Functional and genic modulation of serum lipids and lipoproteins of carotid atherosclerosis in hyperalphalipoproteinemia

Orientador: Eliana Cotta de FariaDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias MedicasResumo: Está bem estabelecida na literatura especializada a associação inversa entre as concentrações plasmáticas de colesterol das lipoproteínas de alta densidade (HDL-C) e a incidência de doença arterial coronariana (DAC). Além de propriedades anti-oxidante, anti-inflamatória e anti-trombótica, a HDL participa do transporte reverso de colesterol, via pela qual o colesterol é captado das lipoproteínas e das membranas células periféricas e transportado ao fígado para sua excreção na forma livre ou de ácidos biliares. A lipase hepática (LH) possui função crucial no transporte reverso do colesterol, por sua atividade lipolítica e pela função de ligante à lipoproteínas facilitando sua captação tissular. A proteína de transferência de ésteres de colesterol (CETP), e mesma importância metabólica, promove a troca de ésteres de colesterol por triglicérides entre a HDL e as lipoproteínas ricas em triglicérides. Mutações nos genes que codificam estas proteínas têm sido muito estudadas para se compreender a função destas no metabolismo lipídico. O modelo experimental da hiperalfalipoproteinemia tem sido utilizado no decorrer dos últimos anos com o intuito de elucidar os mecanismos de ação da HDL e das proteínas reguladoras do seu metabolismo. A hiperalfalipoproteinemia é caracterizada pelo aumento das concentrações de HDL-C e é causada principalmente por deficiências genética de CETP e/ou LH. Os objetivos desta dissertação foram o de se estabelecer à modulação da hiperalfalipoproteinemia sobre os parâmetros antropométricos, bioquímicos, moleculares (¿514C/T do gene da LH e I405V do gene da CETP) e radiológicos (espessura da camada íntima média de carótidas) em uma amostra populacional brasileira. O estudo foi conduzido em 291 voluntários de ambos os sexos, classificados como hiperalfalipoproteinemicos (Hiper-A), HDL-C =68mg/dL, ou controles, HDL-C<68 e =32 mg/dL, de acordo com o valor do percentil 90, obtido em um estudo prévio do Laboratório de Lípides a partir população normolipidêmica. Os polimorfismos LH-514C/T e CETP I405V foram identificados através de técnicas de reação em cadeia polimerase (PCR) e a espessura da camada íntima-média de carótidas (EIM) pela ultra-sonografia de alta resolução. Em um primeiro trabalho observou-se em um sub-grupo de 169 indivíduos, com a medida da EIM, que somente a idade foi correlacionada com a EIM na hiperalfalipoproteinemia, enquanto que em controles houve modulação positiva pela idade, sexo masculino, pressão arterial sistólica, e controversamente com relatos da literatura, com HDL-C. Apesar de Hiper-A possuir um perfil com maior número de fatores de risco cardiovasculares, a semelhança encontrada na EIM de carótidas, assim como, da freqüência de EIM maior que 1 mm poderia, em parte, ser explicada pela grande diferença de modulação entre os grupos apontando para um traço protetor contra a aterosclerose carotídea em hiperalfalipoproteinemia. A ateroproteção reduzida em controles, tanto em homens quanto em mulheres, está de acordo com a observada associação negativa neste grupo entre EIM e a CETP com possível presença de HDL com a composição química alterada (ricas em TG e pobres em ésteres de colesterol), e ocorreu possìvelmente no sub-grupo masculino, com perfil pró-aterogênico evidente. Em um segundo trabalho, no sub-grupo de 169 indivíduos, com a medida da EIM, foi avaliado o efeito do polimorfismo LH-514C/T sobre a espessura da camada íntima-média de carótidas na hiperalfalipoproteinemia. Não se observou nenhuma variação de EIM em ambos os grupos em função deste polimorfismo. Quando comparados os grupos, o genótipo CC do polimorfismo LH-514C/T mostrou apenas tendência a maior EIM de carótidas em hiperalfalipoproteinemia (p<0,09), mas a freqüência de EIM maior que 1 mm foi igual. Em um terceiro trabalho, em 282-291 indivíduos foram avaliadas as semelhanças de freqüências entre os polimorfismos LH-514C/T e CETP I405V na hiperalfalipoproteinemia e normolipidemia. Ambos apresentaram altas freqüências, similares entre grupos e entre o polimorfismo LH-514C/T, CC 39%, CT+TT 61%; e o polimorfismo CETP I405V: II 26%, IV+VV 74% e CTL: CC 40%, CT+TT 60%, II 43% e IV+VV 57%. Descrevemos o polimorfismo LH-514C/T na hiperalfalipoproteinemia os TT vs CC apresentaram cintura menor, concentrações mais baixas de colesterol plasmático (C), fosfolípides (FL), LDL-C, estimativa do tamanho da LDL (LDL-C/ApoB). O polimorfismo CETP I405V na hiperalfalipoproteinemia em VV vs II, mostrou alta pressão arterial