Dissertations / Theses on the topic 'High density lipoproteins'

Chapter 1 provides an overview of the structure, composition, metabolism and clinical significance of the plasma lipoproteins, and specifically focuses on high density lipoprotein (HDL), and its major subfractions (HDL2 and HDL3). Chapter 2 describes the development and validation of a rapid ultracentrifugation method for the isolation of HDL2 and HDL3. This facilitated the examination of HDL subfractions in a variety of contexts, and lead to the discovery of a novel pro-oxidant effect of HDL on the oxidation ofvery low density lipoprotein (VLDL). Chapter 3 explores the role which HDL subfractions may play in endothelial dysfunction, by examining their effect on human coronary artery endothelial cells. Overall, native HDLs reduced the expression of pro-inflammatory molecules; in the oxidised state, HDLs promoted the release ofpro-inflammatory molecules. Chapter 4 describes the involvement of lecithin:cholesterol acyltransferase in lipoprotein oxidation and atherosclerosis. This work identified a duplicitous effect of LCAT during oxidation ofApo B-containing lipoproteins; it acted as a pro-oxidant during VLDL oxidation, but as an antioxidant during oxidation oflow density lipoprotein (LDL). Chapter 5 reports the effect of metfonnin and pioglitazone on the composition and oxidation of HDLz and HDL3, in a group of obese and overweight men. Results demonstrated that pioglitazone exhibited a broader range of effects on HDL subfractions, particularly by increasing the HDLz-to-HDL3 ratio - a change associated with improved reverse cholesterol transport. Chapter 6 presented a clinical case study on inherited cholesterol ester transfer protein (CETP) deficiency. HDL isolated from the proband had an abnormal composition and oxidation profile. Moreover, HDLisolated from the proband had a reduced pro-oxidant effect towards VLDL, confinning the participation ofCETP in this novel reaction ofHDL. Chapter 7 provides a general discussion, and concluding remarks, for the main body of the thesis, including suggestions for future work.

Hepatic lipase (HL) plays a pivotal role in the catabolism of apolipoprotein (apo)B-containing lipoproteins and high density lipoprotein (HDL) particles through its reported catalytic and non-catalytic extracellular functions. The current study tested the hypothesis that HL expression might impair formation and secretion of hepatic derived very low density lipoproteins (VLDL) and apoA-I (nascent HDL). Stable or transient expression of human HL (hHL) in McA-RH7777 cells resulted in decreased incorporation of [3H]glycerol into cell-associated and secreted (VLDL-associated) 3H-triacylglcyerol (TAG) relative to control cells. Stable expression of catalytically-inactive hHL (hHLSG) also resulted in decreased secretion of VLDL-associated 3H-TAG whereas cell-associated 3H-TAG levels were unchanged. Expression of hHL or hHLSG increased cell-associated 35S-apoB100 with relatively no change in secreted 35S-apoB100. Importantly, hHL or hHLSG expression resulted in reduced 3H-TAG associated with the microsomal lumen lipid droplets (LLD), and increased relative expression of ApoB and genes involved in lipogenesis and fatty acyl oxidation. Transient expression of hHL in HL-null primary hepatocytes, mediated by adenoviral gene transfer, resulted in decreased steady-state levels of cell-associated and secreted apoA-I and reduced rates of synthesis and secretion of 35S-apoA-I. HL-null hepatocytes exhibited increased levels of secreted 35S-apoA-I relative to wildtype hepatocytes while cell-associated 35S-apoA-I levels were normal. Transient expression of a hHL chimera (hHLmt), in which the C-terminus of hHL was replaced with mouse HL sequences, exerted an inhibitory effect on apoA-I production similar to that of hHL even though hHLmt was secreted less effectively than hHL with impaired exit from the endoplasmic reticulum (ER) as compared with hHL. In contrast, stable expression of hHL in McA-RH7777 cells resulted in a dose-dependent increase in cell-associated and secreted 35S-apoA-I levels. These studies demonstrate that hHL has an intracellular (but non-catalytic) role in reducing the content of the LLD and ultimately the buoyancy of secreted VLDL particles, and that the N-terminal sequences of ER-residing hHL directly or indirectly modulates the production and secretion of apoA-I (nascent HDL) from hepatocytes.

Reduced high-density lipoprotein cholesterol (HDL-C) concentrations are a major risk factor for cardiovascular disease. Intense interest recently has been in identifying the genetic factors that contribute to an aberrant HDL-C phenotype. Finding genetic factors associated with low HDL-C concentrations in New Zealanders was the major aim of this study, with a similar analysis aimed at finding factors associated with high HDL-C concentrations included. The study began with an investigation of a New Zealand family with HDL-C deficiency. The proband had virtually no HDL-C, and was homozygous for an R1068H mutation in the ABCA1 gene. Nineteen relatives were recruited into the study, and an analysis aimed at finding the underlying cause for the disparate HDL-C phenotypes amongst H1068 carriers initiated. A further investigation into HDL-C levels aimed to find other genetic factors that contribute to HDL-C in New Zealanders. Subjects were selected from those taking part in the Otago Vascular Disease study, and placed into three groups based on their HDL-C lipid measurement: low (n=154), mid (n=105), and high (n=102) HDL-C. Mutations in ABCAI and the apolipoprotein AI gene (APOAI) were identified in subjects with low HDL-C. Polymorphisms in ABCA1 and APOAI were also investigated as contributors to HDL-C concentrations. Seventeen SNPs in functionally relevant regions were genotyped in the three HDL-C groups. Five single polymorphisms and one polymorphism haplotype showed a statistically significant association with HDL-C concentrations. The final analysis investigated plasma lipoprotein compositions in individuals with low HDL-C to identify if other lipoprotein abnormalities concurred. Individuals with HDL-C below 0.65 mmol/L were almost invariably associated with triglyceride-rich VLDL and/or triglyceride-rich LDL. This finding may have relevance for an increased atherosclerotic risk for those with low HDL-C.

[Truncated abstract] The metabolic syndrome is characterized by cardiovascular risk factors including dyslipidemia, insulin resistance, visceral obesity, hypertension and diabetes. The dyslipidemia of the metabolic syndrome includes elevated plasma triglyceride and apolipoprotein (apo) B levels, accumulation of small, dense low-density lipoprotein (LDL) particles and low high-density lipoprotein (HDL) cholesterol concentration. However, the precise mechanisms for this dyslipoproteinemia, specifically low plasma HDL cholesterol, are not well understood. This thesis therefore, focuses on HDL, its structure, function and metabolism. However, lipoprotein metabolism is a complex interconnected system, which includes forward and reverse cholesterol transport pathways. Hence, this thesis also examines and discusses the metabolism of apoB-containing lipoproteins. This thesis tests the general hypothesis that apolipoprotein kinetics are altered in the metabolic syndrome, and that lipid regulating therapies can improve these kinetic abnormalities. The aims were first, to compare and establish the clinical, metabolic and kinetic differences between metabolic syndrome and lean subjects; and second, to determine the regulatory effects of statin therapy, specifically, rosuvastatin on lipoprotein transport in the metabolic syndrome. Five observation statements were derived from the general hypothesis and examined in the studies described below. The findings are presented separately as a series of original publications. Study 1 Twelve men with the metabolic syndrome and ten lean men were studied in a case-control setting. ... These findings explain the HDL raising effects of rosuvastatin in the metabolic syndrome. Collectively, these studies suggest that the dyslipidemia of the metabolic syndrome results from increased production rates of VLDL and LDL particles, reduced fractional catabolic rates of these lipoproteins, together with accelerated catabolism of HDL particles. Treatment with rosuvastatin increases the catabolic rates of all apoB-containing lipoproteins and at a higher dose, decreases LDL apoB production. These effects are consistent with inhibition of cholesterol synthesis leading to an upregulation of LDL receptors. Rosuvastatin decreases the fractional catabolism of HDL particles. The effects of rosuvastatin on HDL kinetics may be related to a reduction in triglyceride concentration and cholesterol ester transfer protein activity. These findings are consistent with the general hypothesis that apolipoprotein kinetics are altered in the metabolic syndrome, and that statin therapy improves these kinetic abnormalities.

