Cette étude vise à définir l'effet des lipoprotéines de basses densité natives (LDL) et oxydées (ox-LDL) sur les fonctions des cellules endothéliales en relation avec les processus physiopathologiques de l'athérosclérose. Le microscope à force atomique (AFM) fut utilisé en combinaison avec les méthodes biochimiques traditionnelles afin d'acquérir de l'information sur les propriétés biomécaniques des cellules endothéliales. L'AFM est un outil permettant l'acquisition d'images et de mesures de forces quantitatives concernant les propriétés viscoélastiques des cellules vivantes selon leur exposition aux LDL ou ox-LDL. L'AFM rassemble localement des informations sur la membrane cellulaire et le cytosquelette des cellules et ce, de manière non invasive. Il est ensuite possible de corréler les résultats obtenus avec les marquages immunohistochimiques afin d'évaluer la réponse cellulaire suite à une exposition à des LDL ou ox-LDL. Ces données recueillies, les protocoles étant au point, il ne restera plus qu'à effectuer les tests avec les antioxydants afin de déterminer les agents et les dosages appropriés permettant une protection salutaire de l'endothélium. Ce travail amène donc de nouvelles connaissances sur les mécanismes moléculaires fondamentaux de la dysfonction endothéliale en vue éventuellement de développer de nouvelles thérapies cytoprotectrices efficaces. Une méthode d'imagerie des LDL a également été mise au point en utilisant l'AFM. Il est maintenant possible d'obtenir des images de bonne qualité permettant aussi de mesurer les dimensions de LDL individuelles. Cette technique pourrait entre autre servir à évaluer des pathologies touchant les LDL comme le diabète.

Atherosclerosis is a disease characterized by cholesterol-rich plaques within the intima of medium and large arteries. Cholesterol deposition is thought to occur by infiltration of low-density lipoprotein (LDL) into lesions followed by uptake into macrophages, generating lipid-loaded “foam cells.” Foam cells can also be generated in vitro by treatment of macrophages with LDL or oxidized LDL (oxLDL). The purpose of the current investigation was to determine the contribution of selective cholesteryl ester (CE) uptake versus whole-particle uptake during LDL-induced foam cell formation in cultured macrophages. Murine bone marrow-derived macrophages (BMMs) exhibited significant cholesterol accumulation when treated with LDL as indicated by quantification of cellular cholesterol and visualization of Oil Red-O-stained neutral lipid droplets. Uptake of LDL cholesterol was determined by measuring uptake of 3H and 125I into BMMs during treatment with [3H]CE/125I-LDL. [3H]CE uptake was linearly related to the LDL concentration at the concentrations used and was much larger than 125I uptake, indicating that the majority of LDL-cholesterol was acquired by nonsaturable, selective CE uptake. This pathway was demonstrated to be independent of whole-particle uptake by showing that inhibition of actin polymerization blocked LDL particle uptake but not selective CE uptake. Analysis by thin-layer chromatography (TLC) indicated that following uptake, [3H]CE was rapidly hydrolyzed into [3H]cholesterol by cells and largely effluxed into the culture medium. In contrast to LDL, studies of [3H]CE/125I-oxLDL uptake demonstrated that CE was acquired from oxLDL by whole-particle uptake with little or no selective CE uptake. Using a series of ten different [3H]CE/125I-oxLDLs oxidized for 0-24 hours, selective [3H]CE uptake was shown to be progressively impaired by LDL oxidation, while 125I-LDL particle uptake was increased as expected. Interestingly, the impairment of selective CE uptake occurred very early in LDL oxidation and this minimally oxidized LDL induced significantly less cholesterol accumulation in BMMs compared to native LDL. Together, these results demonstrate that selective CE uptake is the primary mode of cholesterol uptake from LDL but not oxidized LDL, a finding that has important implications for cholesterol metabolism in atherosclerotic lesions. Future studies seek to identify the molecular components that participate in the macrophage selective CE uptake mechanism.

O efeito do fotossensibilizador na estrutura biológica não é apenas influenciado por suas propriedades fotofísicas, mas também por sua interação específica com biosistemas.Além disso, a localização do fotossensibilizador no tecido tumoral é um importante fator que resulta em diferentes mecanismos de destruição do tumor. Muitos fotossensibilizadores, após administração sistêmica, se ligam às proteínas plasmáticas e com isso são distribuídos em diferentes sítios no organismo. Os fotossensibilizadores hidrofílicos são largamente transportados por albuminas e globilinas e se acumulam preferencialmente no estroma vascular dos tumores. Entretanto, fotossensibilizadores mais hidrofóbicos se ligam às lipoproteínas, principalmente LDL, que promove a entrada do FS na célula através de endocitose mediado por receptor. Sendo assim, a localização do FS depende de sua ligação com as deferentes proteínas plasmáticas, sua farmacocinética e também é influenciada pela diferença entre o tecido normal e tumoral. O tecido tumoral tem pH mais baixo e maior expressão de receptores de LDL do que os tecidos normais, aumentando a seletividade dos FSs as células tumorais. A incorporação de FS hidrofóbicos em lipossomas para a administração sistêmica pode realçar ao transporte deste pelas lipoproteínas. No presente trabalho estudou-se a influência do pH na interação de fotossensibilizadores com lipossomas de DMPC e LDL. Os fotossensibilizadores utilizados nesse estudo foram Photofrin®, Photogem® e Photosan® que são derivados de hematoporfirinas. A metodologia empregada constitui de variação das concentrações de DMPC e LDL para os seguintes valores de pHs 5,0; 7,4 e 9,0, esse último pH utilizou-se somente para DMPC. O complexo FS - DMPC foi obtido por incubação dos FSs na concentração de 10 micro g.mL-1 com diferentes concentrações de DMPC (0 a 400 micro M) por trinta minutos no escuro. Isolou-se o LDL do plasma humano por ultracentrifugação por gradiente de densidade. Após a separação, o complexo FS - LDL foi obtido por incubação (12 horas no escuro) do FS na concentração 10 micro g.mL-1 com diferentes concentrações de LDL (0 a 0,04 micro M). O comportamento desses complexos foi analisado por espectroscopia de absorção ótica e por espectroscopia de fluorescência. / The effect of a photosensitizing compound on