Introduction: Coronary artery bypass grafting surgery is an effective treatment for coronary heart disease for many patients; however, evidence suggests that there are some patients who do not report a good post-operative recovery. Although several studies have begun investigating possible reasons for these observations, little is known about the impact of CABG on quality of life and there still remains a lack of information that can help clinicians identify those people more likely to experience poorer recovery so that interventions can be targeted appropriately. Aims: The overall aim was to investigate barriers to and facilitators of recovery after CABG. Method: Phase 1 was a retrospective qualitative study involving semi-structured interviews with eleven patients who had undergone CABG and with ten health professionals experienced in caring for these patients. Data were analysed using thematic analysis. Phase 2 was a prospective study comprising two components, questionnaire and interview. The questionnaire included measures of quality of life, perceived recovery, demographic and psychosocial variables and was administered prior to surgery and at six and twelve months post-surgery. A sample of ten people who completed questionnaires were interviewed at the same time points and data analysed using framework analysis. Results: Interview data described the patient experience of undergoing CABG and identified components of a good recovery from the patient perspective. Patient and health professional participants identified numerous barriers and facilitators to recovery at three key time points - prior to surgery, during the hospital inpatient stay and post-CABG - and noted the complex inter-relationships between them, thus emphasising the need for a holistic approach to investigating recovery. Questionnaire data described the pattern of psychosocial functioning, quality of life and perceived recovery across the surgical pathway and identified depression and self-efficacy as the main predictors of post-CABG quality of life and perceived recovery. Using interview and questionnaire data a model of recovery is proposed. Conclusions: Findings from this research have identified a complex inter-related network of barriers and facilitators to recovery, suggested the possible mechanisms by which they impact on post-CABG outcome and identified recommendations for clinical practice.

Nine physiological measures were evaluated pre- to post-intervention on subjects participating in a university health promotion program over a seven-month period. Frequency of program attendance and choice of activity were also assessed. Of the 88 employees initially screened, most of the subjects were staff members (n=82, 93%),with a majority being female (n=68, 77%). Significant differences in physiological measures were found pre- to post-intervention between "higher" and "lower" cardiovascular risk participants, primarily due to the type of activity chosen. .The results indicate that health promotion programs at a university are an effective way to have an impact on employees in reducing their cardiovascular risk factors.

Cardiomiopatia dilatada (CMD) é uma doença miocárdica caracterizada por dilatação cardíaca e redução da contratilidade da parede do ventrículo esquerdo ou de ambos os ventrículos, sendo a etiologia de origem genética ou desconhecida. Em suínos existem raros relatos de CMD, sendo frequentemente relacionados com intoxicações por ionóforos ou gossipol. Surtos de CMD de etiologia desconhecida em suínos de rebanhos comerciais no Brasil sugeriram a existência de nova etiologia, possivelmente nutricional. Este estudo teve como objetivo investigar as possíveis causas dos surtos de CMD em suínos, a partir de análises macroscópicas, microscópicas, bioquímicas, cromatográficas, moleculares, imuno-histoquímica (IHQ) e reprodução experimental. E teve como resultado 2 artigos científicos. O primeiro artigo descreve os achados clínicos, patológicos, químicos e toxicológicos de três surtos de CMD em suínos de crescimento, além da reprodução experimental desta condição utilizando a ração de uma das propriedades afetadas. Para o estudo experimental utilizou-se 9 animais divididos em 3 grupo; Grupo 1 recebendo ração suspeita, Grupo 2 metade ração suspeita mais metade de ração controle e o grupo 3 recebeu ração controle. Dois suínos do grupo 1 apresentaram condições clínicas e patológicas semelhantes aos casos naturais após 8 dias de consumo da ração suspeita. Os principais sinais clínicos observados eram tosse e dispneia grave. Na necropsia foram constatados dilatação cardíaca bilateral acentuada, hidrotórax, hidropericárdio, edema pulmonar, ascite e fígado com aspecto de noz moscada. O segundo artigo teve como objetivo fazer a caracterização histológica, histoquímica e imuno-histoquímica das lesões cardíacas em 8 suínos com CMD e compara-las com dois suínos controles. As principais lesões evidenciadas foram atrofia de cardiomiócitos, vacuolização sarcoplasmática, ruptura de miofibras e fibras