RESUMO:Aterosclerose é uma das principais causas de morbilidade e mortalidade no mundo ocidental. É responsável, direta ou indiretamente, pela maior percentagem de gastos com a saúde na maioria dos países europeus. A “teoria lipídica” da aterosclerose, que se baseia na dislipidemia como causa primária para a doença vascular tem algumas implicações práticas importantes: permite a definição de linhas de orientação e protocolos simples e ainda estabelece alvos terapêuticos que podem ser atingidos na maior parte dos casos com a atual intervenção farmacológica. A associação da aterosclerose com o sistema imunológico (a “teoria imunológica”), forneceu por sua vez novas formas de explorar os mecanismos envolvidos e abriu novas perspetivas para um conhecimento mais completo da doença. No entanto, levanta dificuldades evidentes no que diz respeito às possibilidades terapêuticas. De todos os intervenientes no processo aterosclerótico (bioquímicos, imunológicos e anatómicos), as lipoproteínas de elevada densidade (HDL) são atualmente reconhecidas como um dos fatores mais importantes na aterogénese. Isto é baseado no reconhecimento das múltiplas propriedades anti-aterogénicas das HDL como por exemplo: a anti-oxidante, a anti-inflamatória e a antitrombótica, bem como o seu importante papel na melhoraria da função endotelial. Atualmente, é consensual que as funções anti-aterogénicas das HDL vão além do seu papel no transporte reverso do colesterol (RCT) e a importância das HDL no processo aterosclerótico baseia-se não apenas no seu papel protetor impedindo a formação da placa de ateroma, mas também na estabilização destas, prevenindo a sua ruptura e, consequentemente o evento trombótico. Como fundamentais no processo aterosclerótico estão reconhecidos dois principais conjuntos de eventos: um caracterizado por alterações no metabolismo das lipoproteínas que resultam em lipoproteínas pró-inflamatórias e pró-oxidantes que interagem com os componentes celulares da parede arterial e que conduzem à formação da placa de ateroma; o outro evento é a resposta imunológica desencadeada contra um novo conjunto de antigénios que por sua vez leva à produção de citoquinas pró-inflamatórias. Dada a complexidade da HDL e das suas múltiplas funções estas lipoproteínas tornaram-se um potencial alvo para a resposta auto-imune, e cujas consequências podem explicar algumas das associações identificados em estudos clínicos e epidemiológicos. Contudo esta interação entre o sistema imunológico e HDL nunca foi exaustivamente estudada. Portanto, pomos a hipótese de que em condições oxidativas e pró-inflamatórias, um aumento do antigénio (HDL) conduz a um consequente acréscimo na produção de anticorpos anti-HDL (aHDL) responsáveis pela alteração quantitativa e / ou qualitativa das HDL. O conceito de que estes anticorpos podem contribuir tanto para a evolução a longo prazo do processo aterosclerótico, como para o desencadeamento de eventos clínicos pode também explicar a heterogeneidade encontrada em cada doente e nos grandes estudos clínicos, no que diz respeito aos fatores de risco e outcomes clínicos. Para além disso, a confirmação desta hipótese pode permitir explicar porque é que as intervenções terapêuticas atualmente em desenvolvimento para aumentar os níveis de HDL, não conseguem mostrar a tão esperada redução do risco vascular. O objetivo geral desta tese foi identificar e caracterizar a resposta humoral contra os componentes da HDL, e avaliar possíveis mecanismos que possam contribuir para a modificação das propriedades anti-aterogénicas das HDL. Para alcançar este objetivo investigou-se: 1) A presença de anticorpos aHDL em doentes com lúpus eritematoso sistémico (SLE) e em doentes com manifestações clínicas de aterosclerose, como os doentes com doença arterial coronária (CAD), acidente vascular cerebral isquémico (IS) e diabetes tipo 2; 2) Os principais alvos antigénicos dentro do complexo das HDL e a associação entre os títulos de anticorpos aHDL e diferentes características clínicas destas doenças; 3) As modificações das funções normais associadas às HDL, em particular da função anti-oxidante e anti-inflamatória; 4) A atividade biológica dos anticorpos aHDL isolados do soro de doentes através de um conjunto de experiências in vitro de inibição da atividade da paraoxonase 1 (PON1) e da expressão de moléculas de adesão em culturas de células endoteliais. Para tal foi necessário estabelecer um método de isolamento dos anticorpos. Os anticorpos aHDL isolados do soro de doentes foram utilizados de forma a identificar as potenciais alterações dos sistemas celulares utilizados; 5) O efeito de fármacos usados no tratamento das dislipidemias, em particular o ácido nicotínico e as estatinas, na variação dos títulos de anticorpos aHDL através de ensaios clínicos randomizados, controlados com placebo e em dupla ocultação. Os métodos utilizados neste trabalho incluíram: técnicas imunológicas (como por exemplo, enzyme-linked immunoabsorbent assay - ELISA, ensaio imunoturbidimetrico e cromatografia de imuno-afinidade) técnicas bioquímicas (tais como a quantificação de atividade enzimática por espectrofotometria e por luminescência), experiências com cultura de células e citometria de fluxo. Os nossos resultados mostram que: 1) A presença de anticorpos aHDL, e mais especificamente anticorpos contra alguns do seus principais componentes como a apolipoproteína A-I (ApoA-I, principal apolipoproteína presente nas HDL) e a PON1 (o enzima que mais contribui para a propriedade anti-oxidante das HDL), quer em doentes com doenças auto-imunes, como o SLE, quer em doentes com manifestações clínicas de aterosclerose, como CAD, IS e diabetes tipo 2. Os doentes apresentaram títulos de anticorpos IgG aHDL, aApoA-I e aPON1 significativamente mais elevados do que controlos saudáveis com a mesma idade e sexo. 2) A correlação positiva estatisticamente significativa entre os títulos de aHDL e aApoA-I e aPON1 sugere que estes sejam dois dos principais alvos antigénicos dentro do complexo das HDL. Os anticorpos encontrados nestes doentes estão associados com a diminuição da atividade da PON1 e a uma redução da capacidade anti-oxidante total (TAC) do soro, um aumento dos biomarcadores de disfunção endotelial (como por exemplo dos metabolitos do óxido nítrico - NO2- e NO3-, as moléculas de adesão vascular e intracelular - VCAM-1 e ICAM-1 e os níveis de 3-nitrotirosina). Nos doentes com SLE os títulos destes estão associados a um aumento do dano cardiovascular e à atividade global da doença avaliados pelas escalas SLICC/ACR DI e BILAG score, respetivamente. Enquanto que nos doentes com diabetes tipo 2 estes anticorpos estão associados com um aumento dos níveis de glicemia em jejum (FGP) e hemoglobina glicada (HbA1c). 3) Após se ter estabelecido um método de isolamento dos anticorpos que permite isolar quantidades significativas de anticorpos do soro de doentes sem perder a sua especificidade, foi identificada a capacidade dos anticorpos isolados do soro de doentes inibirem de uma forma dependente da concentração a atividade da PON1 até um máximo de 70% no caso dos doentes com SLE e ente 7-52% no caso dos anticorpos isolados de doentes com CAD e IS. 4) O efeito anti-inflamatório das HDL na inibição da produção de VCAM-1 induzida por citoquinas (como o TNF-) foi revertido em mais de 80% pelos anticorpos aHDL isolados do soro de doentes. 5) A angiogenesis induzida por HDL através do aumento do fator de crescimento do endotélio vascular (VEGF) foi anulada em 65% pelos anticorpos aHDL isolados do soro de doentes. 6) Os atuais agentes farmacológicos disponíveis para aumentar as concentrações de HDL-C estão associados a um aumento dos títulos de anticorpos.-------- ABSTRACTAtherosclerosis is the major cause of morbidity and mortality in the western world. It is also responsible, directly or indirectly, for the highest percentage of health costs in most European countries. Despite the use of new technologies for the diagnosis of vascular disease and regardless of the major advances in treatment, the atherosclerosis-related clinical burden is still raising. The “lipid theory” of atherogenesis, which identifies dyslipidemia as the primary cause of this vascular disease has some important practical implications: it allows the definition of simple guidelines and establishes therapeutic targets which can be generally met with current pharmacologic intervention. The association between atherosclerosis an the immune system (the immune concept) has in turn provided new ways of exploring the mechanisms involved in this condition and has opened new perspectives in the understanding of the disease. However, it raises obvious difficulties when it comes to treatment options. Of all the players (biochemical, immunological and anatomical) involved in this matter, high-density lipoproteins (HDL) are currently recognised as one of the most important factors in atherogenesis. This is based on the recognition of HDL's multiple anti-atherogenic properties: anti-oxidant, anti-inflammatory and antithrombotic, as well as its capacity to improve endothelial function. Nowadays, it is widely recognized that the anti-atherogenic functions of HDL go beyond reverse cholesterol transport (RCT), and the importance of HDL is based not just on its ability to reduce atheroma formation but also on its ability to stabilise plaques, therefore preventing their rupture and ultimately thrombosis. Two main set of events have been recognised as fundamental in atherogenesis: one, characterized by lipoprotein metabolism alterations, resulting in pro-inflammatory and pro-oxidative lipoproteins, which interact with the normal cellular elements of the arterial wall leading to atheroma formation; the other, the immune cellular response towards new sets of antigens which lead to the production of pro-inflammatory cytokines. Given to HDL complexity and multiple functions this lipoprotein has became a potential target for an auto-immune response, the consequences of which may explain some of the association identified in epidemiological and clinical studies, though the interaction between the immune system and HDL has never been thoroughly addressed. Therefore, we hypothesized that under oxidative and pro-inflammatory conditions, the increase in the antigen (HDL) would lead to a consequent increase in the production of anti-HDL (aHDL) antibodies be responsible for quantitative and/or qualitative changes of HDL. The concept that these antibodies may contribute either to the long-term evolution of atherosclerosis or to the triggering of clinical events may also explain the heterogeneity found in individual patients and in large cohorts regarding risk factors and clinical outcomes. Moreover this may be a major breakthrough in understanding why therapeutic interventions that increase HDL levels, failed to show the anticipated reduction in vascular risk. The overall aims of this thesis were to identified and characterize the humoral response towards HDL components and to evaluate the possible mechanisms that may contribute to the modifications of the anti-atherogenic properties of HDL. To achieve this objective we investigated: 1) the presence of aHDL antibodies in patients with systemic lupus erythematosus (SLE) and in patients with atherosclerosis-related clinical events, such as coronary artery disease (CAD), ischemic stroke (IS) and type 2 diabetes; 2) the association between the titres of aHDL antibodies and different clinical features of these diseases; 3) the modifications of the anti-atherogenic properties of HDL; 4) the biologic effect of aHDL antibodies isolated from serum of patients on the anti-oxidant and anti-inflammatory properties of HDL; 5) the effect of different pharmacologic treatments for dyslipidemia on the prevalence and activity of aHDL antibodies. The methodologies used in this work included immunologic-related techniques (e.g. enzyme-linked immunoabsorbent assay – ELISA, immunoturbidimetric immunoassay and immunoaffinity chromatography), biochemical techniques (enzymatic assays with quantification by spectrophotometry and luminescence methods), cell culture experiments and flow cytometry. Our results indicate that: 1) The titres of IgG aHDL, anti-apolipoprotein A-I (aApoA-I) and anti-paraoxonase 1 (aPON1) antibodies were higher in patients with SLE, CAD, IS and type 2 diabetes when compared with age and sex matched healthy controls. 2) The antibodies found in these patients were associated with decreased PON1 activity, (the enzyme responsible for most of the anti-oxidant effect of HDL), reduced total anti-oxidant capacity (TAC) of serum and increased biomarkers of endothelial dysfunction (nitric oxide metabolites, adhesion molecules, nitrotyrosine). In patients with SLE the antibody titres were associated with an increase in disease-related cardiovascular damage and activity whereas in patients with type 2 diabetes they were directly related with the fasting glucose plasma (FGP) levels and the glycosylated haemoglobin (HbA1c). 3) The antibodies isolated from serum of our patients, directly inhibited HDL-associated PON1 activity in a dose dependent way ranging from 7 to 52%. 4) The anti-inflammatory effect of HDL, measured by the percentage of inhibition of the cytokine-induced production of vascular adhesion molecules (VCAM-1), was reduced in more than 80% by aHDL antibodies isolated from our patients. 5) The HDL-induced angiogenesis by increasing vascular endothelial growth factor (VEGF) levels was abrogated in 65% by the antibodies isolated from serum of patients. 6) The current available pharmacologic agents for increasing HDL-C concentrations were associated with an increase in the titres of IgG aApoA-I antibodies. This increase was higher in the extended release niacin when compared to statins probably due to their dampening effect on oxidative stress. In conclusion, aHDL antibodies are present in different pathologic conditions. aHDL antibodies represent a family of self-reacting immunoglobulins, of which ApoA-I and PON1 might be the most relevant targets. These antibodies are biologically active, interfering with the HDL anti-oxidant and anti-inflammatory properties and, consequently, with the atherosclerotic process. The pathogenic potential of these antibodies may lead to the identification of a new biomarker for vascular disease, whilst presenting itself as a novel target for a different treatment approach which may redefine the treatment strategies and clinical trials design for HDL interventions in the future.