biological structures is governed not only by its photophysical properties but also by the specificity of its interaction with biosystems. Moreover, localization of the photosensitizer in the tumor tissue is an important factor affecting the outcome as well as mechanism leading to tumor destruction. Following administration, most photosensitizers are bound to blood components and delivered to different sites in the organism. It is generally accepted that hydrophilic photosensitizers are largely transported by albumins and globulins and mainly accumulate in the vascular stroma of tumors. More hydrophobic sensitizers are bound to lipoproteins, which promote drug internalization by cells through endocytosis of the lipoprotein carrier. In this way, uptake and localization depend on the initial plasma binding and the plasma pharmacokinetics of the drug. However, the selective localization of some photosensitizers are influence for the difference between malignant and normal tissues. Notably, the lower pH of the microenvironment usually found in the tumor tissue and the expression of greater number of LDL receptors on the surface of the tumor cells might influence cellular uptake. Delivery to lipoproteins or target tissues may be facilitated and enhanced by the incorporation of lipophilic photosensitizers into liposomes for systemic administration. In the present work we have studied the pH-dependence of the interaction of photosensitizers with DMPC liposomes and low density lipoprotein (LDL). The photosensitizers used in this study are Photofrin®, Photogem® and Photosan®, which are hematoporphyrin derivates. The methodology used to this work, constitute of various concentrations of DMPC liposomes and LDL at different pH values. It were used the 5,0; 7,4 and 9,0 pH values. The DMPC-drug complexes were obtained by incubation of the photosensitizers 10 _g.mL-1 with differents DMPC concentrations for 30 min in the dark. The LDL was isolated from human plasma by sequential density gradient ultracentrifugation. The LDL-drug complexes were obtained by incubation of the photosensitizers 10 _g.mL-1 with differents LDL concentrations. The incubation was performed in a water bath at 20_C for 12 hours in the dark. The comportment of the complexes was analyzed by fluorescence spectroscopy and UV-visible spectroscopy.

Introdução: Dentre as diversas formas modificadas da lipoproteína de baixa densidade (LDL), a LDL eletronegativa, LDL (-), contribui para o processo aterosclerótico, promovendo o recrutamento leucocitário para a intima arterial e modulando a resposta inflamatória. Mimotopos são moléculas que podem se ligar à porção variável de um anticorpo, a enzimas ou a receptores. Os peptídeos mimotopos da LDL (-) são moléculas capazes de mimetizar a conformação de epítopos expostos apenas quando a LDL sofre modificações in vivo. Objetivo: Avaliar os efeitos de peptídeos mimotopos da LDL(-), previamente selecionados pela técnica de phage display e denominados p1A3 e p2C7, em células endoteliais e macrófagos. Metodologia: Avaliou-se a atividade dos peptídeos mimotopos sobre a expressão de RNAm de genes relacionados tanto à ativação quanto à disfunção endotelial, assim como a atividade citotóxica e a captação dos peptídeos por células endoteliais (HUVEC, HAEC e EA.Hy 926). Verificou-se também a atividade pró-inflamatória dos peptídeos em cultura primária de macrófagos murinos derivados da medula óssea (BMDM), investigando-se a ativação do complexo proteico inflamassoma, assim como a internalização dos peptídeos por essas células, utilizando citometria de fluxo e microscopia confocal. Resultados: As células endoteliais e os macrófagos internalizaram os peptídeos mimotopos p1A3 e p2C7. Nos macrófagos houve inibição (50%) da internalização dos peptídeos p1A3 e p2C7 na presença de brefeldina A, indicando que esses peptídeos são majoritariamente captados por endocitose independente de clatrina. Nas células HAEC, observou-se 25% de inibição da captação de ambos os peptídeos se bloqueando o receptor scavenger LOX-1 com anticorpo anti-LOX-1. Quanto à atividade citotóxica, observou-se acentuada redução na atividade mitocondrial das células endoteliais EA.Hy 926 e HAEC apenas com o peptídeo p1A3, não sendo observada citotoxicidade para os macrófagos tanto para p1A3 quanto p2C7 nas concentrações estudadas (12,5 a 200 ug/mL). Nos macrófagos, o peptídeo p2C7 induziu o aumento da expressão de RNAm de genes relacionados com a polarização para o fenótipo M1 (iNOS, TNF-α, IL-1α) e da produção de •NO, assim como, a ativação do sinal 1 (transcricional) do complexo proteico inflamassoma, o que também se observou para a LDL (-). O peptídeo p1A3 modulou a expressão gênica da linhagem HAEC, diminuindo a expressão de RNAm dos genes relacionados à proliferação celular (VEGFr1 e 2, TGFβ, PDGFβ e eNOS). Conclusão: Os peptídeos mimotopos p1A3 e p2C7 interagem com as células endoteliais e macrófagos de maneiras distintas. O p2C7 induz uma resposta pró-inflamatória em macrófagos, mimetizando a LDL (-), sugerindo assim, que esse peptídeo possa atuar como um DAMP (padrão molecular associado ao perigo) importante para o processo inflamatório da placa aterosclerótica. / Introduction: Among the different subtypes of modified low-density lipoprotein (LDL), the electronegative LDL [LDL (-)] contributes to the progression of atherosclerosis, promoting leukocyte recruitment into the arterial intima and modulating the inflammatory response. Mimotopes are molecules capable of binding to the variable portion of an antibody, to enzymes or receptors. The mimotope peptides of LDL (-) are molecules mimicking the conformation of epitopes exposed only when LDL undergoes in vivo changes. Objective: To evaluate the effects of two mimotope peptides of LDL (-), previously selected the by phage display and called p1A3 and p2C7, on endothelial cells and macrophages. Methodology: We evaluated the activity of the mimotope peptides on gene mRNA expression related to both the activation and endothelial dysfunction as well as the cytotoxic activity and uptake of peptides by endothelial cells (HUVEC, HAEC and EA.Hy 926). It was also investigated the pro-inflammatory activity of the peptides on murine primary macrophage derived from bone marrow (BMDM), evaluating the activation of the protein complex inflammasome, as well as the internalization of peptides by these cells by flow cytometry and confocal microscopy. Results: The endothelial cells and macrophages internalized both peptides. In macrophages, brefeldin A inhibited the internalization of p1A3 and p2C7 around 50%, indicating that these peptides are mostly taken up by clathrin-independent bulk endocytosis. In HAEC it was observed a 25 % inhibition of the uptake of both peptides by blocking the LOX scavenger receptor with an anti-LOX-1 antibody. In relation to cytotoxicity, a reduction on mitochondrial activity was induced by p1A3 peptide only in endothelial cells (EA.Hy 926 and HAEC). No cytotoxic activity was observed for p2C7 peptide in macrophages or endothelial cells at the studied concentration range (12.5 to 200 µg/mL). In macrophages, the p2C7 peptide increased mRNA expression of genes related to the polarization to M1 phenotype (iNOS, TNF-α and IL-1α) and of •NO production, as well as, protein complex signal 1 activation (transcriptional) what was also observed for LDL (-). The p1A3 peptide modulated the expression of genes related with cell proliferation (VEGFr1 e 2, TGF?