sangüínea e menores concentrações plasmáticas de HDL2TG e HDL3TG. O genótipo IV teve maiores concentrações plasmáticas de ApoAI e pressão arterial diastólica quando comparado com o genótipo II. Em resumo esta dissertação aponta para efeitos ateroprotetores ou neutros da hiperalfalipoproteinemia em uma amostra de população brasileira sobre a aterosclerose carotídea, inclusive no polimorfismo LH -514C/T. Os polimorfismos LH-514C/T e CETP I405V foram muito semelhantes com relação aos lípides e lipoproteínas séricos, mas não às proteínas reguladoras, oferecendo modulação protetora na hiperalfalipoproteinemiaAbstract: There is an inverse relationship between plasma concentration of high-density lipoprotein (HDL-C) and the risk of coronary arterial disease (CAD). Beyond anti-oxidant, anti-inflammatory and anti-thrombotic properties, HDL plays a role on the reverse cholesterol transport, where cholesterol is taken from lipoproteins and peripheral cells to the liver for excretion. Hepatic lipase (HL) plays a key role in this process, by its lipolitic activities and ligand functions. Cholesterol ester transfer protein (CETP), of equal metabolic importance, facilitates the exchange of cholesterol ester and triglycerides between HDL and triglyceride rich-lipoproteins. Mutations and polymorphisms of these enzymes have being studied in order to evaluate its activity and metabolic consequences. Hyperalphalipoproteinemia (Hyper-A) has being used in the latest years with the purpose of evaluating the anti and pro-atherogenic mechanisms of HDL and of regulating proteins. The aim of this work was to establish the modulation of hyperalphalipoproteinemia in relation to controls on the anthropometric, biochemical, radiological and molecular manifestations. This study was conducted on 291 volunteers, classified as Hyper-A, HDL-C=68mg/dL and controls, HDL-C <68 e 32 mg/dL according to the percentile 90th, obtained from a local normolipidemic population study. We determined clinic data, lipid, lipoproteins and radiological parameters of volunteers. The HL-514C/T and CETP I405V polymorphism were determined by polymerase chain reaction methods. The carotid intima-media thickness measurements were performed high performance ultrasound. We showed in the first manuscript that although possessing a higher risk coronary vascular disease profile the similarity found in carotid could in part be explained by the striking differences in its modulation between the two groups, indicating a protective trait against carotid atherosclerosis in hyperalphalipoproteinemia. In the Hyper-A population, was only correlated with age, while in controls had a positive correlation with age, male sex, systolic blood pressure, and surprisingly with HDL-C. This dissociation between IMT and HDL-C could be accounted for by a small HDL particle number in CTL. In the manuscript 2, the ¿514C/T polymorphism did not contribute to variations in the carotid IMT and Hyper-A did not modulate the IMT variations, contrary to Rundek et al., (2002) who investigated the ¿514C/T polymorphism on variations in the carotid IMT in 87 stroke-free subjects suggested that CC genotypes had increase of carotid IMT, FMT and HALP. The HL-514C/T e CETP I405V polymorphisms, were no associate, were highly prevalent in the two groups but were not associated with HDL-C. In Hyper-A, LH-514C/T induced lower plasma cholesterol (C), phospholipids (PL), LDL-C and LDL size (LDL-C/ApoB). In Hyper-A CETP I405V decreased blood pressure, reduced TG in HDL subfractions 2 and 3 of (HDL2TG and HDL3TG) and increase ApoAI. The HL -514C/T polymorphism in Hyper-A the TT vs CC had lower waist hip-circumference, cholesterol (C) concentrations, phospholipids (PL), LDL-C and estimated size particle by LDL-C/ApoB. The genotype TT was different between 2 groups: in Hyper-A with relation the CTL, had lower HL, estimated size particle by TG/HDL-C and higher HDL2C, HDL3C, HDL3TG, ApoAI and C concentrations and had higher C, estimated size particle by LDL-C/ApoB, ApoAI, HDL2C, HDL3C and estimated size particle by TG/HDL-C. The CETP I405V polymorphism in Hyper-A, the VV vs II had higher Systolic Blood Pressure and lower HDL2TG e HDL3TG concentrations. The IV genotype had higher ApoAI concentration and Diastolic Blood Pressure. In Hyper A, the VV genotype had higher HDL2C, HDL3C, ApoAI, e TG concentrations and reduced concentration of VLDL- and estimated size particle of LDL by TG/HDL-C. In summary, this work indicates an athero-protector and neutral effect on the carotid atherosclerosis in Hyper-A between HL-514C/T and CETP I405V polymorphisms both modulated for plasma lipids more atheroprotectiveMestradoCiencias BasicasMestre em Clinica Medic

