Density gradient ultracentrifugation (DGU) is a powerful method for analyzing lipoprotein particles in great detail. It yields considerable amounts of information regarding the density distribution of these particles when coupled with fluorometric analysis and is an invaluable tool in determining their relative abundance. This union allows relationships between subclasses of lipoproteins to be established that gives researchers a more focused path to aid them in developing methods to predict the early onset of coronary artery disease (CAD). The research presented here focuses on the pairing of DGU with post-separatory techniques including matrix-assisted laser desorption mass spectrometry (MALDI-MS), liquid chromatography mass spectrometry (LC-MS), capillary electrophoresis (CE), isoelectric focusing (IEF) and apoptosis studies involving cell cultures. It is becoming clearer that cholesterol concentrations themselves do not provide sufficient data to assess the quality of cardiovascular health. As a result, research is becoming more focused on identifying better markers that may be indicative of development of CAD in a patient. Of specific interest is group of particles known as high density lipoproteins (HDL). Classically, this molecule is considered the “good cholesterol”, but literature from the last decade suggests that there may be atherogenic variants to this group. By utilizing DGU as a preparatory method for secondary analyses, new dimensions can be added to the density distribution analysis to allow a better determination of markers of cardiovascular health. The aim of this work is to utilize the principles involved with these various techniques to develop a comprehensive set of methods to aid in the detection of potential risk markers. In this study, the properties of metal ion complexes of EDTA as solute systems for analysis of lipoproteins by DGU are analyzed. We show that by varying the complexing ion and counter-ion of these metal-ion complexes, we gain the ability to control the separation of lipoprotein subclasses for subsequent analyses. Qualitative and quantitative data is presented that describes the analysis of different density regions of HDL for apolipoprotein content. Trends between control and atherogenic samples are also described and a clinical link between the biological activity of these regions and the chemical analysis is discussed.

Urotensin-II (U-II) and its receptor UT are upregulated in the pathological setting of various cardiovascular diseases including atherosclerosis. However, their exact role in atherosclerosis remains to be determined. In the present study, we hypothesized that selective overexpression of UT in an SMC-specific fashion would increase atherosclerotic lesion formation in a hypercholesterolemic mouse model. The objectives were to demonstrate the role of UT in this mouse model of atherosclerosis, and to elucidate some of the mechanism involved in the process. We used four strains of mice; wildtype (WT), UT+ (a transgenic strain expressing human UT driven by the alpha-SM22 promoter), ApoE knockout (ko), and UT+/ApoE ko. All animals were fed a high-fat diet for 12 weeks. Western blot analysis revealed a significant increase in UT expression in UT+ and ApoE ko mice (P<0.05). Serum cholesterol and triglyceride levels were significantly increased in ApoE ko and in UT+/ApoE ko but not in UT + mice when compared to wild type mice (P<0.0001). Analysis of aortas showed a significant increase in atherosclerotic lesion in the UT +, ApoE ko and UT+/ApoE ko compared to WT mice (P<0.05). Oral administration of the UT receptor antagonist SB-657510A for 10 weeks in a group of ApoE ko mice fed a high fat diet resulted in a significant reduction of lesion (P<0.001). Immunohistochemistry revealed the presence of strong expression of UT and U-II proteins in the atheroma of UT+, ApoE ko and UT+/ApoE ko mice, particularly in foam cells. SB-657510A also significantly reduced ACAT-1 protein expression in the atherosclerotic lesion of ApoE ko mice (P<0.05). The present findings suggest that the use of UT receptor antagonists may reduce lesion formation through reduced foam cell formation and lipid uptake, demonstrating an important role for UT in the pathogenesis of atherosclerosis.

No description available.