β, PDGFβ and eNOS) in HAEC. Conclusion: The mimotope peptides p2C7 and p1A3 interact with the endothelial cells and macrophages by different ways. The p2C7 induces a pro-inflammatory response in macrophages, mimicking LDL (-), thus suggesting that this peptide can act as a DAMP (Damage-associated molecular pattern) important in the inflammatory process of atherosclerotic plaque.

Introdução: A doença cardiovascular é a principal causa de morbimortalidade precoce em todo o mundo, e responde por grande parte dos gastos dos recursos destinados aos programas de políticas públicas. Neste contexto, a dieta representa uma importante ferramenta na redução dos fatores de risco cardiovasculares. Tendo em vista que inúmeros estudos mostram que o consumo de ômega 9 ou alimento fonte modifica positivamente diversos fatores de risco cardiovascular clássicos, se torna importante avaliar seu efeito sobre propriedades físico-químicas da LDL e da HDL, marcadores cardiometabólicos e oxidativos em indivíduos brasileiros com diferentes níveis de risco cardiovascular. Objetivo: Avaliar o efeito do consumo de azeite de oliva sobre parâmetros cardiometabólicos clássicos e novos em indivíduos com diferentes níveis de risco cardiovascular. Métodos: O estudo foi do tipo clínico prospectivo, aleatorizado, placebo controlado, duplo cego baseado em intervenção nutricional. Indivíduos de ambos os sexos, distribuídos em grupos azeite de oliva (AO) e placebo (PL) receberam durante 8 semanas 3 g/d de azeite de oliva ou placebo. Todos os indivíduos foram classificados quanto ao risco cardiovascular, seguindo os critérios estabelecidos pelo Escore de Risco de Framingham (ERF). Nos momentos basal, T=4S e T=8S foram determinados o perfil clínico, antecedentes familiares de doenças, pressão arterial, consumo alimentar e nível de atividade física. A partir do plasma ou soro, obtidos após 12 h de jejum, foram determinados o perfil lipídico, as apolipoproteínas, o tamanho da HDL e da LDL, o conteúdo de LDL(-) e de NEFAS e atividade da paraoxonase. A aderência à intervenção foi monitorada por meios diretos (marcadores bioquímicos) e indiretos (registro de intercorrências). Resultados: O azeite de oliva foi efetivo em reduzir concentração de triacilglicerois dos indivíduos em alto risco cardiovascular (p=0,023 no T=4S e p=0,049 no T=8S) e a de LDL-C dos indivíduos com risco cardiovascular intermediário (p=0,045 no T=8S) no atual estudo. Observou-se também redução significativa na LDL(-), quando a amostra foi estratificada pelo ERF. Demais parâmetros permaneceram inalterados em função do tempo da intervenção e do ERF. Conclusão: Baixa suplementação (3 g/d) de azeite de oliva promoveu redução dos triacilglicerois, LDL-C e da LDL(-). Portanto, recomenda-se a incorporação de azeite de oliva na dieta brasileira ainda que em baixas doses. Sugere-se também que estudos adicionais usando doses maiores sejam realizados no sentido de identificar potenciais benefícios cardioprotetores adicionais associados ao consumo de azeite de oliva. / Introduction: Cardiovascular disease is the leading cause of premature morbidity and mortality worldwide, and accounts for a large part of the costs of resources devoted to public policy programs. In this context, the diet is an important tool in managing and reducing the risk of cardiovascular disease. Given that numerous studies show that consumption of omega 9 or food source changes positively several classical cardiovascular risk factors, it becomes important to evaluate its effect on physicochemical properties of LDL and HDL, cardiometabolic and oxidative markers in Brazilian individuals with different levels of cardiovascular risk. Objective: This study aimed to evaluate the effect of consuming olive oil on classical and new cardiometabolic properties in individuals with different levels of cardiovascular risk. Methods: It was a clinical, prospective, randomized, placebo controlled, double blind study based on nutritional intervention. Individuals of both sexes, divided into groups olive oil (AO) and placebo (PL) for 8 weeks received 3 g/d of olive oil or placebo. All subjects were classified for cardiovascular risk following the criteria established by the Framingham Risk Score (FRS). At baseline period, T = 4W and T = 8W the clinical profile, the family history of diseases, blood pressure, food consumption and physical activity level were determined. From plasma or serum obtained after 12 h of fasting lipid profile, apolipoproteins, the size of LDL and HDL, LDL (-) and NEFAS content, and activity of paraoxonase were determined. Adherence to the intervention was monitored by direct means (biochemical markers) and indirect (register of complications). Results: The olive oil was effective in reducing the concentration of triacylglycerol of individuals at high cardiovascular risk (p = 0.023 at T=4W and p=0.049 at T=8W) and LDL-C in individuals with intermediate cardiovascular risk (p=0.045 at T=8W) in the current study. It was also observed a significant reduction in LDL (-) when the sample was divided by the FRS. However, changes in other parameters were not detected when comparing the intervention group and the placebo group. Conclusion: Even at low dosage, olive oil has proved to be beneficial in reducing triglycerides, LDL-C and LDL (-).It is therefore recommended the incorporation of olive oil in the Brazilian diet even in low doses. It is suggested that future studies to use higher doses in order to check additional benefits associated with olive oil consumption.