Cardiovascular diseases are the main cause of death worldwide and a relevant source of economic cost and physical disability. The Mediterranean Diet, associated with a high intake of virgin olive oil, has been shown to be protective against the development of cardiovascular diseases. Adherence to the Mediterranean Diet and the consumption of virgin olive oil induce this protection by improving several cardiovascular risk factors, such as the lipid profile. These two dietary interventions are known to increase cholesterol levels in high-density lipoproteins (HDLs) and decrease cholesterol concentrations in low-density lipoproteins (LDLs). However, it is becoming increasingly more accepted that the information provided by HDL and LDL cholesterol levels is quite limited. On the one hand, the biological functions of HDLs may reflect the anti-atherogenic role of the lipoprotein better than HDL cholesterol levels. On the other hand, several LDL characteristics beyond LDL cholesterol levels, such as the pro- atherogenic LDL traits (LDL size, oxidation, composition, etc.), may be more informative with respect to the unexplained cardiovascular risk of an individual. Nevertheless, despite their growing relevance, very few randomized controlled trials have examined the effects of healthy lifestyle modifications on these properties. The aim of the present thesis project was to assess whether adherence to the Mediterranean Diet or the consumption of virgin olive oil was able to increase HDL functionality and decrease LDL pro-atherogenic traits in humans. Samples in the present project were obtained from two randomized controlled trials: the EUROLIVE Study (Effects of Olive Oil Consumption on Oxidative Damage in European Populations) and the PREDIMED Study (Effects of Mediterranean Diet on the Primary Prevention of Cardiovascular Disease). In both cases, we isolated HDLs and LDLs in different sub-samples from volunteers in order to perform a complete battery of determinations related to HDL function and LDL atherogenicity. The consumption of virgin olive oil increased the main HDL function: its cholesterol efflux capacity. An increase in the content of olive oil phenolic compounds in HDL, as well as the enhancement of HDL composition and size, may explain the functional improvement. Regarding the Mediterranean Diet, increasing adherence to this dietary pattern improves the four main HDL functions: cholesterol efflux capacity, HDL role in other steps of reverse cholesterol transport, HDL antioxidant activities, and HDL vasodilatory capacity. An improvement in HDL oxidation, size, and composition may also justify HDL enhanced function. In addition, the consumption of virgin olive oil decreased LDL levels and LDL atherogenicity (increasing LDL size and resistance against oxidation). Adherence to the Mediterranean Diet also improved LDL pro-atherogenic traits, by increasing LDL size and resistance against oxidation, decreasing LDL oxidation in vivo, improving LDL composition, and decreasing LDL cytotoxicity in macrophages. In conclusion, the present PhD thesis project shows that the consumption of virgin olive oil and adherence to the Mediterranean Diet improved HDL function and LDL pro- atherogenic traits in humans in two randomized controlled trials. Our findings provide two novel mechanisms to explain part of the benefits of these healthy diet interventions, and support previous evidence concerning the cardioprotective role of virgin olive oil and the Mediterranean Diet in humans.
Les malalties cardiovasculars són la principal causa de mort en el món i una rellevant font de despesa social i discapacitat física. La Dieta Mediterrània, associada a un alt consum d’oli d’oliva verge, ha demostrat ser protectora davant el desenvolupament de malalties cardiovasculars. L’adherència a una Dieta Mediterrània o la ingesta d’oli d’oliva verge indueixen aquesta protecció millorant nombrosos factors de risc cardiovascular, com el perfil lipídic. Ambdues intervencions són capaces d’incrementar els nivells de colesterol en lipoproteïnes d’alta densitat (HDL, en les seves sigles en anglès) i de disminuir les concentracions de colesterol en lipoproteïnes de baixa densitat (LDL, en les seves sigles en anglès). Però, cada vegada s’accepta més que la informació proporcionada pels nivells de colesterol HDL i LDL és limitada. Per un costat, l’anàlisi de les funcions biològiques de les HDLs han reflectit el paper anti- aterogènic de la lipoproteïna millor que els nivells de colesterol HDL. Per un altre, certes característiques de les LDLs més enllà dels seus nivells de colesterol, com els trets pro-aterogènics de les LDLs (grandària, oxidació, composició, etc.), podrien ser més informatives del risc cardiovascular residual dels individus. No obstant això, i malgrat la seva creixent rellevància, molt pocs assajos clínics aleatoritzats i controlats han estudiat els efectes protectors d’intervencions dietètiques saludables sobre aquestes propietats. L’objectiu del present projecte de tesi va ser determinar si l’adherència a una Dieta Mediterrània o el consum d’oli d’oliva verge era capaç d’incrementar la funcionalitat de les HDLs o de disminuir l’aterogenicitat de les LDLs en humans. Les mostres del present projecte van procedir de dos assajos clínics aleatoritzats i controlats: l’estudi EUROLIVE (Effects of Olive Oil Consumption on Oxidative Damage in European Populations) i l’estudi PREDIMED (Effects of Mediterranean Diet on the Primary Prevention of Cardiovascular Disease). En ambdós casos, vam aïllar les HDLs i LDLs de diferents sub-mostres de voluntaris i vam realitzar una bateria completa de determinacions relacionades amb la funció d’HDL i l’aterogenicitat de LDL. El consum d’oli d’oliva verge va incrementar la principal funció d’HDL, la capacitat d’eflux de colesterol. Un increment en el contingut de metabòlits dels compostos fenòlics de l’oli d’oliva a les HDLs, així com unes millors composició i grandària, podrien justificar l’anterior millora funcional. Respecte a la Dieta Mediterrània, augmentar l’adherència a aquest patró dietètic va millorar les quatre funcions principals de les HDLs: la capacitat d’eflux de colesterol, el rol de les HDLs en altres punts del transport revers de colesterol, la capacitat antioxidant de les HDLs i la funció vasoprotectora de les lipoproteïnes. Una millora a la oxidació, composició i grandària de les HDLs també podria justificar la millora funcional de les HDLs. A més, el consum d’oli d’oliva verge va disminuir els nivells de LDL i l’aterogenicitat de les mateixes (augmentant la grandària i la resistència davant l’oxidació de les lipoproteïnes). L’adherència a una Dieta Mediterrània també va millorar les característiques pro-aterogèniques de les lipoproteïnes, augmentant la grandària i la resistència a l’oxidació de les lipoproteïnes, disminuint la seva oxidació in vivo, millorant la seva composició i disminuint la seva citotoxicitat en macròfags. En conclusió, el present projecte de tesi demostra que el consum d’oli d’oliva verge o l’adherència a una Dieta Mediterrània millora la funció de les HDLs i l’aterogenicitat de les LDLs en humans en dos assajos clínics aleatoritzats. Els nostres resultats proporcionen dos nous mecanismes per explicar part dels beneficis d’aquestes intervencions dietètiques saludables i recolzen les evidències prèvies que indiquen el rol cardioprotector de l’oli d’oliva verge i la Dieta Mediterrània en humans.

ApoA-I and HDL readily displace cell surface-bound HL and stimulate TG-hydrolysis by HL. To evaluate the regulatory effect of HDL structure on HL displacement, cell culture experiments using the native and reconstituted HDL were undertaken. Structural features of HDL such as size, density, and chemical composition were found to be important regulators of HL displacement. The larger, more buoyant HDL were stimulatory to HL displacement, whereas the smaller denser particles were inhibitory. Apolipoprotein and lipid composition had a direct regulatory role in HL displacement. Apolipoprotein A-II increased HL displacement significantly. Apolipoprotein C-I also increased the displacement, but in a lesser degree than apolipoprotein A-II. Phospholipid content of HDL was inhibitory regardless of the electrostatic charge of the HDL particle. The triglyceride component of HDL had the most significant inhibitory role in HL displacement and blocked the displacement almost completely. In addition, TG-enriched HDL fractions from hypertriglyceridemic/hypercholesterolemic subjects were unable to displace HL from the cell surface. In summary, these results show that the structure and composition of HDL particles in plasma are central to regulation of HL displacement and, thereby, regulate the hydrolytic activity of HL.

Errata attached to inside front cover. Bibliography: leaves 112-166. This thesis is concerned with the interaction of the plasma factor, phospholipid transfer protein (PLTP) with HDL. The studies described in this thesis define the mechanism of the remodelling of reconstitute HDL (rHDL) by PLTP and the reasons why triglyceride enrichment of rHDL enhances the remodelling process. Other studies describe why triglyceride enrichment of rHDL enhances PLTP-mediated phospholipid transfers. In conclusion, this thesis describes the mechanism of the PLTP-mediated remodelling of rHDL and presents reasons why the remodelling is enhanced in particles that are enriched with triglyceride.