com padrão ondulado evidenciadas nas colorações de hematoxilina e eosina (HE), Tricrômico de Masson e Picrosírius. Na análise imuno-histoquímica utilizando o anticorpo anti-desmina houve uma imunomarcação reduzida ou inexistente em áreas com lesões histopatológicas. A imuno-histoquímica anti-desmina demonstrou ser uma importante ferramenta diagnóstica para caracterização de lesões de CMD em suínos. / Dilated cardiomyopathy (DCM) is a myocardial disease characterized by cardiac dilatation and reduced contractility of the left ventricular wall or both ventricles, the etiology of which is genetic or unknown. In pigs there are rare reports of DCM and are often related to ionosphere or gossypol poisoning. DCM outbreaks of unknown etiology in swine from herds in Brazil suggested the existence of a new, possibly nutritional, etiology. This study aimed to investigate the possible causes of DCM in pigs through macroscopic, microscopic, biochemical, chromatographical, molecular and immunohistochemical (IHC) evaluations, as well as the experimental reproduction of the disease. The project resulted in 2 scientific papers. The first article describes the clinical, pathological, chemical and toxicological findings of three DCM outbreaks in grower pigs, in addition to the experimental reproduction of this condition using the ration of one of the affected farms. For the experimental trial, 9 animals were divided into 3 groups; Group 1 received suspected ration only, Group 2 was fed a diet composed of half suspected ration plus half control ration, and group 3 received control ration only. Two pigs from group 1 presented clinical and pathological conditions similar to the natural cases after 8 days of consumption of the suspected ration. The main clinical signs observed were cough and severe dyspnea. At necropsy, bilateral cardiac dilatation, hydrothorax, hydropericardium, pulmonary edema, ascites and liver with the appearance of nutmeg were observed. The second article aimed to perform the histological, histochemical and immunohistochemical characterization of the cardiac lesions in 8 pigs with DCM and compare it with two control pigs. The main lesions evidenced were cardiomyocyte atrophy, sarcoplasmic vacuolization, rupture of myofibers and fibers with corrugated pattern evidenced in the staining of hematoxylin and eosin (HE), Masson's trichrome (MT) and Picrosírius (PS). Immunohistochemistry analysis using the anti-desmin antibody showed reduced or non-existent immunostaining in areas with histopathological lesions. The anti-desmin IHC proved to be an important tool for the diagnosis and characterization of DCM lesions in pigs.

Introdução: A associação positiva entre circunferência da cintura (CC) e hipertensão arterial sistêmica (HAS), diabete melito (DM) e doença cardíaca (DC) é bem estabelecida e avaliada a partir de valores que evidenciaram essa associação em população adulta, porém, essa relação deve ser investigada em indivíduos idosos, devido às alterações fisiológicas desse grupo populacional. Objetivo: Analisar a associação entre valores de CC, mensurados em 2000, e HAS, DM E DC, referidas em 2006, e identificar valores de CC, com melhor capacidade discriminatória do risco para desenvolvimento dessas doenças, em idosos residentes no município de São Paulo. Métodos: Trata-se de estudo longitudinal com utilização de dados do Estudo SABE: Saúde, Bem-estar e Envelhecimento, realizado no município de São Paulo, em 2000 e em 2006. A população de estudo foi composta por idosos que não referiram HAS, DM e DC, em 2000, e que apresentavam valor de CC. As variáveis de estudo foram CC (variável contínua), índice de massa corporal (< 28 kg/m 2 e 28 kg/m 2 ), sexo, grupo etário (60 a 74 e 75) e etnia (caucasiana e outras), dados de 2000, e HAS, DM e DC referidas, em 2006. Para verificar a associação, utilizou-se o teste Rao & Scott (p<0,05) para amostra complexa, e regressão logística múltipla. A área sob a curva (AUC) ROC (Receiver Operating Caracteristics) foi utilizada para estimar o desempenho dos valores de CC, em discriminar corretamente idosos, segundo a referência ou não das doenças associadas à CC. Os valores críticos de CC foram identificados pelos maiores valores de razão de verossimilhança positiva, mantendo razão de verossimilhança negativa igual a zero. Para avaliar a capacidade discriminatória dos valores de CC identificados no presente estudo e dos valores de CC usualmente utilizados (CC 80 ou 88 cm, para mulheres, e CC 94 ou 102 cm, para homens), foi feita a comparação das AUC e respectivos intervalos de confiança de 95 por cento . Todos os cálculos foram realizados pelo programa estatístico Stata versão 10.0. Resultados: Foram analisados 405 idosos, dos quais 26,9 por cento referiram HAS, 4,1 por cento DM e 10,0 por cento DC. A CC, em 2000, manteve-se como fator de risco para referência de DM, em 2006, independente do sexo, grupo etário, etnia e índice de massa corporal (OR 1,10; IC 95 por cento 1,05-1,16; p=0,000; p do modelo=0,000). A AUC mostrou desempenho