by Lo Hoi Ka Elka. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 172-181). / Abstracts in English and Chinese. / ABSTRACT --- p.i / 摘要 --- p.iv / LIST OF TABLES AND FIGURES --- p.viii / ABBREVIATIONS --- p.xiv / Chapter CHAPTER 1 : --- INTRODUCTION / Chapter 1.1. --- DIFFERENT TREATMENTS OF THE CANCER THERAPY --- p.1 / Chapter 1.2. --- THE SIDE EFFECTS OF CANCER TREATMENT / Chapter 1.2.1. --- Surgery --- p.1 / Chapter 1.2.2. --- Radiotherapy --- p.2 / Chapter 1.2.3. --- Chemotherapy --- p.2 / Chapter 1.3. --- THE CHARACTERISTICS OF DOXORUBICIN (DOX) / Chapter 1.3.1. --- The structure of Dox --- p.6 / Chapter 1.3.2. --- The actions of Dox --- p.8 / Chapter 1.3.3. --- The adverse side effect of Dox --- p.8 / Chapter 1.4. --- THE RATIONALE OF USING LOW DENSITY LIPOPROTEIN (LDL) AS A TARGET CARRIER IN CANCER THERAPY / Chapter 1.4.1. --- The correlation between cholesterol and cancer --- p.9 / Chapter 1.4.2. --- Low density lipoprotein (LDL) as a target carrier --- p.11 / Chapter 1.4.3. --- The down and up regulation of LDL receptors --- p.14 / Chapter 1.4.4. --- The characteristics of Fuctus Craegus (FC) --- p.15 / Chapter 1.5. --- DIFFERENT METHODS OF THE PREPARATION OF THE LOW DENSITY LIPOPROTEIN-DRUG (LDL- DRUG) --- p.18 / Chapter 1.6. --- THE CHARACTERISTICS OF LOW DENSITY LIPOPROTEIN (LDL) / Chapter 1.6.1. --- The structure of LDL --- p.20 / Chapter 1.6.2. --- The metabolic pathway of LDL in human bodies --- p.23 / Chapter 1.7. --- THE MULTIDRUGS RESISTANCE IN TUMOR CELLS --- p.25 / Chapter 1.7.1. --- The mechanism of multidrug resistance --- p.27 / Chapter 1.7.2. --- The structure of P-glycoprotein --- p.27 / Chapter 1.7.3. --- The mechanism of P-glycoprotein --- p.30 / Chapter 1.8. --- COMBINED TREATMENT WITH HYPERTHERMIA --- p.31 / Chapter 1.9. --- AIM OF THE STUDY --- p.33 / Chapter CHAPTER 2 : --- MATERIALS AND METHODS / Chapter 2.1. --- MATERIALS / Chapter 2.1.1. --- Animals --- p.34 / Chapter 2.1.2. --- Buffers --- p.34 / Chapter 2.1.3. --- Cell culture reagents --- p.36 / Chapter 2.1.4. --- Chemicals --- p.38 / Chapter 2.1.5. --- Culture of cells --- p.40 / Chapter 2.2. --- METHODS / Chapter 2.2.1. --- In vitro studies / Chapter 2.2.1.1. --- "LDL, doxorubicin complex formation" --- p.41 / Chapter 2.2.1.2. --- Determination of the concentration of LDL-Dox --- p.42 / Chapter 2.2.1.3. --- In vitro cytotoxicity --- p.43 / Chapter 2.2.1.4. --- The cytotoxicity of the combined treatment with anticancer drugs --- p.44 / Chapter 2.2.1.5. --- The preparation of Fructus Crataegus (FC) --- p.46 / Chapter 2.2.1.6. --- Western blot --- p.47 / Chapter 2.2.1.7. --- Flow cytometry --- p.49 / Chapter 2.2.1.8. --- Confocal laser scanning microscopy --- p.52 / Chapter 2.2.2. --- In vivo studies / Chapter 2.2.2.1. --- Subcutaneous injection of R-HepG2 cells in nude mouse --- p.55 / Chapter 2.2.2.2. --- Treatment schedules --- p.55 / Chapter 2.2.2.3. --- Assay of investigating of the myocardial injury --- p.56 / Chapter 2.2.2.4. --- Tissue preparation procedure for light microscope (LM) --- p.57 / Chapter 2.2.3. --- Statistical analysis in our research --- p.59 / Chapter CHAPTER 3 : --- RESULTS / Chapter 3.1. --- in vitro STUDIES / Chapter 3.1.1. --- The preparation of low density lipoprotein-doxorubicin (LDL-Dox) --- p.60 / Chapter 3.1.2. --- Studies on human hepatoma cells line (HepG2 cells) / Chapter 3.1.2.1. --- The comparison of Dox and LDL-Dox accumulated in HepG2 cells --- p.63 / Chapter 3.1.2.2. --- Confocal laser scanning microscopic (CLSM) studies on the accumulation of Dox and LDL-Dox in HepG2 cells --- p.65 / Chapter 3.1.2.3. --- The comparsion of the cytotoxicity of Dox and LDL-Dox on HepG2 cells --- p.67 / Chapter 3.1.2.4. --- The comparison of the cytotoxicty of Dox and LDL-Dox with and without hyperthermia on HepG2 cells --- p.73 / Chapter 3.1.2.5. --- The comparison of accumulation of Dox and LDL-Dox in HepG2 cells treated with and without combination of with hyperthermia --- p.77 / Chapter 3.1.2.6. --- Confocal laser scanning microscopic (CLSM) studies on the accumulation of Dox and LDL-Dox in HepG2 treated cells with and without hyperthermia --- p.80 / Chapter 3.1.2.7. --- Modulation of LDL receptors on HepG2 cells------Up- regulation of LDL receptors by Fructus Craegtus (FC) / Chapter 3.1.2.7.1. --- The comparsion of LDL receptor expression on HepG2 cells after Fructus Craegtus (FC) pre-treatment --- p.83 / Chapter 3.1.2.7.2. --- The comparison of accumulation of LDL-Dox accumulated in HepG2 cells pre-treated with and without Fructus Craegtus (FC) --- p.85 / Chapter 3.1.2.7.3. --- Confocal laser scanning microscopic (CLSM) studies on the accumulation of LDL-Doxin HepG2 cells after Fructus Craegtus (FC) pre- treatment --- p.88 / Chapter 3.1.2.7.4. --- Cytotoxicity of combined treatment with LDL-Dox and Fructus Craegtus (FC) --- p.91 / Chapter 3.1.3. --- Studies on multidrug human resistant hepatoma cell line (R-HepG2 