An array of analytical methods including density gradient ultracentrifugation, capillary electrophoresis, and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), were utilized to analyze serum high density lipoprotein (HDL) subfractions from two cohorts of normolipidemic individuals, which included subjects with diagnosed coronary artery disease (CAD), and angiographically proven non-CAD controls. These methods collectively provided characteristic information about the two populations of individuals including composition, electrophoretic mobilities, molecular weights, isoforms, and post-translational modifications of HDL apolipoproteins. This information proved useful in identifying potential biomarkers for CAD risk, and understanding the biological functions of a novel atherogenic HDL phenotype in individuals with CAD. Through the implementation of the aforementioned methodologies, new isoforms of apoC-I were identified. MALDI-MS, detected a shifting of approximately 90 Da in the mass to charge ratios corresponding to apoC-I peaks in the serum subfractions from all CAD cohort patients. This shifting was not observed in the non-CAD cohort, which displayed apoC-I peaks in accordance with the known mass of this protein. In addition to the shifting observed in the CAD cohort, some CAD patients showed further modifications of apoC-I that were indicative of oxidative processes. Interestingly, one patient, who has not been diagnosed with CAD, and has a family history of the disease, contained the apoC-I isoforms. This feature could underlie this subject’s known family history of CAD, and serve as an initial screening that could indicate the future development of CAD in this individual. Through collaborative work with Johns Hopkins University, it was initially observed that apoC-I enriched HDL induced apoptosis of aortic smooth muscle cells. Conversely, apoC-I depleted HDL induced minimal to no apoptosis, which led to the hypothesis that apoC-I is a contributor to atherogenic HDL and is a potential risk factor for CAD. Further collaborative work with Johns Hopkins assessed the apoptosis levels induced by HDL from both cohorts of patients. A distinct difference in apoptosis was identified between the two cohorts. High density lipoprotein subfractions from subjects in the CAD cohort, all of which contained the apoC-I isoforms, induced marked apoptosis compared to the non-CAD controls. These results further supported the hypothesis that apoC-I compromises the functionality of HDL and showed that through the induction of apoptosis, apoC-I can contribute to the destabilization of atherosclerotic plaque and the acceleration of CAD.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-07-26T12:55:35Z No. of bitstreams: 1 Leonor Fernandes Teixeira Amaral Remodelamento...2015.pdf: 447268 bytes, checksum: f7454eb0efdd04147e5c7a0370bd8d82 (MD5) / Approved for entry into archive by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2016-07-26T13:04:59Z (GMT) No. of bitstreams: 1 Leonor Fernandes Teixeira Amaral Remodelamento...2015.pdf: 447268 bytes, checksum: f7454eb0efdd04147e5c7a0370bd8d82 (MD5) / Made available in DSpace on 2016-07-26T13:04:59Z (GMT). No. of bitstreams: 1 Leonor Fernandes Teixeira Amaral Remodelamento...2015.pdf: 447268 bytes, checksum: f7454eb0efdd04147e5c7a0370bd8d82 (MD5) Previous issue date: 2015-08-28 / Fundação Gonçalo Moniz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / INTRODUÇÃO: as doenças cardiovasculares acometem milhares de pessoas no mundo, sendo a doença aterosclerótica a de maior morbimortalidade. Além disso, a aterosclerose pode manifestar-se precocemente dada a presença de dislipidemias, processos inflamatórios e alterações metabólicas como a diabetes. OBJETIVO: avaliar se existem diferenças no remodelamento da HDL e atividade antioxidante entre pacientes diabéticos e não diabéticos com doença aterosclerótica. Ainda, identificar, quantificar e estimar biomarcadores relacionados ao remodelamento de partículas lipoprotéicas e ao risco cardiovascular em função da concentração de colesterol na HDL, colesterol livre total, LDL-C, apoB, apoA-I, atividade da paraoxonase 1 (PON1), razões de risco como TG/HDL-C, LDL-C/ApoB, HDL-C/apoA-I, PON1/apoA-I, apoA-I/ApoB e tamanho estimado de partículas de HDL, LDL, glicemia, insulina e HbA1c. MÉTODOS: foram selecionados por conveniência 69 pacientes do sexo masculino, entre 18 e 75 anos, oriundos da enfermaria de cardiologia do Hospital Ana Neri, subdivididos em dois subgrupos: diabéticos e não diabéticos, ambos, com doença aterosclerótica coronária. Foram utilizadas metodologias enzimáticas, imunoturbidimétricas e nefelometricas nesse estudo. RESULTADOS: dos achados da comparação direta entre os grupos apenas a glicemia de jejum foi significativamente diferente (Teste t; p<0,05). Embora não significante o valor do colesterol não esterificado (CL) foi, em média, quatro vezes maior nos diabéticos quando comparado aos não diabéticos. A análise de correlação linear mostrou achados importantes do ponto de vista fisiológico, como correlação positiva entre CL e HDL-C (r=0,617; p<0,01083) e razão apoA-I/apoB e insulina (r=0,489; p<0,02095) nos diabéticos, e correlação negativa entre PON1/apoA-I com CL (r=-0,499; p<0,0065) e HDL-C com HbA1c (r=-0,444; p<0,0324) nos pacientes não diabéticos. CONCLUSÃO: Os achados desse estudo mostram que o cálculo das razões utilizadas para a análise de risco cardiovascular foram importantes indicadores quando correlacionados com marcadores séricos sugestivos de risco cardiovascular na população masculina diabética deste estudo. / Introduction: cardiovascular diseases affect thousands of people around the world, and atherosclerotic disease is the one with the greatest morbidity and mortality. Furthermore, atherosclerosis may manifest early by the presence of dyslipidemia, inflammatory processes and metabolic disorders such as diabetes. Objective: to assess whether there are differences between HDL remodeling and antioxidant activity from diabetic and nondiabetic patients with coronary artery disease. Also, identify, quantify and evaluate biomarkers related to lipoprotein particles remodeling and cardiovascular risk depending on HDL cholesterol concentration, total free cholesterol, LDL-C, apoB, apoA-I, paraoxonase activity 1 (PON1), and risk ratios like TG/HDL-C, LDL-C/ApoB, HDLC/ apoA-I, PON1/apoA-I, apoA-I/ApoB, HDL and LDL estimated particles size, glucose, insulin and HbA1c. Methods: we selected by convenience 69 male patients between 18 and 75 years, from the Cardiology Unit of Hospital Ana Neri, they were subdivided into two groups: diabetic and non-diabetic patients, both with coronary atherosclerosis. In these study were used enzymatic, immunoturbidimetric and nephelometric methodologies. Results: From the findings of the direct comparison between groups only fasting glucose was significantly different (t test; p <0.05). Although not significant, the value of non-esterified cholesterol (CL) was on average, four times higher in diabetics when compared to non-diabetics. Linear correlation analysis showed significant findings, from a physiological point of view, as positive correlation between CL and HDL-C (r = 0.617, p <0.01083) and apoA-I ratio/apoB and insulin (r = 0.489, p <0.02095) in diabetics, and negative correlation between PON1/apoA-I with CL (r = -0.499; p <0.0065) and HDL-C with HbA1c (r = -0.444; p <0.0324) in patients non-diabetic. Conclusion: the findings shows that the calculated ratio´s used for cardiovascular risk analysis were important indicators when correlated to serum markers suggestive of cardiovascular risk in the study diabetic male population.