This study was designed to investigate the mechanism(s) underlying the hypercatabolism of high density lipoprotein in Tangier disease (TD). Initially, the metabolism of normal HDL incubated in Tangier plasma in vitro was examined. Sufficient normal human HDL was added to TD plasma to raise the concentration of HDL-cholesterol to within normal levels. During incubation the concentration of HDL-cholesterol in the TD plasma fell by up to 50% in a time dependent manner. This was not seen in control samples treated in a similar manner. The loss of HDL-cholesterol in the TD could be completely accounted for by the loss of HDL-cholesteryl ester and was accompanied by a 2.3-fold increase in the concentration of HDL-triglyceride. These observations could not be accounted for by lecithin: cholesterol acytransferase activity, cholesteryl ester hydrolysis, or the triglyceride level in the TD plasma. However, preliminary evidence suggested that the activity of cholesteryl ester transfer protein in TD plasma is responsible for the changes in HDL-lipid composition. The resulting triglyceride-rich, cholesteryl-poor HDL was shown to have a normal affinity for the human skin fibroblast HDL receptor. However, this finding does not exclude other pathways of HDL catabolism that may contribute to the rapid turnover of modified HDL in TD plasma. The metabolism of normal HDL by TD fibroblasts and monocytes in vitro was also studied in an attempt to identify a cellular defect of HDL metabolism in TD. However, both TD fibroblasts and monocytes were normal with respect to their ability to bind/internalize and degrade normal HDL invitro. It is concluded that the hypercatabolism of normal HDL in TD involves alterations of HDL-lipid and protein composition prior to removal from the plasma component. Thus, these studies support the hypothesis that the defect in TD resides in the plasma and not in the cells of these patients.
Medicine, Faculty of
Pathology and Laboratory Medicine, Department of
Graduate

Dissertação de Mestrado Integrado em Medicina Veterinária
Envolvido em múltiplas funções da homeostasia do corpo, o colesterol, depois de absorvido no lúmen intestinal e processado nos enterócitos, entra na corrente sanguínea onde é transportado sob a forma de lipoproteínas as quais se classificam de acordo com a sua densidade, resultante da quantidade e do tipo de apoproteínas e de colesterol que as constituem, em quilomicras, lipoproteínas de muito baixa densidade (VLDL), lipoproteínas de densidade intermédia (IDL), lipoproteínas de baixa densidade (LDL) e lipoproteínas de alta densidade (HDL), sendo esta última a fracção predominante no cão. É sabido que os valores de colesterol e o perfil das lipoproteínas plasmáticas variam em função de um conjunto de factores intrínsecos e extrínsecos ao indivíduo. O presente estudo utilizou uma amostra de 20 indivíduos (n=20) da espécie Canis lupus familiaris, e teve por objectivos: 1) Caracterizar os indivíduos incluídos no estudo quanto ao sexo, raça, idade e condição corporal; 2) Determinar a relação entre os valores plasmáticos de COL e as variáveis consideradas em 1); 3) Determinar a relação entre os valores plasmáticos de HDL-C e as variáveis consideradas em 1); 4) Determinar a relação entre os valores plasmáticos de COL e HDL-C. Os resultados obtidos permitiram concluir que na amostra a média dos valores de COL foi de 223,20±85,54mg/dL, encontrando-se a maioria dos indivíduos (95%) numa situação de normocolesterolémia. Os indivíduos mais velhos, os mais obesos e as fêmeas inteiras, foram os que apresentaram os valores de COL mais elevados. Para o HDL-C, a média foi de 86,02±10,37mg/dL, tendo sido os valores mais elevados registados em fêmeas inteiras, e os mais baixos nos indivíduos obesos. Os testes estatísticos realizados não permitiram contudo excluir a hipótese nula da ausência de diferenças nos valores de COL e de HDL-C, considerando os parâmetros sexo, raça e condição corporal para ambos e ainda, para o HDL-C, o factor idade.
ABSTRACT - Preliminary characterization of plasmatic cholesterol values in dogs based on gender, age, breed and body condition - Playing a role in multiple functions of the body homeostasis, the cholesterol, after being absorbed at intestinal lumen and processed by the enterocyte, reaches bloodstream, where its transport is performed by lipoproteins, classified by their density, which result from its constitution in apoproteins and cholesterol, in chylomicrons, very-low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), low-density lipoproteins (LDL) and high-density lipoproteins (HDL), being this last one the predominant fraction in the dog. It is well known that total cholesterol and the profile of plasmatic lipoproteins vary in function of a set of factors, intrinsic and extrinsic to the individuals. The present study used a sample of 20 individuals of Canis lupus familiaris specie, to achieve the following objectives: 1) characterize the individuals included according to their gender, breed, age and body condition; 2) determine a relation between the plasmatic values of COL and the variables considered at 1); 3) determine the existence or not of a relation between the plasmatic values of HDL-C and the variables considered at 1); 4) determine the existence or not of a relation between the plasmatic values of COL and HDL-C. The results showed that the mean of COL values of the sample was 223,20±85,54mg/dL, and the majority of the individuals (95%) were in a situation of normocolesterolemia. The older individuals, the more obese and intact females showed the highest COL values. Concerning the HDL-C, the mean was 86,02±10,37mg/dL, the highest values were seen in intact females, and the lowest values were found in obese individuals. Nevertheless, the statistical tests to whom the sample was submitted did not allowed to exclude the nule hypothesis of the absence of differences in the values of COL and HDL-C considering gender, breed and body condition for both and still, for HDL-C, the age.

Thesis (Ph.D.)--Tufts University, 2004.
Adviser: Alice H. Lichtenstein. Submitted to the School of Nutrition Science and Policy. Includes bibliographical references. Access restricted to members of the Tufts University community. Also available via the World Wide Web;

Three studies were conducted to determine whether the elevated plasma and HDL cholesterol levels observed in copper-deficient rats could be explained by the interaction of ¹²⁵I-HDL subfractions with liver membrane preparations in vitro. Rats from all studies were randomly divided into two dietary treatments, copper-deficient and adequate (0.7 mg and 8.0 mg Cu/kg diet, respectively). Deionized water and diet were provided ad libitum. After eight weeks, rats were exsanguinated, membranes prepared from livers, and plasma high density lipoproteins (HDL) isolated by ultracentrifugation and agarose column chromatography. Heparin-Sepharose affinity chromatography was used to isolate specific subfractions of HDL. The HDL subfractions derived from rats of each dietary treatment were iodinated and bound to either crude liver membranes or purified liver plasma membranes prepared from rats of both treatment groups. Total binding data and computer derived estimates (K(d) and B(max)) were used to compare differences between treatments. Binding data from all experiments conformed to a one-site model. In all cases, binding was saturable and EDTA and pronase insensitive. Treatment differences were observed in Study I (¹²⁵I-apo E-free HDL binding to crude liver membranes). Significantly lower total binding and B(max) were observed when lipoproteins and membranes from copper-deficient animals were used in the assay. Competition experiments from Studies II and III demonstrate that the different HDL subfractions competed effectively with one another for binding sites, indicating that apo E is not a determinant in binding of rat ¹²⁵I-HDL subfractions to purified liver plasma membranes.

Background Over the past few years, results refuting the causal role of HDL cholesterol (HDL-c) have been reported by a number of randomized controlled trials (RCTs) testing different ways of modifying HDL-c. Results from Mendelian randomization studies showed no difference in cardiovascular disease (CVD) risk among individuals with genetically different serum levels of HDL-c. The causal role of HDL-c in CVD is thus uncertain, raising the question as to whether HDL-c is a worthwhile target of public health interventions and medical treatments. The objective of these meta-analyses is to explore whether changes in HDL-c are symptomatic of prior causes instead of being a causal factor for CVD by first identifying two possible candidates—androgens and zinc—for the investigation of associations. Experimental evidence would then be investigated for whether either of them might underlie (i.e. confound) the observed association of HDL-c with CVD risk factors. Methods This study followed the PRISMA statement. A literature search was conducted through PUBMED, MEDLINE, EMBASE, and Cochrane Central Register of Controlled Trials. Keywords of “androgens/testosterone”, “HDL”, “high-density lipoprotein”, “lipid”, “cholesterol”, “lipoprotein”, “CVD”, “cardiovascular”, “heart”, “cardiovascular disease” were used with the search period limited to January 2000 – June 2013 with only human RCTs conducted and reported in English. For locating studies concerning the effect of zinc, the keyword “zinc” was used instead of “androgens/testosterone”. Inclusion and exclusion criteria were applied during study screening and selection. The Cochrane Collaboration’s tool for assessing risk of bias was used for quality assessment. Heterogeneity across included studies was measured using I2 statistic and publication bias was assessed via funnel plots and the Begg’s Rank Correlation test. The “trim and fill” method was also used for the correction of funnel plot asymmetry. The meta-analyses were performed using The Comprehensive R Archive Network Program (Version R 3.0.0), using the function “metacont” from the “meta” package, where the pooled intervention effects were displayed using forest plots, with inverse variance weighting and random effects model. Results A total of twelve and ten RCTs were identified and included in the meta-analyses of androgens and zinc respectively. There were no consistent beneficial effects of androgens on CVD observed, as the results from CVD surrogate markers were inconclusive, despite showing significant overall reduction in HDL-c levels. However, as current findings suggest that lower HDL-c levels are associated with higher cardiovascular risk, it is possible that androgens may increase that risk by influencing HDL metabolism. On the other hand, zinc was associated with healthier CVD profile. This supports the notion of zinc as a cardioprotective agent. Nonetheless, conclusion failed to be drawn concerning the effect of zinc on HDL-c as there were contradictory results across included studies. Conclusion The meta-analyses suggest that androgens could be a factor which lowers HDL-c and thus increases cardiovascular risk, rather than HDL-c being the direct causative agent. This research may serve as a template for more extensive search for other potentially better candidates in this new study focus in cardiovascular epidemiology.
published_or_final_version
Community Medicine
Master
Master of Public Health

Magister Pharmaceuticae - MPharm
Cardiovascular diseases (CVDs) such as ischaemic heart diseases, heart failure and stroke remain a major cause of death globally. Various deep-rooted factors influence CVD development; these include but are not limited to elevated blood lipids, high blood pressure, obesity and diabetes. A considerable number of proteins are involved directly and indirectly in the transport, maintenance and elimination of plasma lipids, including high and low-density lipoprotein cholesterol (HDL-C and LDL-C). There are several mechanisms involved in the removal of LDL particles from systemic circulation. One such mechanism is associated with the gene that encodes proprotein convertase subtilisin/kexin type 9 (PCSK9), which has become an exciting therapeutic target for the reduction of residual risk of CVDs. Currently, statins are the mainstay treatment to reduce LDL-C, and a need exists to further develop more effective LDL-C-lowering drugs that might supplement statins. This study was aimed at contributing to the generation of knowledge regarding the effect of niacin in reducing LDL levels through PCSK9 interaction.