cells) / Chapter 3.1.3.1. --- The overexpression level of P-glycoprotein in resistant cell line R-HepG2 --- p.93 / Chapter 3.1.3.2. --- The comparison of Dox and LDL-Dox accumulated in R- HepG2 cells --- p.95 / Chapter 3.1.3.3. --- Confocal laser scanning microscopic (CLSM) studies on the accumulation of Dox and LDL-Dox in R-HepG2 cells --- p.97 / Chapter 3.1.3.4. --- The comparsion of the cytotoxicity of Dox and LDL-Dox on R-HepG2 cells --- p.99 / Chapter 3.1.3.5. --- The comparison of the cytotoxicty of Dox and LDL-Dox with and without hyperthermia on R-HepG2 cells --- p.109 / Chapter 3.1.3.6. --- The comparison of the accumulation of Dox and LDL- Dox in R-HepG2 cells treated in combination with hyperthermia --- p.113 / Chapter 3.1.3.7. --- Confocal laser scanning microscopic (CLSM) studies on the accumulation of Dox and LDL-Dox in R-HepG2 cells with and without hyperthermia --- p.117 / Chapter 3.1.3.8. --- Modulation of LDL receptors on R-HepG2 cells ------ Up-regulation of LDL receptors by Fructus Craegtus (FC) / Chapter 3.1.3.8.1. --- The comparsion of LDL receptor expression on R-HepG2 cells after Fructus Craegtus (FC) pre-treatment --- p.120 / Chapter 3.1.3.8.2. --- The comparsion of the accumulation of LDL- Dox in R-HepG2 cells after Fructus Craegtus (FC) pre-treatment --- p.122 / Chapter 3.1.3.8.3. --- Confocal laser scanning microscopic (CLSM) studies in the accumulation of LDL-Dox by Fructus Craegtus pre-treatment in R-HepG2 cells --- p.125 / Chapter 3.1.3.8.4. --- The comparison of cytotoxicity of combined treatment with LDL-Dox and Fructus Craegtus (FC) in R-HepG2 cells --- p.128 / Chapter 3.2. --- in vivo STUDIES / Chapter 3.2.1. --- The comparison of Dox and LDL-Dox on reducing the tumor sizes and weight in nude mice bearing R-HepG2 cells / Chapter 3.2.1.1. --- The comparison of Dox and LDL-Dox on reducing the tumor size in nude mice bearing R-HepG2 cells --- p.130 / Chapter 3.2.1.2. --- The comparison of Dox and LDL-Dox on reducing the tumor weight in nude mice bearing R-HepG2 cells --- p.138 / Chapter 3.2.2. --- Myocardial injury measured by Lactate dehydrogenase (LDH) activity in nude mice bearing R-HepG2 cells treated with Dox and LDL-Dox --- p.140 / Chapter 3.2.3. --- Myocardial injury measured by Creatine kinase (CK) activity in nude mice bearing R-HepG2 cells treated with Dox and LDL-Dox --- p.143 / Chapter 3.2.4. --- Histological studies of heart of nude mice bearing R-HepG2 cells treated with Dox and LDL-Dox / Chapter 3.2.4.1. --- Heart section of nude mice --- p.146 / Chapter 3.2.4.2. --- Heart section of nude mice bearing R-HepG2 cells --- p.148 / Chapter 3.2.4.3. --- Heart section of lmg/kg Dox treated nude mice bearing R- HepG2 cells --- p.150 / Chapter 3.2.4.4. --- Heart section of 2mg/kg Dox treated nude mice bearing R- HepG2 cells --- p.152 / Chapter 3.2.4.5. --- Heart section of lmg/kg LDL-Dox treated nude mice bearing R-HepG2 cells --- p.154 / Chapter CHAPTER 4 --- : DISCUSSION / Chapter 4.1. --- in vitro STUDIES / Chapter 4.1.1. --- The cytotoxicity of Dox and LDL-Dox on HepG2 cells and R- HepG2 cells --- p.156 / Chapter 4.1.2. --- The combined treatment on HepG2 cells and R-HepG2 cells --- p.157 / Chapter 4.1.3. --- The modulation of LDL-R expression --- p.159 / Chapter 4.2. --- in vivo STUDIES --- p.162 / Chapter CHAPTER 5 --- : CONCLUSION / Chapter 5.1. --- CONCLUSION / Chapter 5.1.1. --- In vitro studies --- p.167 / Chapter 5.1.2. --- In vivo studies --- p.169 / Chapter 5.2. --- FUTURE PROSPECTIVE --- p.170 / REFERENCES --- p.172

L'athérosclérose est une complication cardiovasculaire majeure des maladies liées à une augmentation du stress oxydatif, comme le diabète de type 2 et le syndrome métabolique. Dans ces situations, les lipoprotéines de faible densité (LDL) subissent une oxydation et leur forte absorption induit une accumulation de cholestérol dans les macrophages sous-endothéliaux. D'autre part, les LDL oxydées sont enrichies en produits d'oxydation du cholestérol appelés oxystérols, dont certains sont impliqués dans la capacité des LDL oxydées à induire un stress oxydant cellulaire et une cytotoxicité, principalement par apoptose. Le Bis(Monoacylglycéro)Phosphate (BMP) est un phospholipide unique, localisé préférentiellement dans les endosomes tardifs, compartiment cellulaire clef dans le métabolisme du cholestérol dérivé des LDL. Lors de travaux antérieurs, l'équipe a démontré le rôle prépondérant du BMP dans la régulation de l'homéostasie du cholestérol dans les macrophages. L'objectif de ce travail a été de décrypter les mécanismes moléculaires intervenant dans le trafic intracellulaire du cholestérol. Ainsi, le BMP régule l'efflux de cholestérol par les HDL (high density lipoproteins) grâce à des mécanismes impliquant les LXRs (liver X receptors) et les transporteurs ABCA1/ABCG1 (ATP binding cassette-type A1/G1). De plus, notre étude indique que le BMP exerce également un rôle