Objective: In obese patients, we hypothesized physical exercise (PE) to affect lipids rather through its intrahepatic fat accumulation-lowering effect, associated with a decrease of total body fat (Fat%) and even weight (Mass), than through muscle work and an improvement of physical fitness. Design and setting: Thirty seven sedentary, non-diabetic women (BMI median 34.8 kg/m2) from our out- patient department were tested. Elimination criteria: recent weight reduction, lipid-influencing or heart rate-modifying medication. Interventions: PE protocol: 60 min supervised trainings, intensity at 65% of VO2max, modified by the Clamped Heart Rate test. Median (M) of total training hours was 34 during 115 days . Main Outcome Measurements: an effect of PE on total cholesterol (CH), triacylglycerols, HDL- cholesterol (HDL-C), LDL-cholesterol (LDL-C), index of atherogenity (IA), atherogenic index of plasma (AIP), maximum peak oxygen consumption (VO2max), Mass, body mass index (BMI), waist circumference (Waist) and Fat%. Results: Statistically significant differences at start (_s) and at the end (_e) of PE (p<0.05): AIP -0.049, Mass -3.6 (kg), BMI -1.7 (kg/m2), Waist -2.5 (cm), Fat% -2.5, VO2max 2.92 (l.min-1.kg-1), W 16.4 (Watt). Correlation coefficients, Pearson's between Gaussian distributed (Gd-v) variables and...

Objective: In obese patients, we hypothesized physical exercise (PE) to affect lipids rather through its intrahepatic fat accumulation-lowering effect, associated with a decrease of total body fat (Fat%) and even weight (Mass), than through muscle work and an improvement of physical fitness. Design and setting: Thirty seven sedentary, non-diabetic women (BMI median 34.8 kg/m2) from our out- patient department were tested. Elimination criteria: recent weight reduction, lipid-influencing or heart rate-modifying medication. Interventions: PE protocol: 60 min supervised trainings, intensity at 65% of VO2max, modified by the Clamped Heart Rate test. Median (M) of total training hours was 34 during 115 days . Main Outcome Measurements: an effect of PE on total cholesterol (CH), triacylglycerols, HDL- cholesterol (HDL-C), LDL-cholesterol (LDL-C), index of atherogenity (IA), atherogenic index of plasma (AIP), maximum peak oxygen consumption (VO2max), Mass, body mass index (BMI), waist circumference (Waist) and Fat%. Results: Statistically significant differences at start (_s) and at the end (_e) of PE (p<0.05): AIP -0.049, Mass -3.6 (kg), BMI -1.7 (kg/m2), Waist -2.5 (cm), Fat% -2.5, VO2max 2.92 (l.min-1.kg-1), W 16.4 (Watt). Correlation coefficients, Pearson's between Gaussian distributed (Gd-v) variables and...

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Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal