natural chylomicron and remnant submitted to changes in lipoprotein lipase action.

Objetivo: comparar o perfil lipídico de em crianças e adolescentes, com e sem inflamação, atendidas num pronto atendimento geral de pediatria de um hospital universitário de nível de atendimento secundário, segundo estado nutricional, sexo e idade. Métodos: Estudo transversal, realizado entre outubro de 2012 e agosto de 2013, avaliou 124 crianças e adolescentes (3 meses a 14 anos de idade) em atendimento na unidade de emergência do Hospital Universitário da Universidade de São Paulo, com queixa relacionada a processo inflamatório/infeccioso. Os pacientes foram separados em dois grupos de acordo com os níveis de proteína C reativa (PCR): grupo I se maior ou igual a 5 mg/L, e grupo II se menor que 5mg/L. Dosagens de colesterol total, lipoproteína de alta densidade (HDL) e baixa densidade (LDL), triglicerídeos e albumina foram comparadas entre os dois grupos, levando em conta o estado nutricional (avaliado através de medidas antropométricas), gênero e idade. Resultado: A mediana de idade foi de 51 meses, com maioria dos pacientes classificados como eutróficos (76,5%). Do total da amostra, 34,7% dos pacientes apresentaram colesterol total e/ou triglicerídeos alterados e 67% apresentaram baixos níveis de HDL. Não houve diferença significativa do perfil lipídico entre os dois grupos de pacientes separados de acordo com PCR. Dentre os pacientes com PCR >= 5mg/L, a PCR apresentou correlação inversa com HDL [r= (-)0,363 e p=0,001], com LDL [r= (-) 0,235 e p=0,034], com albumina [r= (-) 0,308 e p=0,005] e correlação direta com TG (r=0,426 e p > 0,001). Na analise de regressão linear, se evidenciou que para cada aumento de 1mg/L nos valores da PCR espera-se uma redução média de 0,072 mg/dL da HDL, de 0,083 mg/dL da LDL, de 0,002g/dL de albumina, e um aumento médio de 0,564 mg/dL do triglicerídeo. Conclusão: Pacientes com processo inflamatório apresentam alterações nos níveis séricos do HDL, LDL e triglicerídeos que se relacionam com o grau de inflamação, de forma independente do estado nutricional / Aim: To compare the lipid profile in children and adolescents with and without inflammation, met a ready general pediatric service of a university hospital secondary care level, according to nutritional status, gender and age. Methods: Cross-sectional study conducted between October 2012 and August 2013, assessed 124 children and adolescents (3 months to 14 years old) in the emergency department of the University Hospital of the University of São Paulo, with reports of inflammatory/ infectious process. The patients were divided into two groups according to the C reactive protein (CRP) levels: group I is higher than or equal to 5 mg/L, and Group II was lower than 5 mg/L. Total cholesterol, high density lipoprotein (HDL) and low density lipoprotein (LDL), triglycerides and albumin were compared between the two groups, taking into account the nutritional status (assessed by anthropometric measurements), gender and age. Results: The median age was 51 months, with patients mostly classified as well-nourished (76.5%). Of the overall sample, 34.7% of patients had total cholesterol and/or triglycerides altered and 67% had low levels of HDL. There was no significant difference in lipid profile between the two groups of PCR. For the patients with CPR > 5mg/L, CPR presented an inverse correlation with HDL [r = (-) 0.363 and p = 0.001], with LDL [r = (-) 0.235 and p = 0.034], with [r = albumin (-) 0.308 and p = 0.005] and direct correlation with TG (r = 0.426 and p < 0.001). Linear regression analysis it became clear that for each increase of 1 mg/L in the values of CRP expected an average reduction of 0,072 mg/dL of HDL, the 0,083 mg/dL of LDL, the 0,002 g /dL albumin, and an average increase of 0,564 mg/dL of triglycerides. Conclusion: Patients with an inflammatory process exhibit changes in the serum levels of the lipids HDL, LDL and TG that are related to the degree of inflammation. These changes occurred regardless of nutritional status