protecteur contre l'effet pro-apoptotique des LDL oxydées via la réduction de la production intracellulaire d'oxystérols. Comme une partie du trafic intracellulaire des stérols au sein des macrophages est régulé par OSBP (oxysterol binding protein) et ses protéines dérivées, les ORPs (OSBP-related proteins), nous montrons dans ce rapport que l'action de protection du BMP contre les effets cytotoxiques des oxystérols est fortement diminuée dans des cellules où la protéine ORP11 est supprimée, suggérant que le BMP exerce son rôle protecteur via un mécanisme utilisant la fonction d'ORP11 dans le transport intracellulaire de stérols / Atherosclerosis is a major cardiovascular complication in increased oxidative stress-related diseases such as type 2 diabetes and metabolic syndrome. In these situations, the low density lipoproteins (LDL) undergo oxidation and their high uptake induces cholesterol accumulation in subendothelial macrophages. On the other hand, oxidized LDL are enriched in cholesterol oxidation products called oxysterols, some of them are involved in the ability of oxidized LDL to induce cellular oxidative stress and cytotoxicity, mainly by apoptosis. Bis(Monoacylglycero)Phosphate (BMP) is a unique phospholipid localized preferentially in late endosomes, a central cellular compartment of LDL-cholesterol metabolism. In previous work, the team demonstrated the leading role of BMP in regulation of cholesterol homeostasis in macrophages. The aim of this work was to characterize the molecular mechanisms involved in the intracellular trafficking of cholesterol. Thus, BMP regulates cholesterol efflux to HDL (high density lipoproteins) by mechanisms involving liver X receptors (LXRs) and ABCA1/ABCG1 (ATP binding cassette-type A1/G1) transporters. Moreover, we report BMP also exerts a protective role against the pro-apoptotic effect of oxidized LDL via a reduced production of intracellular pro-apoptotic oxysterols.As part of macrophage intracellular sterol traffic is regulated by oxysterol binding protein (OSBP) and OSBP-related proteins (ORPs), we show that protective action of BMP against cytotoxic oxysterol effects in ORP11-silenced cells, was markedly abrogated, suggesting BMP exerts its protective role via a mechanism involving the function of ORP11 in intracellular sterol transport

Introdução A Síndrome Metabólica (SM) representa um conjunto de fatores que determinam maior risco para Doença Cardiovascular (DCV), devido principalmente à Resistência à Insulina (RI) e ao estado inflamatório promovido pelo tecido adiposo branco hipertrofiado. Nesse contexto, a condição de dislipidemia favorece o desenvolvimento da aterosclerose, devido a que maiores concentrações de lipoproteína de baixa densidade (LDL) no plasma podem propiciar modificações nessas partículas. Essas modificações podem ter origem oxidativa ou não oxidativa e impactar nas características aterogênicas da LDL. O ômega três tem mostrado efeitos hipolipemiantes e anti-inflamatórios, que podem reduzir o risco cardiovascular de indivíduos com SM. Objetivo:: avaliar o efeito da suplementação de 3g de &#969;-3 por um período de oito semanas sobre a concentração plasmática das partículas de LDL eletronegativas [LDL(-)] e seus anticorpos, assim como monitorar possíveis mudanças nas concentrações das subfrações das LDL. Metodologia: Foram incluídos 115 participantes de ambos os sexos, entre 30 e 74 anos, com SM, separados nos grupos de intervenção com &#969;-3 (n=58) e controle com &#969;-9 (n=57). Foram realizadas análises de perfil lipídico e por meio de ELISA foram avaliadas a concentração de LDL(-) e de antiLDL(-) no plasma dos participantes. O tamanho das subfrações de LDL foi realizado no sistema Lipoprint. O efeito do tempo e das intervenções foi testado por meio do programa SPSS versão 20.0, adotando-se o concentração de significância de p<0,05. Resultados: A suplementação de &#969;-3 teve efeito significativo na redução do % de massa grassa (%MG) e na pressão arterial sistólica (4%) e diastólica (5%). Em relação ao perfil lipídico, ambas intervenções tiveram efeitos significativos de redução de colesterol total (CT), LDL-c, não HDL (nHDL) e triacilglicerois (TG). As concentrações plasmáticas de LDL(-) dos participantes que tomaram ômega 3 apresentaram redução de 21%, enquanto que os que tomaram ômega 9 tiveram redução de 1%. Tal redução foi significativa em relação ao tempo de intervenção (p<0,05), mas não quando o efeito da internveção foi avaliado. Perfil semelhante foi observado para a razão LDL(-)/antiLDL(-), observou-se redução de 22% no grupo ômega 3 e redução de somente 3% no grupo ômega 9. Conclusão: A intervenção com ômega 3 promoveu redução na concentração plasmática de LDL(-), porém não modificou a concentração do anticorpo antiLDL(-) nem o tamanho das subfrações da LDL. / Introduction: The Metabolic Syndrome (MetS) is a combination of factors that