satisfatório dos valores críticos de CC, na discriminação da referência de DM, para mulheres e homens, de 60-74 anos. Os valores críticos de CC, identificados foram 87 cm para mulheres e 99 cm para homens, os quais apresentaram melhor desempenho segundo o valor de AUC, em comparação aos valores usualmente utilizados. Conclusão: Os valores críticos de CC, identificados no presente estudo, para indivíduos de 60-74 anos, apresentaram melhor capacidade discriminatória da referência de DM, em 6 anos, quando comparado aos valores de CC usualmente utilizados / Background: The positive association between waist circumference (WC) and hypertension (H), diabetes mellitus (DM) and heart disease (HD) is well established and evaluated from critic values that showed this association in adults, however, this relationship must be investigated in the elderly due to physiological changes in this population group. Objective: To analyze the association between WC values, measured in 2000, and H, DM and HD, reported in 2006, and identify WC values, with better discriminatory capacity of the risk for developing these diseases, in elderly residents in the municipality of São Paulo. Methods: This was a longitudinal study using data from the SABE Survey: Health, Wellness and Aging, held in São Paulo, in 2000 and 2006. The study population was comprised of elderly individuals who did not report H, DM and HD in 2000, and who had WC value. The study variables were WC (continuous variable), body mass index (<28 kg/m and 28 kg/m 2 ), sex, age group (60 to 74 and 75), and ethnicity (caucasian and others), information of 2000, and H, DM and HD, reported in 2006. To verify the association, the Rao & Scott test for complex sample was used (p <0.05), and multiple logistic regression. The area under the ROC (Receiver Operating Caracteristics) curve (AUC) was used to estimate the performance of WC values in correctly discriminating elderly, according to the reference or not of diseases associated with WC. WC critical values were identified by the highest positive likelihood ratio, and negative likelihood ratio equal to zero. To assess the discriminatory capacity of WC values identified in this study and the WC values commonly used (WC 80 ou 88 cm, for women, e WC 94 ou 102 cm, for men) was made the comparison of AUC and confidence intervals of 95 per cent . All calculations carried out by using Stata version 10.0. Results: Four hundred five elderly people were analyzed, of which 26.9 per cent reported H, 4.1 per cent DM and 10.0 per cent HD. The WC value in 2000, was a risk factor for DM reference, in 2006, regardless of sex, age group, ethnicity and body mass index (OR 1.10, 95 per cent CI 1.05-1.16, p=0.000, p model=0.000). The AUC showed satisfactory performance of WC critical values, in discriminating the DM reference to women and men of 60-74 years. The WC critical values identified were 87 cm, for women, and 99 cm, for men, which showed better performance according to the AUC value compared to the WC values commonly used. Conclusion: The WC critical values identified in this study, for individuals of 60-74 years, showed better discriminatory ability of reference of DM, in period of 6 years, compared to the WC values commonly used

Heterotaxy refers to the abnormal arrangement of internal organs in relation to each other. Model organism studies have shown that functions of more than eighty genes are required for normal asymmetric left-right organ development. CRELD1 has been shown to be necessary for proper heart development and mutations in CRELD1 are known to increase risk of cardiac atrioventricular septal defects (AVSD). AVSD is the most common form of heart defect associated with heterotaxy, and we have previously shown that some individuals with heterotaxy-related AVSD have mutations in CRELD1. Therefore, we propose to examine the CRELD1 gene in a large sample of patients with heterotaxy syndrome. Our goal was to determine if mutations in CRELD1 are associated with other manifestations of heterotaxy or if they only coincide with AVSD. To achieve this aim, a sample size of 126 patients with heterotaxy collected by Dr. Belmont, Baylor college of Medicine, Texas, with approximately 66% of the heterotaxy population with different types of heart defects, were used for this study. Ten exons, promoter regions, and regulatory elements in the introns of CRELD1 gene were sequenced and analyzed. In this study three different heterozygous missense mutations in CRELD1 were identified in three unrelated individuals. These three individuals were diagnosed with different forms of heart defects in addition to AVSD. All three mutations were identified in highly conserved regions of CRELD1 possibly altering the CRELD1 properties. This demonstrates that mutations in CRELD1 may increase the susceptibility of AVSD in heterotaxy population. This information can help us to find factors effecting disease susceptibility in heterotaxy patients since the heart defects are a complex trait with incomplete penetrance.

Atherothrombosis can induce acute myocardial infarction and stroke by progressive stenosis of a blood vessel lumen to full occlusion. The goal of this research is to determine what shear rates are pertinent to an occluding blood vessel, the rate of thrombus growth relative to wall shear rates, and to develop a predictive model for estimating length of time to thrombus occlusion for a given atherosclerotic lesion. Computational studies of severely stenotic idealized vessels were performed to investigate the wall shear rates that may exist. The study shows that maximum shear rates in severe short stenoses were found to exceed 250,000 1/s (9,500 dynes/cm2). We utilize an in vitro experiment consisting of blood flow through a collagen coated stenosis to study the rate of thrombus growth. Growth is monitored through light microscopy and a camera. Computational fluid dynamics are used to determine shear rates along the thrombus surface as it grows. We found a strong positive correlation between thrombus growth rates and shear rates up to 6,000 1/s after a log-log transformation (r=0.85, p<0.0001). Growth rates at pathologic shear rates were typically 2-4 times greater than for physiologic shear rates below 400 s-1. To determine whether transport or kinetic binding limits the rate of thrombus growth, a computational model of platelet transport was developed. The model allows for thrombus growth by occluding computational cells. We show that thrombus is transport rate-limited for shear rates below 6,000 1/s, while it is more likely to be kinetic rate-limited for higher shear rates. Predictions of occlusion times based on the model demonstrate that increases in stenosis severity results in decreased time to occlusion.

La disfunción ventricular, las arritmias ventriculares o la isquemia residual son factores que condicionan la supervivencia y la aparición de nuevos accidentes coronarios tras el infarto de miocardio. Para conocer el riesgo de un determinado paciente y valorar la indicación de las técnicas de revascularización se han utilizado diferentes pruebas diagnósticas; sin embargo existe cierta controversia sobre si se deben practicar de forma sistemática. Los estudios realizados hasta el momento no son concluyentes y sugieren que las pruebas incruentas no aportan suficiente información pronóstica adicional a la obtenida de variables clínicas y electrocardiográficas y su realización no justifica el aumento de coste que producen. El presente estudio se planteó con los siguientes objetivos: (1) Identificar las variables clínicas que permiten conocer el pronóstico tras la fase aguda del infarto de miocardio. (2) Conocer los parámetros obtenidos mediante pruebas incruentas que aportan información pronóstica independiente, complementando la obtenida de las variables clínicas. (3) Determinar si la información obtenida del cateterismo mejora la predicción pronóstica realizada a partir de las variables clínicas y de las exploraciones incruentas. (4) Establecer la mejor pauta diagnóstica utilizando diversas pruebas. MATERIAL Y METODOS: De 495 pacientes consecutivos ingresados en la Unidad Coronaria con el diagn6stico de infarto agudo de miocardio, 295 tenían menos de 65 años, sobrevivieron a la fase aguda del infarto y aceptaron ser incluidos en el estudio. De ellos 7 se perdieron en el seguimiento, por lo que la población estudiada se compone de 288 pacientes, de los cuales 16 fallecieron durante el seguimiento de un año. A su ingreso se recogieron datos sobre su historia previa y sobre la exploración física. Se practicó un electrocardiograma diariamente y se iniciaron las determinaciones de enzimas cardiacos. Entre el 4º y 5º día se practicó un ecocardiograma en modo M y bidimensional (231 pacientes), entre los días 8º y 10º se realizó un registro del electrocardiograma de 24 horas (204 pacientes), una ventriculografía isotópica con tecnecio para el cálculo de la fracción de eyección (120 pacientes) y un cateterismo cardiaco izquierdo que incluyó la medición de presiones endocavitarias, la práctica de angiografía en dos proyecciones y coronariografía selectiva. Al mes del alta se realizó una prueba de esfuerzo en bicicleta ergométrica limitada por síntomas y una gammagrafía con talio-201 (193 pacientes). Los objetivos del análisis estadístico fueron la identificación de las variables capaces de predecir la muerte por un lado y la isquemia grave por otro. Para cada uno de los objetivos se practicó un análisis univariado estudiando un total de 63 variables. A continuación se realizaron diversos análisis de regresión logística de tipo escalonado, incluyendo en el primero las variables clínicas y comparando en los siguientes las variables clínicas con las variables obtenidas de las diversas pruebas diagnósticas. Asimismo se trazaron curvas ROC de sensibilidad Y especificidad para cada una de las ecuaciones de regresión. RESULTADOS. Predicción de muerte: Las variables clínicas que aportaron información pronóstica independiente fueron la existencia de diabetes, bloqueo bifascicular e infarto de miocardio previo al actual. Al añadir a estas variables la fracción de eyección isotópica, ésta tuvo valor pronóstico independiente