Orientadores: Andrei Carvalho Sposito, Eliana Cotta de Faria
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: Doenças cardiovasculares constituem a principal causa de mortalidade no Brasil e no mundo. Baixas concentrações de HDL - colesterol são consideradas um fator de risco cardiovascular independente. Esta relação inversa tem sido atribuída às diferentes propriedades protetoras da HDL, dentre as quais podemos destacar seu papel no transporte reverso de colesterol, por meio do efluxo de colesterol, sua habilidade de inibir a agregação plaquetária, e suas atividades antioxidantes e anti-inflamatórias. No entanto, estudos recentes indicam que baixas concentrações de HDL - colesterol constituem um preditor significante de doença aterosclerótica somente em indivíduos assintomáticos e que a capacidade de efluxo de colesterol é um melhor preditor de carga aterosclerótica do que HDL - colesterol. Em conjunto, estes achados sugerem que a função da HDL poderia ser um fator chave na relação entre concentrações de HDL - colesterol e o desenvolvimento de doença aterosclerótica. Se o for, alterações das funções da HDL podem discernir melhor, entre os indivíduos com hipoalfalipoproteinemia, aqueles com maior propensão a desenvolver doença aterosclerótica. Assim, investigamos, em indivíduos sem doença cardiovascular previamente manifesta, se funções da HDL estão associadas a concentrações de HDL - colesterol e carga aterosclerótica. Participantes foram classificados em HDL - colesterol baixo (LH; HDL-C? 32 mg/dL; n=33), intermediário (IH; HDL-C= 40-67 mg/dL; n=33), ou alto (HH; HDL-C?78mg/dL; n=35). Nós avaliamos composição química da HDL, tamanho da partícula, capacidade de efluxo de colesterol, atividade antioxidante, susceptibilidade à oxidação, atividade anti-inflamatória, e habilidade de inibir agregação plaquetária. O grupo LH foi associado à espessura intimo-medial de carótidas (IMT) aumentada (p?0,001), maior conteúdo de triglicérides (4±2% vs. 4±2% em IH e 3±1% em HH, p?0,001), menor conteúdo de fosfolípides (12±4% vs. 14±5% em IH e 13±3% em HH, p=0,035), menor tamanho de partículas (7,33±0,33nm vs. 7,72±0,45nm em IH e 8,49±0,42nm em HH, p?0,001) e menor capacidade de efluxo de colesterol celular (9±3 % vs. 12±3 % em IH e 11±4 % em HH, p?0,001). Indivíduos HH apresentaram menor atividade antioxidante (37(53)% vs. 48(35)% em IH e 55(41)% em LH, p=0,003), maior suscetibilidade à oxidação (57±22% vs. 45±20% em IH e 46±25% em LH, p=0,017) e maior habilidade de inibir agregação plaquetária (45±25% vs. 31±18% em IH e 37±24 % em LH, p=0,0026). Indivíduos com IMT acima de 1 mm apresentaram partículas com menor tamanho (7,55±0,49 nm vs. 7,89±0,64 nm, p?0,001), atividade antioxidante (37(23)% vs. 49(42)%, p=0,018) e capacidade de efluxo de colesterol (31±14% vs. 40±14%, p=0,02). Nenhuma diferença foi encontrada para as outras características ou propriedades funcionais da HDL. Nós concluímos que, em um contexto de prevenção primária, o menor tamanho da partícula, o conteúdo reduzido de fosfolípides, e capacidade de efluxo de colesterol diminuída são relacionados com ambos LH e magnitude da doença aterosclerótica subclínica. Nestes indivíduos, estas características podem explicar a associação entre HDL - colesterol e o desenvolvimento da doença aterosclerótica.
Abstract: Cardiovascular diseases are the main cause of death in Brazil and worldwide. Low HDL-C levels are considered an independent cardiovascular risk factor. This inverse relationship has been attributed to different protective properties described for HDL, such as its role in the reverse cholesterol transport, through cholesterol efflux, its ability to inhibit platelet aggregation, and its antioxidant and anti-inflammatory effects. However, recent studies indicate that low HDL-cholesterol is a significant predictor of atherosclerotic disease in healthy individuals and that cholesterol efflux capacity is a better predictor of carotid atherosclerotic burden as compared to HDL-cholesterol. Altogether these findings have suggested that HDL function would be the key factor for the link between HDL-cholesterol concentration and the subclinical disease in a primary prevention setting. If so, changes in HDL function could help to discriminate, among individuals with hypoalphalipoproteinemia, those who are prone to develop atherosclerotic disease. Hence, in a primary prevention setting, we investigated whether HDL dysfunction is associated with HDL-cholesterol concentration and atherosclerotic burden. Participants were classified as low (LH; HDL-C? 32 mg/dL; n=33), intermediate (IH; HDL-C= 40-67 mg/dL; n=33), or high HDL-cholesterol (HH; HDL-C?78mg/dL; n=35). We measured HDL chemical composition, particle size, cholesterol efflux capacity, antioxidant activity, susceptibility to oxidation, anti-inflammatory activity, and ability to inhibit platelet aggregation. LH was associated to enhanced carotid intima-media thickness (IMT;p?0.001), high HDL triglyceride (4±2% vs. 4±2% in IH and 3±1% in HH, p?0.001), low HDL-phospholipids (12±4% vs. 14±5% in IH and 13±3% in HH, p=0.035), decreased particle size (7.33±0.33nm vs. 7.72±0.45nm in IH and 8.49±0.42nm in HH, p?0.001) and reduced cholesterol efflux capacity (9±3 % vs. 12±3 % in IH and 11±4 % in HH, p?0.001). The HH group presented reduced antioxidant activity (37(53)% vs. 48(35)% in IH and 55(41)% in LH, p=0.003), and increased susceptibility to oxidation (57±22% vs. 45±20% in IH and 46±25% in LH, p=0.017) and ability to inhibit platelet aggregation (45±25% vs. 31±18% in IH and 37±24 % in LH, p=0.0026). Carotid IMT>1mm was associated with reduced HDL size (7.55±0.49 nm vs. 7.89±0.64 nm, p?0.001), antioxidant activity (37(23)% vs. 49(42)%, p=0.018), and cholesterol efflux capacity (31±14% vs. 40±14%, p=0.02). No differences were found for the other HDL characteristics or functional properties. We conclude that in a primary prevention setting, small particle size, reduced HDL-phospholipids content, and diminished cholesterol efflux capacity are related to both LH and carotid IMT. In these individuals, these characteristics may underlie the association between HDL-cholesterol and atherosclerotic burden
Doutorado
Ciencias Biomedicas
Doutora em Ciências Médicas

Les macrophages jouent un rôle clé dans l'athérosclérose. Après la captation massive des LDL oxydées (oxLDL), les macrophages sous-endothéliaux sont chargés en cholestérol et se transforment ainsi en cellules spumeuses qui contribuent à la formation de la plaque d'athérome. Les oxystérols, produits d'oxydation du cholestérol, sont retrouvés en quantité importante dans les oxLDL. Au niveau cellulaire, ils sont impliqués dans la régulation de l'homéostasie du cholestérol, l'induction du stress oxydatif cellulaire et de la cytotoxicité. Notre travail montre que le cholestérol, associé aux LDL et d'origine cellulaire, est fortement oxydé par les macrophages lors d'une exposition aux oxLDL. Les principaux produits d'oxydation sont le 7-cétocholesterol et 7α/β-hydroxycholestérol. De plus, nous démontrons que ces oxystérols sont exportés hors des macrophages via les HDL, mais pas l'apoA1. Nous avons aussi caractérisé les oxystérols formés dans les HDL suite à des modifications oxydatives et les HDL issues de patients diabétiques. Nous avons en outre montré que ces modifications sont associées à une diminution de la capacité des HDL à exporter les oxystérols
Macrophages play a key role in atherosclerosis. After massive uptake of oxidized LDL (oxLDL), subendothelial macrophages are overloaded with cholesterol thereby leading to the formation of foam cells, which is one characteristic of atherogenesis. Oxysterols, the oxidation products of cholesterol, are one of major components of oxLDL; they are involved in the regulation of cholesterol homeostasis, induction of cellular oxidative stress and cytotoxicity. Our works show that both LDL derived-cholesterol and cellular cholesterol can be strongly oxidized in human THP1 and murine RAW macrophages, especially during exposure of oxLDL. The major oxidative products are 7-ketocholesterol and 7α/β-hydroxycholesterol. Moreover, we demonstrate that both oxysterols derived from LDL cholesterol and cellular cholesterol can be exported to HDL, whereas not to apoA1. Then, we studied the functionality of modified HDL and diabetic HDL on oxysterols efflux. A decrease of oxysterols efflux was observed with oxidized and glycoxidized HDL. Compared to the HDL of healthy controls, the HDL of diabetic subjects are less efficient to efflux oxysterols. Taken together, the increased production of oxysterols in presence of oxLDL and their lower efflux by modified HDL support the detrimental role of these oxidative compounds in pathophysiological conditions like diabetes

Thesis (Ph. D.)--University of Sydney, 2009.
Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Discipline of Pathology, Faculty of Medicine. Title from title screen (viewed Sept. 30, 2009) Includes bibliography. Also available in print form.