determine increased risk for Cardiovascular Diseases (CVD), mainly because of Insulin Resistance (IR) and the inflammatory state promoted by the hypertrophied white adipose tissue. Under this background, the dyslipidemic condition goes together with the developing of atherosclerosis, meaning that higher concentrations of low density lipoprotein (LDL) in plasma promote modifications from different sources in these particles and may have an impact over LDL subfractions, turning them more atherogenic. Omega three has proved hypolipidemic and anti-inflammatory effects, which may reduce cardiovascular risk in MetS individuals. Objetive: evaluate the effect of 3g of fish oil supplementation, 60% EPA and DHA during an eight week period over plasma concentrations of electronegative LDL particles [LDL(-)] and its antibodies, as well as monitoring possible changes in LDL subfractions. Methods: A total number of 115 participants, both sexes, between 30 and 74 years of age, with MetS, were included and separated in the intervention group receiving omega 3 (n=58) and the placebo group with omega 9 (n=57). Biochemical analyses were carried out using ELISA for LDL(-) and antiLDL(-) in plasma. Particle sizes were measured using the Lipoprint system. The effects of time and intervention were analyzed using the statistical program SPSS 20.0, assuming p<0.05 as significant. Results: Omega 3 supplementation had a significant effect in the reduction of fat mass percentage (FM%) and blood pressure, showing a 4% decrease for systolic and 5% decrease for diastolic pressures respectively. Considering the lipid profile, both interventions had significant effects reducing total cholesterol (TC), LDL-c, not HDL (nHDL) and tryacylglycerides (TG). LDL(-) plasma concentrations from the omega 3 group showed 21% reduction while the omega 9 group had only 1% reduction. Such difference was significant considering time (p<0,05), but not while comparing the intervention effect. There was also a change in the antiLDL(-) antibodies; reducing 2% in the omega 3 group and increasing 1% in the omega 9 group. However, those differences were not significant. Similar effects were observed in LDL(-)/antiLDL(-) ratio, showing 22% decrease in the omega 3 group and only 3% in the omega 9 group. Conclusion: Omega 3 intervention promoted a significant reduction in plasmatic LDL(-), however, it didn\'t modify LDL subfraction distribution.

Anticorpos (Ac) anti-oxLDL estão envolvidos no desenvolvimento da lesão aterosclerótica. Induzimos a produção desses Ac através da imunização com oxLDL. Avaliamos os perfis lipídico e de Ac anti-oxLDL e anti-pepD (pepD é um peptídeo derivado da apoB). Animais apoE-/- possuem maiores níveis de lípides sorológicos em comparação com animais C57Bl/6, exceção da HDL. A imunização oxLDL não alterou este perfil. A imunização aumentou os títulos dos Ac IgM, IgG e IgG1 anti-oxLDL. Animais C57Bl/6, mas não os apoE-/- tiveram aumento dos Ac IgM anti-pepD após a imunização. Acreditamos que o aumento de anticorpos IgG1 deve-se ao adjuvante usado. A aterosclerose pode ocorrer com maior freqüência, em indivíduos portadores de doenças renais crônicas. Para estudar essa interferência submetemos os animais a um modelo de isquemia e reperfusão renal (I/R). A I/R promoveu aumento da concentração de TG, podendo agravar a aterosclerose. O aumento de Ac IgG anti-oxLDL promovidos pela I/R sugere que o processo inflamatório desencadeado por este procedimento, aumenta a oxidação de LDL. Desta forma, concluímos que a I/R pode ser considerado um procedimento pró-aterosclerótico que não pode ser revertido pela imunização com oxLDL. / Oxidized LDL (oxLDL) antibodies (Ab) are involved in the development of the atherosclerotic lesion. We induced the formation of such Ab through immunization with oxLDL. We evaluate the lipids and antibody (anti-oxLDL and anti-pepD pep D is a peptide derived from apolipoprotein B) profile. The apoE-/- mice used have higher seric levels of most lipids than C57Bl/6 animals, the exception being HDL. The oxLDL immunization does not change this profile. The immunization increased anti-oxLDL Ab e IgM, IgG and IgG1 titers. Anti-pepD IgM Ab increased with immunization in C57Bl/6 animals only, and not in apoE-/-. We believe the increased IgG1 Ab due to the adjuvant used. Atherosclerosis has a higher incidence in individuals with chronic renal conditions. In order to study such interference, we submitted the animals to a renal ischemia and reperfusion model (I/R). The I/R procedure increased TG concentration, what can make atherosclerosis more severe. The increase in IgG anti-oxLDL antibody caused by I/R suggests that the inflammation process set by the procedure increases LDL oxidation. This way, we conclude that the I/R can be considered a pro-atherosclerotic procedure. The oxLDL immunization was not able to revert the atherogenic effect caused by I/R model.