Evaluation of the genetic expression of inflammatory mediators in mononuclear cells from deep venous thrombosis patients

Orientador: Joyce Maria Annichino BizzacchiTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: A trombose venosa é definida como a oclusão de um vaso do sistema venoso. Três fatores básicos para a formação de um trombo no interior dos vasos são: alteração do fluxo sangüíneo, da parede vascular e/ou dos elementos sangüíneos. A trombose venosa e a embolia pulmonar, que ocorre como uma complicação subseqüente representa uma causa importante de morbidade e mortalidade em pacientes hospitalizados. A freqüência da trombose venosa foi estimada em aproximadamente 1/1000 na população em geral. Na maior parte dos casos existe uma tendência para trombose determinada pela presença de um fator causal herdado ou adquirida, ou pela interação desses fatores, bem como por variações genéticas que determinam alterações nos níveis das proteínas pró-coagulantes e anticoagulantes. Dados da literatura têm sugerido a associação de mecanismos inflamatórios com a fisiopatologia da trombose venosa profunda (TVP). Os monócitos, estimulados por citocinas ou endotoxinas, expressam fator tecidual, o maior indutor da coagulação sangüínea, e que também tem a função de sinalização para a mobilidade celular e vascular. Os leucócitos apresentam receptores capazes de ligar e ativar o fator X da coagulação, servindo como via alternativa para a formação de trombina. As plaquetas podem aderir ao endotélio intacto e através da liberação de mediadores e citocinas como interleucina (IL)-1 e Fator de necrose tumoral-a (TNF-a), induzindo a expressão de moléculas de adesão e fator tecidual pela célula endotelial. Com base nestes dados e considerando que a migração leucocitária é um dos principais eventos que caracterizam o processo inflamatório, justificamos a escolha de células mononucleares (monócitos e linfócitos) como células centrais do nosso estudo. Utilizando técnicas de separação de células mononucleares por centrifugação em gradiente de "Ficoll-Hypaque", extração do Ácido ribonucléico (RNA) total, hibridação em Ácido desoxiribonucléico complementar (cDNA) -Microarray, e validação usando a reação em cadeia da polimerase em tempo real quantitativo (qRT-PCR) avaliamos do perfil de expressão gênica de alguns mediadores inflamatórios nessas células e a possível relação com a trombose venosa. Neste trabalho, usando a tecnologia de Microarray encontramos 60 induzidos e 56 genes reprimidos diferencialmente expressos nos pacientes com TVP, estes genes que estavam relacionados à resposta imune, inflamação, proteólise e transcrição. Destes genes diferencialmente expressos, selecionamos nove relacionados com inflamação para validação usando a técnica de qRT-PCR. Destes, somente houve aumento de expressão do gene da caspase 4 (CASP4) nos pacientes com TVP, sendo que, esta diferença se manteve no subgrupo com TVP espontâneo. Neste mesmo subgrupo, também foi verificado o aumento da expressão no gene Elong factor 1, alpha 2(EEF1A2)Abstract: Venous thrombosis is defined as a vessel occlusion of the venous system. Three basic factors for the formation of a thrombus inside the vessel are: changes in blood flow, vascular wall and / or blood elements. Venous thrombosis and pulmonary embolism, which occurs as a subsequent complication is a major cause of morbidity and mortality in hospitalized patients. The frequency of venous thrombosis was estimated to be approximately 1 / 1000 in the general population. In most cases there is a tendency for thrombosis determined by the presence of a causal factor inherited or acquired, or by the interaction of these factors, as well as genetic variations that determine changes in protein levels of procoagulants and anticoagulants. Literature data have suggested the association of inflammatory mechanisms in the pathophysiology of deep venous thrombosis (DVT). Monocytes, stimulated by cytokines or endotoxin, express tissue factor, the greater inducer of blood coagulation, and also have the function of signaling for cell motility and vascular. Leukocytes have receptors that can bind and activate coagulation factor X, serving as an alternative route for the formation of thrombin. Platelets can adhere to intact endothelium and through the release of mediators and cytokines such as interleukin (IL)-1 and Tumor necrosis factor-a (TNF-a), inducing expression of adhesion molecules and tissue factor by endothelial cells. Based on these data and considering that leukocyte migration is one of the main events that characterize the inflammatory process, we justify the choice of mononuclear cells (monocytes and lymphocytes) as the central cells of our study. Using techniques of separation of mononuclear cells by gradient centrifugation "Ficoll-Hypaque," extraction of total RNA, cDNA-Microarray hybridization, and validation using real time qPCR assessed the gene expression profile of some inflammatory mediators in these cells and the possible relationship with venous thrombosis. In this work using Microarray technology we found 60 genes upregulated and 56 downrelated differentially expressed in patients with DVT. Genes that were related to immune response, inflammation, proteolysis and transcription. Of these differentially expressed genes, we selected nine genes that were related with inflammation to validation using qRT- PCR technique. Just one of then, the caspase 4 (CASP4) genes was differentially increased in DVT patients, this increase kept in patients with spontaneous DVT and an increased of Elong factor 1, alpha 2 (EEF1A2) gene tooDoutoradoCiencias BiomedicasDoutora em Ciências Médica