Abdominal Aortic Aneurysms (AAAs) are multi-genic, slow growing, degenerative vascular lesions resulting in focal aortic dilations. Following diagnosis, growth is monitored and the aneurysm surgically repaired when dilatation reaches 5.0 - 5.5 cm. Pharmacological treatments regressing or reducing growth rate remain a clinical ideal. Epidemiological evidence shows an inverse correlation between plasma High Density Lipoproteins (HDLs) and incidence of AAA, suggesting a role in the pathoaetiology This thesis investigates the role of HDLs in experimental AAA. Reconstituted HDL (rHDL - CSL-111) prevented AAA formation and reduced established AAA in mouse models. I used a hepatospecific adenoviral vector AdA-l; to induce production of human ApoA-l, increasing plasma HDL particles. Whilst infection with AdA-l significantly increased circulating human ApoA-l, it did not reduce established Ang Il-induced AAA or atheroma at the aortic root. Due to the complexity of HDL particles I elected to concentrate on mimicking its anti-inflammatory effects, rather than investigate alternate methods of increasing HDLs. One of HDL’s key anti-inflammatory effects is the prevention of Toll Like Receptor 4 (TLR4)-mediated inflammation, implicated in AAA development. I attempted to prevent AAA formation with a novel small molecule TLR4 antagonist, IAXO-102. IAXO-102 reduced TLR4-mediated aortic inflammation after three days of Ang II infusion; prevented AAA formation, and early rupture. Cytokines produced by TLR4-mediated inflammation stimulate other pro- inflammatory signalling pathways, such as the CD40/TRAF6 pathway. I investigated the effect of a CD40/TRAF6 inhibitor (687702), on experimental AAA. Following 3 days of Ang II infusion, treatment with Compound 6877002 made no significant difference to inflammatory markers associated with AAA; and no difference to vascular macrophage infiltration, a key cell mediator in AAA development. x HDLs are known to inhibit TLR4-mediated inflammation through up-regulation of Activating Transcription Factor 3 (ATF3), I investigated rHDLs manipulation of this negative feedback loop in HUVEC, and found stimulating HUVEC with rHDL, as well as with the TLR4 ligand LPS, induces ATF3 expression. Whilst surprisingly IAXO-102 did not prevent LPS stimulated ATF3 expression, 6877002 did, suggesting CD40/TRAF6 is essential for ATF3 expression. Through investigating the mechanisms in which HDL prevents AAA, I have highlighted the importance of TLR4 mediated inflammation in AAA and identified a potential pharmaceutical target.

Research has reported that increased levels of plasma TC are directly related, while low levels of plasma HDL-C are inversely related, to coronary heart disease. Regular physical exercise has been suggested as a method for reducing plasma TC and increasing plasma HDL-C. Thirty-one healthy, sedentary women (ages 18-30) were studied to determine the effects of a jogging, weight training, or a combined jogging and weight training program on plasma total cholesterol, high-density lipoproteins, body composition. Experimental subjects were randomly assigned to the treatment conditions. The subjects trained three days a week for nine weeks. The R group ran for 30 minutes a session at 75% predicted maximum HR. The W group trained with weights utilizing exercises to strengthen all major muscle groups for one hour at 60% one repetition maximum the first 3 weeks and 75% one repetition maximum weeks 4 - 9. The RW group ran for 25 minutes a session at 75% predicted maximum HR, then lifted weights using the leg-strengthening exercises for 30 minutes, similar to the W group. Preceding and following the treatment period, plasma TC, HDL-C, body weight, and percent body fat was assessed for all four groups. Plasma TC was not significantly altered, although a downward trend was observed for all three treatment groups. Plasma HDL-C did not change over the treatment period for any group. The plasma TC/HDL-C ratio changed significantly among groups over the treatment period, with the R group decreasing their ratio from 3.5 to 2.9 (p < .05). No changes were noted In percent body fat, fat-free mass, or body weight for any of the groups. The Pearson product-moment correlations performed between the changes in blood lipids and the changes in body composition found no significant relationships. The results of this study indicate that an exercise program consisting of endurance training for 30 minutes, 3 times per week, or weight training for one hour, 3 times per week, or a combination aerobic/weight training program 3 times per week is not adequate to significantly improve plasma TC or HDL-C in young females over a nine week period. However, significant improvements may be made in the plasma TC/HDL-C ratio which may decrease the risk for CHD.
Master of Science

Abstract Phosphatidylethanol (PEth) is an abnormal phospholipid formed only in the presence of ethanol. Ethanol causes changes in the concentration and composition of plasma lipoproteins and it also influences the enzymes and transfer proteins that modify lipoproteins in plasma. PEth might be one of these changes brought on by ethanol in the circulation. The present study was designed to investigate whether qualitative changes in high density lipoprotein (HDL) phospholipids caused by ethanol can mediate the beneficial effects of alcohol on atherosclerosis, and to investigate the transfer of PEth between lipoproteins and the effects of PEth on the charge of lipoprotein particles. PEth was shown to be transferred from low density lipoproteins (LDL) to HDL particles mainly by transfer proteins other than cholesteryl ester transfer protein (CETP). The transfer of PEth between lipoproteins enables the redistribution of PEth between lipoproteins in plasma. The results of this study provide evidence that PEth in HDL particles stimulates the vascular endothelial growth factor (VEGF) secretion from vascular wall cells. The increase in the secretion was mediated through protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) signalling pathways. PEth-containing HDL particles were able to increase the VEGF secretion in rats in vivo. Similar effects were also observed when rats were given HDL particles isolated from the plasma of alcoholics. The PEth-induced change in the electrical charge of lipoproteins may affect the binding of lipoproteins to their receptors and binding proteins. The effects of PEth on the secretion of VEGF from the endothelial cells were shown to be mediated through HDL receptor. The changes in HDL particles caused by phosphatidylethanol may modify the metabolism of lipoproteins and lipid-mediated signalling pathways regulating VEGF in vascular wall cells.

Two studies were conducted to determine how HDL cholesteryl ester and apoprotein catabolism might contribute to the observed hypercholesterolemia of copper-deficient rats. Weanling male Sprague-Dawley rats were divided into two dietary treatments; copper-adequate (control, 5-7 mg Cu/kg diet) and copper-deficient (0.6-0.8 mg Cu/kg diet). Deionized water and diet were provided ad libitum. Dietary copper deficiency resulted in enlarged intravascular pools of HDL cholesteryl esters and total protein. HDL were isolated from rats of both treatment groups, radiolabeled, and injected into animals of the respective groups. In Study I, HDL apoproteins were labeled by iodination, whereas HDL in Study II were doubly labeled by additionally incorporating into the particle core [³H]cholesteryl linoleyl ether, which served as a nondegradable analog of HDL cholesteryl ester. At specific time intervals up to 12 hours after injection, blood and tissue samples were removed and analyzed for radioactivity. Plasma disappearance curves indicated that HDL cholesteryl esters were preferentially catabolized 1.6-fold faster than HDL protein in controls and 2.5-fold faster in copper-deficient animals. Clearance of individual apoproteins did not occur at significantly different rates in either treatment group. Absolute mass removal of HDL cholesteryl ester and total protein from the plasma was significantly increased in copper-deficient rats. Virtually all of the increased removal of HDL cholesteryl ester was attributed to the liver, whereas most of the increased uptake of HDL protein was attributed to the bulk tissues and not the liver. Since previous studies indicate that copper deficiency may not result in increased cholesterol excretion, these data suggest that cholesteryl esters delivered to the liver of copper-deficient rats are possibly reassembled into new HDL particles at an increased rate. The observed hypercholesterolemia in this animal model, then, appears to be the result of an imbalance in the net flux of cholesterol between the tissues and the plasma.

Abstract The effect of alcohol intake on the pathophysiology of atherosclerotic cardiovascular disease is controversial, especially with respect to heavy alcohol intake. The pathobiology behind atherosclerosis is a complex and multiparametric phenomenon, therefore a self-organizing map (SOM), an unsupervised learning based artificial neural network technique, was applied in the present work. This study was carried out to investigate the effect of heavy alcohol intake on the pathophysiology of atherosclerosis, including several lipoproteins and adiponectin, an adipocyte-derived cytokine that may ameliorate atherosclerosis. Firstly, the effect of heavy alcohol intake on the capacity of HDL and its subclasses (HDL2 and HDL3) to mediate cholesterol efflux from macrophages was studied. Secondly, data of ultracentrifugally isolated lipoproteins were fed into SOM analysis to investigate whether this method can find diverse lipoprotein phenotypes from the heterogeneous lipoprotein data. Thirdly, the aforementioned method was applied to multivariate data of alcohol drinkers to study whether distinct metabolic profiles are associated to heavy alcohol consumption. The results revealed that HDL2, not HDL3, of heavy alcohol drinkers had an enhanced capacity to remove cholesterol from macrophages when compared with control persons. SOM analysis enhanced the ultracentrifugally based lipoprotein data and depicted several novel lipoprotein phenotypes. In addition, lipoprotein-based SOM analysis found two distinct metabolic profiles in heavy alcohol drinkers: an anti-atherogenic and a metabolic syndrome-like profile with opposite metabolic features, such as characteristics of lipoproteins, plasma concentration of adiponectin and prevalence of metabolic syndrome. These profiles also tended to differ in their CV risk. In conclusion, the enhanced cholesterol efflux capacity of HDL2 in heavy drinkers is an anti-atherogenic change linked to alcohol drinking. However, clinically it may be important to be aware that although heavy alcohol drinkers have a low LDL-C level, they differ in their other lipoprotein measures, forming distinct phenotypes with potentially different CV risks. Finally, SOM analysis of ultracentrifugally based lipoprotein data generates in silico classification of lipoprotein particles and thereby offers a new tool for lipoprotein research
Tiivistelmä Alkoholinkäytön vaikutus ateroskleroottisen sydän- ja verisuonitaudin patofysiologiaan on kiistanalainen, etenkin runsaan alkoholinkäytön kohdalla. Koska patobiologia ateroskleroosin taustalla on monimutkainen ilmiö, tässä työssä sovellettiin menetelmänä itseorganisoituvaa karttaa, joka on ohjaamattomaan oppimiseen perustuva neuroverkkomalli. Tutkimuksen tavoitteena oli selvittää runsaan alkoholinkäytön vaikutusta ateroskleroosin patofysiologisiin merkkiaineisiin, mukaan lukien useita lipoproteiineja sekä adiponektiini, rasvasoluperäinen sytokiini, joka voi lievittää ateroskleroosia. Ensimmäisessä osatyössä tutkittiin runsaan alkoholinkäytön vaikutusta HDL:n ja sen alafraktioiden (HDL2 ja HDL3) kykyyn poistaa kolesterolia makrofageista. Toisessa osatyössä ultrasentrifuugaukseen perustuva lipoproteiiniaineisto syötettiin itseorganisoituvaan karttaan. Työssä selvitettiin löytäisikö menetelmä erilaisia lipoproteiinifenotyyppejä heterogeenisestä aineistosta. Kolmannessa osatyössä em. menetelmää sovellettiin monimuuttuja-aineistoon, joka koostui runsaasti alkoholia käyttävistä ja verrokeista. Tutkittiin, liittyykö runsaaseen alkoholinkäyttöön erilaisia metabolisia profiileja. Tulokset osoittivat, että suurkuluttajien HDL2-hiukkasen kolesterolinpoistokyky makrofageista oli suurempi kuin verrokeilla. Itseorganisoituvaan karttaan perustuva lipoproteiinien luokittelumenetelmä löysi useita uusia lipoproteiinifenotyyppejä. Lisäksi, em. menetelmä löysi suurkuluttajilta kaksi erilaista metabolista profiilia: anti-aterogeeninen ja metabolisen syndrooman kaltainen. Näillä oli vastakkaiset metaboliset piirteet, kuten lipoproteiinien ominaisuudet, adiponektiinin pitoisuus plasmassa ja metabolisen syndrooman esiintyvyys. Profiileihin liittyi mahdollisesti myös erilainen sydän- ja verisuonitautiriski. Tutkimus osoittaa, että alkoholin suurkuluttajilla havaittu parempi HDL2:n kyky poistaa kolesterolia soluista on anti-aterogeeninen muutos, joka liittyy alkoholin käyttöön. Kliinisesti voi olla merkittävää, että vaikka alkoholin suurkuluttajilla oli pieni LDL-C pitoisuus, he jakaantuivat muiden lipoproteiiniperäisten muuttujien perusteella kahteen eri fenotyyppiryhmään, joihin liittyi erilainen sydäntautiriski. Lisäksi itseorganisoituva kartta loi ultrasentrifugoinnilla eristetyille lipoproteiineille in silico -luokittelun, joten se tarjoaa uuden työkalun lipoproteiinitutkimukseen