A hipercolesterolemia familiar (HF) é uma doença autossômica dominante causada por mutações no gene do receptor de LDL (LDLR) (cromossomo 19p13.1 - p13.3), que alteram parcialmente ou totalmente a função do LDLR. A HF é também uma das doenças genéticas mais comuns com freqüências estimadas de heterozigotos e homozigotos de 1/500 e 1/1.000.000, respectivamente. Manifesta-se com altos níveis de LDL colesterol, arco corneal, xantomas tendíneos e sintomas prematuros de doença coronariana.. A grande heterogeneidade observada na manifestação clínica desta doença pode ser explicada, ao menos parcialmente, pelo amplo espectro de mutações no gene do LDLR. O presente estudo teve por objetivo a caracterização molecular do gene LDLR em pacientes com HF do Rio Grande do Sul (RS), Brasil. Para isso, foram obtidas amostras de DNA de 40 indivíduos provenientes de cinco macrorregiões do Estado, representando seis diferentes populações de ascendência européia, para a realização do seqüenciamento direto do gene do LDLR, com posterior análise por meio das ferramentas de bioinformática. Quinze mutações pontuais foram identificadas no gene do LDLR, a saber: c.408C>T (D115D), c.1616C>T (P518L), c.1773C>T (N570N) e c.2243A>G (D727G) na região codificadora, IVS6+36G>A, IVS6+171G>A, IVS11+56C>T, IVS11- 69G>T, IVS11-55A>C, IVS15-136A>G, IVS16+46C>T e IVS17-42A>G na região intrônica, e *52G>A, *105T>G e *141G>A na região 3\'-UTR. Destas, oito ainda não foram descritas na literatura (três situadas nos exons, quatro nos introns e uma na região 3\'-UTR). A mutação*52G>A foi previamente identificada em pacientes com HF da região Sudeste do Brasil, sugerindo que possa exercer um importante efeito na patogênese da HF em pacientes brasileiros. Em relação às macrorregiões do RS, os portugueses, italianos e espanhóis apresentaram o maior número de mutações dentre os grupos étnicos analisados. Assim, os resultados obtidos confirmam que existe um amplo de espectro de mutações no gene do LDLR. As mutações nas regiões intrônicas precisam ser investigadas sobre seu efeito potencial no desenvolvimento de HF. Considerando que este é o primeiro estudo que teve por objetivo a caracterização molecular de pacientes com HF no RS, novos estudos que visem a elucidação das bases moleculares da HF devem ser realizados, a fim de obter uma melhor caracterização genética desta doença no Brasil. / Familial hypercholesterolemia (FH) is an autosomal dominant disorder caused by mutations in the low-density lipoprotein receptor (LDLR) gene (chromosome 19p13.1 - p13.3), which alter partially or totally the LDLR function. FH is also one of the most common inherited disorders with frequencies of heterozygotes and homozygotes estimated to be 1/500 and 1/1.000.000, respectively. Affected individuals display high levels of LDL cholesterol, arcus corneae, tendon xanthomas and premature symptomatic coronary heart disease. The extensive heterogeneity observed in the clinical manifestation of this disorder may be explained, at least partially, by the broad spectrum of mutations identified in the LDLR gene. The present study had as the main goal the molecular characterization of the LDLR gene in patients with FH from Rio Grande do Sul (RS) State, Brazil. For this, DNA samples were obtained from 40 individuals living in five macroregions of RS, representing six different isolated populations of European ascendancy. The LDLR gene was subjected to the direct sequencing with further analysis through bioinformatics tools. Fifteen punctual mutations were identified in the LDLR gene, namely: c.408C>T (D115D), c.1616C>T (P518L), c.1773C>T (N570N) and c.2243A>G (D727G) in the coding region, IVS6+36G>A, IVS6+171G>A, IVS11+56C>T, IVS11-69G>T, IVS11-55A>C, IVS15-136A>G, IVS16+46C>T and IVS17-42A>G in the intronic region, and *52G>A, *105T>G and *141G>A in the 3\'-UTR region. Of these, eight were not yet described in the literature (three situated in exons, four in introns and one in 3\'- UTR region). The *52G>A mutation was previously identified in FH patients from Southeast Brazil, suggesting that it can exert an important effect in the pathogenesis of FH in Brazilian patients. In relation to the macroregions of Rio Grande do Sul, Portuguese, Italian and Spanish subjects carried the highest number of mutations among the ethnic groups analyzed. Thus, the results obtained confirm the existence of a broad spectrum of mutations in the LDLR gene. The mutations in intronic regions need to be investigated in relation to its potential effect in the development of FH. Taking into account that this is the first study that had as the goal the molecular characterization of FH patients in RS, further studies aimed at elucidating the molecular bases of FH should be performed, in order to obtain the better characterization of this disease in Brazil.