así como también la presencia de más de 10 extrasístoles ventriculares por hora o taquicardia ventricular en el Holter, la presión arterial sistólica máxima alcanzada en la ergometría, la fracción de eyección angiográfica y el número de vasos coronarios afectados. Al realizar un análisis de regresión logística incluyendo las variables clínicas y las variables derivadas de las pruebas incruentas, tan sólo la diabetes y la fracción de eyección alcanzaron valor pronóstico independiente. Al comparar todas las pruebas simultáneamente, la diabetes, la fracción de eyección y el número de vasos fueron las variables con valor pronóstico independiente de muerte cardiaca. Predicción de isquemia grave: Se consideró como segundo objetivo del análisis la predicción de eventos isquémicos graves no letales considerando como tales la necesidad de revascularización antes del alta, la angina inestable que requiriera reingreso, o el reinfarto no letal. Del presente análisis se excluyeron 7 pacientes sometidos a cirugía por presentar lesiones del tronco común y 15 pacientes que fallecieron sin eventos isquémicos previos. Ninguna de las variables derivadas de las exploraciones no invasivas aportó información pronóstica de eventos isquémicos graves. El análisis de regresión logística mostró que las dos variables clínicas con valor predictivo independiente eran la presencia de angina post-infarto precoz y la existencia de infarto previo. Al añadir la regresión la variable “número de vasos afectados” ésta aportó información independiente. CONCLUSIONES: (1) Las variables clínicas con valor predictivo independiente de muerte cardiaca fueron la diabetes, el bloqueo bifascicular y el infarto previo. (2) De entre las pruebas incruentas analizadas el ecocardiograma y la gammagrafía cardiaca con talio no aportaron información pronóstica independiente de muerte. Asimismo, la información obtenida del Holter y la ergometría fue redundante con la aportada por la fracción de eyección. (3) La información obtenida del cateterismo cardiaco contribuyó de forma independiente a la predicción del riesgo, mejorando la información obtenida de las variables clínicas y de las pruebas no incruentas. (4) Las únicas variables que contribuyeron de forma independiente a la predicción de accidentes isquémicos no letales fueron la angina post-infarto precoz, el infarto previo y el número de vasos. / The value of non-invasive methods in adding useful information to establish the prognosis in survivors of myocardial infarction is still under debate. Therefore we undertook the present investigation to analyze if non-invasive tests and cardiac catheterization added independent prognostic information to simple clinical variables. In 288 survivors of a myocardial infarction (MI) a cardiac catheterization was performed the 10th day after admission. An M-mode and bidimensional echocardiogram was obtained in 231 patients, left ventricular technetium ventriculography in 120 patients and Holter recording in 204 patients. One month after discharge, patients underwent a symptom limited exercise test with ergometric bicycle and an exercise and rest thallium gammagraphy. The first end-point was cardiac death. The variables that showed significant differences in the univariate analysis were entered in successive stepwise logistic regression analysis including the clinical variables and the results of one different test each time. Among the clinical variables, the presence of diabetes mellitus, the existence of bifascicular bundle branch block or previous myocardial infarction showed independent prognostic value for death. Isotopic ejection fraction, ventricular arrhythmias in the Holter, the systolic pressure during exercise test, the angiographic ejection fraction and the number of diseased vessels also showed independent prognostic value. When the clinical variables and all the non-invasive tests were analyzed together, the presence of diabetes mellitus and the ejection fraction were the only variables that showed independent prognostic value. When the variables "number of diseased vessels" was included in the analysis, it also showed independent value. The second end-point was the presence of severe non-fatal ischemia during the follow-up. Among the clinical variables, the early post-myocardial infarction angina and the existence of a previous myocardial infarction were the only ones that showed independent predictive value of severe ischemia. From the tests performed, on1y the number of diseased vessels added independent prognostic value to the equation obtained from the clinical variables. In conclusion the practice of a technetium ventriculography, exercise test or Holter can add some prognostic information for cardiac death to the one obtained from the clinical variables, although the practice of several tests did not improve the prediction. On the other hand non-invasive tests do not seem useful in predicting new several non-fatal ischemic events.

Doctor of Philosophy(PhD) / 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.

Doctor of Philosophy(PhD) / 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.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.