Enzymatic and lipid transfer reactions involved in reverse cholesterol transport were studied in HDL deficient plasma systems. Fasting plasma samples were obtained from control and cholesterol fed guinea pigs as well as from a fish eye disease patient and were used to localize the enzyme LCAT among plasma lipoproteins (VLDL, LDL, and HDL). In both guinea pig and fish eye disease patient plasma, the LCAT activity was found in association with the HDL type particles. Cholesterol feeding in guinea pigs altered the properties of lipoprotein substrates for LCAT resulting in some changes, specifically: 1) decreased fractional rate of plasma cholesterol esterification and, 2) lower transfer of free cholesterol (FC) and esterified cholesterol (CE) within the lipoprotein fractions.

Atherosclerosis manifests a state of increased oxidative stress characterized by comparable lipid and protein oxidation in the affected arterial wall. While oxidative modification of low density lipoprotein (LDL) has been extensively studied, increasing attention has been focused recently on oxidation of high-density lipoproteins (HDL) and its functional consequences in relation to atherosclerosis. Oxidative modification is thought to generate “dysfunctional” HDL that has lost anti-atherosclerotic activities, including the ability to remove cholesterol from lipid-laden cells. Therefore, there has been much interest in the detection of oxidized HDL. Unfortunately, available methods to detect oxidized HDL are limited at present, in part because oxidative modification of HDL is a complex process and ‘oxidized HDL’ is not a chemically defined entity. What is known however is that conversion of methionine (Met) residues of apolipoprotein (apo) A-I to methionine sulfoxide (MetO) is a process that occurs commonly as HDL undergoes oxidative modification. For example, human apoA-I+16 (containing MetO86 or MetO112) and apoA-I+32 (MetO86 plus MetO112) are generated when apoA-I reacts with lipid hydroperoxides formed as a consequence of the lipoprotein being exposed to 1e−oxidants. The formation of MetO in apoA−I induced by 2e−oxidants (i.e., hydrogen peroxide, hypochlorous acid or myeloperoxidase/hydrogen peroxide/chloride system) is associated with an impaired ability of the apolipoprotein to facilitate reactions relevant to reverse cholesterol transport. In addition, a previous study has suggested the plasma content of apoA-I+32 to be increased in certain subjects that have an increased risk to develop cardiovascular disease (CVD). Moreover, the MetO content in circulating, HDL−associated apoA−I is elevated in type 1 diabetes, a disorder commonly associated with increased oxidative stress and a risk factor for atherosclerosis. Therefore, in the present study, an existing HPLC method was applied to HDL samples from the Fletcher−Challenge study, a nested case control study, to test the potential usefulness of MetO-containing apoA-I as a marker of oxidative stress and/or CVD in a general population. Plasma samples whose HDL contained detectable apoA-I+16 and/or apoA-I+32 had significantly elevated levels of F2-isoprostanes, a marker of in vivo lipid oxidation, consistent with MetO-containing apoA-I being a useful marker of in vivo protein oxidation. Despite this however, there was no significant difference between controls and cases in their concentrations of HDL apoA-I+16 and apoA-I+32 or F2-isoprostanes, suggesting that markers of protein and lipid oxidation are not associated with the risk of coronary heart disease (CHD) in this general population. A limitation of the Fletcher−Challenge study was that only 22% of the 534 HDL samples analyzed contained apoA-I+16 and/or apoA-I+32. In addition, the HPLC−based method used is expensive and time−consuming and may lack the sensitivity needed for apolipoproteins to clinical studies. Thus, a mouse monoclonal anti-human apoA-I+32 antibody (MOA−1) was raised using HPLC−purified apoA-I+32 as immunogen. A sensitive ELISA was then developed using a commercial anti-human apoA-I monoclonal antibody as capture and biotinylated MOA−1 as detection antibody, respectively. The assay detected lipid−free HPLC−purified human apoA-I+32 in a concentration-dependent manner and with a significantly lower limit of detection (i.e., 3 ng/mL) than the HPLC method (1 μg/mL). The ELISA also detected lipid-free apoA-I modified by 2e-oxidants (hydrogen peroxide, hypochlorous acid, peroxynitrite), and HDL oxidized by 1e- or 2e-oxidants and present in buffer or human plasma. Moreover, the extent of recognition of MetO by MOA−1 increased with increasing numbers of MetO in apoA−I, as assessed by the experiments with H2O2−oxidized forms of apoA−I mutants, in which one, two or three Met residues were replaced with Leu. Their detection was concentration-dependent, reproducible, and exhibited a linear response over a physiologically plausible range of concentrations of oxidized HDL. In contrast, MOA-I failed to recognize native apoA-I, native apoA-II, apoA-I modified by hydroxyl radicals or metal ions, or LDL modified by 2e-oxidants. Furthermore, MOA−1 did not detect other Met−containing proteins oxidized by either hypochlorous acid or hydrogen peroxide. Taken together, the results showed that recognition of oxidized proteins by MOA−1 is limited to MetO contained in apoA−I. Finally, in a pilot study, plasma samples obtained from subjects with coronary artery disease (CAD) proven by angiography, and samples from CAD patients undergoing percutaneous coronary intervention (PCI) were analyzed by the ELISA. The preliminary data obtained showed elevated levels of MetO-containing apoA-I in plasma samples of CAD patients compared to those of corresponding control subjects. Unexpectedly, levels of MetOcontaining apoA-I decreased PCI compared to before PCI. A possible explanation for these results is that HDL−associated apoA−I become displaced by acute phase proteins, such as serum amyloid A, in response to PCI. In summary, the ELISA developed here specifically detects apoA-I containing MetO in HDL and human plasma. As such it may provide a useful tool for investigating the relationship between oxidized HDL and CAD.

Apolipoprotein L1 (APOL1) is a human-specific serum protein bound to high-density lipoprotein (HDL) particles. This protein allows human resistance to infection by African trypanosomes except for two subspecies, Trypanosoma brucei rhodesiense and T. b. gambiense, the causative agents of sleeping sickness or African trypanosomiasis. This disease infects 20 000 people in sub-Saharan Africa and without treatment, infection is almost always fatal. T. b. rhodesiense resists APOL1 through direct protein neutralization by the Serum Resistance-Associated (SRA) protein. T. b. gambiense does not express SRA, and its mechanism of resistance to APOL1 is orchestrated upon a recently characterized multifactorial defense mechanism.

The mechanism by which the human serum sensitive parasites are killed following APOL1 uptake is described as the result of the lysosomal swelling induced by the generation of ionic pores within the lysosomal membrane.

We show here that preventing the osmotic lysosomal swelling in a hyperosmotic culture condition does not prevent the cell death. In addition, APOL1 appears to trigger some programmed cell death events in the cell such as a fast mitochondrial depolarization followed by a DNA laddering and fragmentation. Furthermore, we show an implication of the endonuclease G (TbEndoG), known to be a key actor in the regulation of cell death process and a kinesin (TbKIFC1), which might be the transporter of APOL1 for the endosomes to the mitochondrion.

In addition, by producing different recombinant human APOL proteins in E. coli and test their activity on T. brucei, we were able to show that APOL3, an other member of the APOL family, also possesses a trypanolytic activity like APOL1 beneath the fact it is not a secreted protein. APOL3 does not only kill T. b. brucei but is also able to lyse APOL1-resistant subspecies such as rhodesiense and gambiense, in vitro and confirmed in vivo when the recombinant APOL3 were injected in infected mice. A beginning of an action mechanism is described herein showing a pH-independent activity for this protein oppositely to APOL1, conferring its specificity.

It is thus conceivable to use this recombinant protein as a first step of a potent curative agent against gambiense or rhodesiense since the few currently available drugs for treatment of African trypanosomiasis, that are outdated, show problems with toxicity and resistance.

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L’ Apolipoprotéine L1 (APOL1) est une protéine sérique humaine associée aux lipoprotéines de haute densité (HDL). Cette protéine confère la résistance à l'infection des trypanosomes africains à l'exception des deux sous-espèces, Trypanosoma brucei rhodesiense et T. b. gambiense, les agents responsables de la maladie du sommeil ou trypanosomiase africaine. Cette maladie infecte 20 000 personnes en Afrique sub-saharienne et en l'absence de traitement, l'infection est presque toujours mortelle. T. b. rhodesiense résiste à l’APOL1 grâce à une neutralisation directe d’APOL1 par une protéine appelé SRA (Serum Resistant-Associated). T. b. gambiense n'exprime pas SRA, et sa résistance à l’APOL1 est orchestrée par un mécanisme de défense multifactorielle récemment caractérisé 1.