Characterization of PCL and Chitosan Nanoparticles as Carriers of Enoxaparin and Its Antithrombotic Effect in Animal Models of Venous Thrombosis

This study was based on the preparation, characterization, and animal in vivo experiments performed to evaluate nanoparticles of poly(ɛ-caprolactone) (PCL) and chitosan as carriers of enoxaparin. The nanoparticles were characterized and presented satisfactory results in terms of size, polydispersity, and encapsulation efficiency. Anticoagulant activity of the nanoparticles was maintained for 14 hours when the administration was subcutaneous; however no activity was observed after oral administration. There was a significant reduction in thrombus size, in vivo, for both free and encapsulated enoxaparin in comparison with the control group after subcutaneous administration. Oral administration results however were indifferent. In conclusion, the double emulsion method w/o/w was efficient for enoxaparin encapsulation, producing spherical nanoparticles with high encapsulation efficiency. For in vivo studies, the encapsulated enoxaparin showed a sustained anticoagulant activity for a higher period of time compared to free enoxaparin, with an antithrombotic effect when administered subcutaneously

Characterization of PCL and chitosan nanoparticles as carriers of enoxaparin and its antithrombotic effect in animal models of venous thrombosis

This study was based on the preparation, characterization, and animal in vivo experiments performed to evaluate nanoparticles of poly(epsilon-caprolactone) (PCL) and chitosan as carriers of enoxaparin. Thenanoparticles were characterized and presented satisfactory results in terms of size, polydispersity, and encapsulation efficiency. Anticoagulant activity of the nanoparticles was maintained for 14 hours when the administration was subcutaneous; however no activity was observed after oral administration. There was a significant reduction in thrombus size, in vivo, for both free and encapsulated enoxaparin in comparison with the control group after subcutaneous administration. Oral administration results however were indifferent. In conclusion, the double emulsion method w/o/w was efficient for enoxaparin encapsulation, producing spherical nanoparticles with high encapsulation efficiency. For in vivo studies, the encapsulated enoxaparin showed a sustained anticoagulant activity for a higher period of time compared to free enoxaparin, with an antithrombotic effect when administered subcutaneously2017CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPsem informaçã

Laboratory Evaluation Of Patients With Undiagnosed Bleeding Disorders.

The evaluation of patients with a bleeding tendency represents a challenge as the routinely available tests for evaluating bleeding disorders are limited, complicating the laboratory determination of the clinically observed bleeding tendency. As a result, some bleeding disorders remain undiagnosed. The aim of the study was to evaluate whether global coagulation tests would contribute to the laboratory analysis of patients with undiagnosed bleeding disorders. Patients were evaluated for coagulation and fibrinolysis activities by thrombin generation test and euglobulin lysis time. In addition, plasma activity of factor XIII, plasminogen, α-2 antiplasmin, plasminogen activator inhibitor-1, and thrombin-activatable fibrinolysis inhibitor was also obtained. Forty-five patients were included. Eight per cent presented a mild bleeding disorder and 20% a moderate bleeding disorder. The thrombin generation test results were similar between patients and controls. Euglobulin lysis time results, however, were lower in patients than in controls, both before (median 175 vs. 250 min, respectively; P = 0.003) and after (median 145 vs. 115 min, respectively; P ≤ 0.001) arm constriction, suggesting that they were experiencing hyperfibrinolysis. Interestingly, patients' median thrombin-activatable fibrinolysis inhibitor activity was higher than in controls (21.2 vs. 19.46 μg/ml; P = 0.016). However, plasminogen, α-2 antiplasmin, plasminogen activator inhibitor-1, and factor XIII activities did not differ between the groups. Global coagulation and fibrinolysis tests proved to be limited in detecting the hemostatic disorders in some patients with a relevant bleeding tendency and may not be adequate to address their bleeding risk. Bleeding scores are currently the available medical approach for the evaluation of these patients