Le mécanisme par lequel les parasites sensibles au sérum humain sont tués suivant l’entrée de l’APOL1 est décrit comme le résultat d’un gonflement du lysosome induit par la génération de pores ioniques à l'intérieur de la membrane lysosomiale2. Nous montrons ici que le gonflement osmotique du lysosome peut être empêché en condition de culture hyper osmotique, sans néanmoins empêcher la mort de la cellule. En outre, l’APOL1 semble déclencher des événements de mort cellulaire programmée dans la cellule, tels qu’une dépolarisation mitochondriale rapide suivie d'une fragmentation de l’ADN. De plus, nous montrons une implication de l'endonucléase G (TbEndoG), connu pour être un acteur clé dans la régulation du processus de mort cellulaire et d’une kinésine (TbKIFC1) qui pourrait avoir le rôle de transporter l’APOL1 des endosomes vers la mitochondrie.

Nous avons également pu montrer que l’APOL3, un autre membre de la famille des APOLs humaines, possède tout comme l’APOL1, une activité trypanolytique bien que cette protéine ne soit pas sécrétée en condition physiologique. De manière intéressante, l’APOL3 ne tue pas seulement T. b. brucei, mais est également capable de tuer les sous-espèces résistantes à l’APOL1 tels que rhodesiense et gambiense, in vitro et in vivo lorsque de l’APOL3 recombinante est injectée dans des souris infectées. La spécificité d’action de l’APOL3 pourrait être liée à une indépendance au pH, au contraire de l’APOL1. Il pourrait être envisagé d'utiliser cette protéine recombinante comme agent curatif contre gambiense ou rhodesiense du fait que les médicaments actuellement disponibles pour le traitement de la trypanosomiase africaine montrent des problèmes de toxicité et de résistance.

Doctorat en Sciences
info:eu-repo/semantics/nonPublished

Recent research has shown that the combination of high triglyceride (TG) levels and low high density lipoprotein (HDL) levels, significantly increases the incidence of coronary artery disease (CAD). The incidence of CAD is also increased in abdominally obese individuals. To assess differences in postprandial TG clearance patterns between abdominally obese (AO) and controls (C), fourteen healthy, normolipidemic males (seven controls and seven abdominally obese) completed an oral fat loading test (78 grams of fat). Blood samples were collected every hour for eight hours. Abdominally obese individuals had significantly greater TG values, significantly lower total HDL and HDL2 values and significantly greater area under the TG curve (p = 0.03). Time to reach peak TG and time to reach baseline TG values did not differ between the two groups, even though fewer AO individuals reached baseline within eight hours. The data from the present investigation indicate that increased time to clear TG in AO individuals may be one pathway that increases the incidence of CAD in this group.
School of Physical Education

Cardiovascular disease remains the first cause of death worldwide. Pharmacological trials and Mendelian randomization studies have failed to unequivocally prove an inverse association between HDL-C levels and the risk of cardiac outcomes. Interest is now focused on the atheroprotective functions of the HDL particle. The aim of the present thesis was to evaluate the predictive role for cardiovascular diseases of a battery of HDL properties and components. Moreover, we evaluated the relationships among major CVD risk factors, 10-year CVD risk, and HDL-functionality and low-density lipoprotein (LDL) atherogenicity. We conducted a meta-analysis to obtain an updated record of the available published literature in this field. To assess the prognostic value for CVD of a set of novel surrogates of HDL-functionality and composition, we analyzed cholesterol efflux capacity (CEC), HDL oxidative-inflammatory index (HOII), platelet activating factor acetylhydrolase (PAF-AH) activity bound to HDL, and the concentrations of apolipoprotein (Apo) A-I and ApoA-IV, serum amyloid A (SAA), component Complement C3, and sphingosine-1-phosphate (S1P) in ApoB depleted plasmain a nested case-control study with volunteers at high cardiovascular risk from the PreDiMed Study (Effects of Mediterranean Diet on the Primary Prevention of Cardiovascular Disease). Samples were matched in a 1:2 fashion by age, sex, body mass index, intervention group, and follow-up time in the study at event occurrence. Finally, we analyzed the relationships among HDL function, LDL atherogenicity, and the risk for cardiovascular disease and classical risk factors. We used data from two representative subsamples of volunteers from the PreDiMed trial. Meta-analysis of data from the 25 studies revealed an inverse association for cholesterol efflux and antioxidant/anti-inflammatory capacities with the risk of major adverse cardiac outcomes. The association was confirmed for the risk of all-cause mortality and cholesterol efflux and antioxidant capacity. Findings from the case-control study display HDL-related function and its main protein, ApoA-I, as promising biomarkers of cardiovascular outcomes. Higher CEC, HOII, and ApoA-I levels are strong, independent surrogates of acute coronary syndrome (ACS). Moreover, S1P almost reached statistical significance, with a p-value=0.056, in the model including HDL-C as confounder. CEC and ApoA-I were both associated with a greater risk for acute myocardial infarction (AMI) independent of classical cardiovascular risk factors. Finally, higher HOII and lower ApoA-I (irrespective of type 2 diabetes mellitus, hypercholesterolemia, hypertension, and smoking habit) increased the risk of unstable angina (UA). High cardiovascular risk was related to low HLD-C and ApoA-I, poor CEC, and a dysfunctional HDL profile (higher content in triglyceride, more oxidized and smaller in size) and with high levels of ApoB and pro-atherogenic LDL (smaller and less resistant to oxidation). Volunteers with type-2 diabetes had low levels of HDL-C, ApoA-I, low-density lipoprotein cholesterol (LDL-C), and ApoB, and smaller, more oxidized LDL. Systemic hypercholesterolemia was related to greater HDL-C, LDL-C, ApoA-I, and ApoB levels and to small HDL size, higher CETP activity, and lower HDL capacity to esterify cholesterol. Each increase of 1 kg/m2 in body mass index was associated with low HDL-C, lower size of both HDL and LDL, and decreased HDL capacity to esterify cholesterol. Finally, men presented lower HDL-C and ApoA-I levels, greater HDL oxidation and HDL impaired vasodilatory capacity, and higher cholesterol content in LDL particles. Age was related to greater triglyceride content in the HDL core. In summary, the meta-analysis study showed that CEC, and the anti-inflammatory and antioxidant capacities of HDL, are inverse predictors of CVD. The association persisted with CEC and antioxidant capacity for all-cause mortality risk. These results were further confirmed in the case-cohort study, in which CEC at baseline showed a strong and independent inverse association with the risk of ACS and AMI. ApoA-I had the ability to predict ACS, AMI, and US. HOII proved to be a predictive index for ACS and UA. Finally, high cardiovascular risk scores and classical risk factors for cardiovascular disease were associated with impaired HDL regarding its components, particle size, and function, and with a more pro-atherogenic LDL.

The kidney is believed to play a major role in the clearance and re-absorption of high density lipoprotein (HDL) particles from the blood. Experiments were undertaken to explore the specific sites of renal HDL metabolism in vivo and to investigate in vitro the factors that regulate the renal re-absorption of HDL by HKC-8 human proximal tubule (PT) cells. Perfusion of a rabbit renal artery with [3H]cholesteryl ester (CE) and 125I-protein labeled HDL particles showed that the kidneys are capable of filtering both apolipoprotein A-I (apoA-I) and whole HDL. A fluorescent microscopic study with the HKC-8 cells showed that the PT cells can bind and take up HDL particles. (Abstract shortened by UMI.)

Les maladies cardiovasculaires (CV) représentent la première cause de mortalité lors de l'insuffisance rénale chronique (IRC). Cette morbidité apparat précocement lors de l'IRC et ne peut être explique par les facteurs de risque traditionnels. Le stress oxydant (SO), composante du cortège métabolique de l'IRC, représente un facteur de risque non traditionnel intriqué avec l'inflammation et la malnutrition. Le but de ce travail a été d'étudier la place du SO dans la survenue des complications CV au cours de l'IRC sur modale animal, puis de comparer le profil protéomique et la fonctionnalité des HDL in vitro entre sujets hémodialysés (HD) et témoins. Le SO au niveau CV a été étudié dans un modèle animal (adénine) d'IRC associé à la malnutrition. L'activité de la NADPH oxydase cardiaque est triple, alors que les activités des complexes de la chaîne respiratoire mitochondriale et de la SOD sont normales. Cette surproduction d'anion super oxyde est associé à une surexpression de l'ostéopontine et du pro-collagène de type I. L'étude protéomique des HDL de sujets HD et témoins a permis de préciser les anomalies qualitatives associées à la baisse des HDL induite par l'IRC. Les propriétés anti-oxydantes des HDL de ces mêmes sujets ont été étudiées in vitro sur un modèle d'oxydation des LDL au cuivre et sur un modèle cellulaire d'activation de la NADPH oxydase. En comparaison aux témoins, les HDL des sujets HD perdent leur capacité de protection des LDL contre l'oxydation. Par contre, la modulation de la NADPH oxydase sur modèle cellulaire est conservée avec les HDL de sujets HD mais serait moindre en présence d'une forte inflammation systémique. Ces résultats suggèrent que le SO est au cœur des complications cardiaques au cours de l'IRC. Parmi les mécanismes de défense endogènes, les propriétés anti-oxydantes des HDL sont en partie altérées chez le sujet HD
Cardiovascular (CV) diseases are the first cause of mortality during chronic kidney disease (CKD) and cannot only be explained by traditional risk factors (age, gender, dyslipidemia, hypertension). Oxidative stress, which has been associated with CKD, appears as a non-traditional risk factor closely interconnected with inflammation and malnutrition.This study aimed at investigating oxidative stress in CV complications in uremic rats. Then, HDL proteomic profile and in vitro functionality of HDL were compared between hemodialyzed (HD) patients and control subjects.First, an animal model of CKD associated with malnutrition, the adenine-fed rats, was set up in order to study CV oxidative stress. NADPH oxidase activity was increased three-fold, but the maximal activity of mitochondrial respiratory chain complexes and SOD were not different between groups. Superoxide anion output was associated with accumulation of osteopontin and of pro-collagen type I. In a second part, HDL proteomic study from HD and control subjects was performed to characterize qualitative modifications associated with the decrease in HDL observed in CKD. HDL anti-oxidative activities from these subjects were studied in vitro in a model of copper-induced LDL oxidation and in a cellular model of NADPH oxidase activation. Compared to control, HDL from HD patients failed to protect LDL oxidation. By contrast, HDL modulation of NADPH activity is maintained in HD patients but could be impaired by elevated inflammation.These results suggest that oxidative stress is a key event in cardiac complications during CKD. Among protective endogenous mechanisms, HDL anti-oxidative properties could be impaired in HD patients