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Atherosclerotic plaque ruptures have been determined as the most common underlying cause of acute coronary syndromes and stroke. Currently, the standard of care for plaque rupture risk is based on the amount of luminal stenosis presented in a particular vessel; however, X-ray angiographic studies have shown that plaques at risk of rupture generally show <50% luminal narrowing. These findings explicate the need for other, more accurate methods of identifying problem lesions prior to the rupture event. Unfortunately, the study of thrombotic events and vulnerable plaque lesions in humans is difficult due to the spontaneity of rupture and the lengthy time course of disease progression. To further the understanding of plaque rupture risk in light of vulnerability detection, a rabbit model of atherothrombosis was used in conjunction with magnetic resonance imaging (MRI). MRI has been validated as a suitable imaging modality for in vivo, non-invasive detection of atherosclerosis and has provided quantitative predictors of plaques at risk of rupture. Additionally, the rabbit model has been shown, histologically, to present 6 of the 8 human plaque types classified by the American Heart Association. The first portion of this dissertation work focuses on using MRI to serially image rabbits undergoing the atherosclerotic protocol in order to assess rupture risk at the various time points. Previous work has determined that an increase in the vessel remodeling ratio (which hides a large plaque in the vessel wall) and contrast uptake (which indicates inflammation) are both characteristics of increased rupture risk. By obtaining these parameters at various time points in the disease progression, it was possible to determine when a certain plaque displays a heightened risk of rupture. The second portion of this work tested the efficacy of a pro-resolving molecule, lipoxin (an endogenous molecule), in reducing atherosclerotic disease state, specifically rupture with a luminal thrombus. Using chronic administration of this molecule in the same rabbit model of atherosclerosis yielded a faint reduction in atherosclerotic severity based on the parameters of decreased vessel lipid content and decreased thrombotic events presented in the treated group.

Atherosclerosis is a disease of both lipids and inflammatory immune cells. More specifically, elevated plasma levels of low-density lipoproteins (LDL) ultimately lead to migration of circulating monocytes into the artery wall. Lipid-loaded monocytes proliferate and become macrophage foam cells, the hallmark of atherosclerotic lesions. A proposed mechanism for the protective effects of high-density lipoprotein (HDL) is apolipoprotein A-I (apoA-I) acting as a mediator of cholesterol efflux from cells and subsequent foam cell regression. To better understand the biological changes stimulated by apoA-I treatment, differential gene expression analysis of microarray data was performed on spleen cells from mice treated with recombinant HDL (rHDL). LDL receptor null (LDLr-/-) and LDL receptor and apoA-I null (LDLr-/-, apoA-I-/-) mice were fed a Western diet consisting of 0.2% cholesterol and 42% calories as fat (HF) for a total of 12 weeks. After six weeks of diet, a subset of mice for each genotype was subcutaneously injected with 200 micrograms of rHDL (protein weight) three times a week for the remaining six weeks. The control group of mice was subcutaneously injected with 200 micrograms of bovine serum albumin (BSA). Spleen cell RNA was isolated, purified, and analyzed via Illumina BeadArray Microarray Technology. Individual differential gene expression analysis that contrasted treated to non-treated groups for each genotype was performed. LDLr-/-, apoA-I-/- rHDL treated mice showed 281 significantly differentially expressed genes compared to non-treated mice while LDLr-/- mice had 1502 such genes. Of the significant genes, 189 intersected across both genotypes. In LDLr-/-, apoA-I-/-, 73 of these were up-regulated and 116 were down-regulated. LDLr-/- similarly showed 71 of the intersected genes to be up-regulated and 118 to be down-regulated. One-directional gene set pathway analysis was also performed. LDLr-/-, apoA-I-/- treated mice revealed 49 significant pathways while LDLr-/- showed a total of 63. Of these, 21 were up-regulated and 14 were down-regulated in both genotypes. Of the overrepresented, up-regulated pathways, eight of the top ten most significant ones were related to immune cells. Major functions involved receptor, adhesion, and chemokine signaling. Overall, preliminary analysis suggests apoA-I treatment induces similar gene expression changes across different genotypes in mouse spleen cells.

Systemic inflammatory networks and local signaling cascades trigger culprit pathogenic mechanisms relating clinical cardiovascular disease (CVD) risk factors to atherosclerotic plaque progression and rupture. Imaging vascular inflammation affords a valuable marker of atherosclerotic disease activity to reveal important mechanistic insights for CVD research, to quantify early anti-inflammatory effects of new atherosclerosis drugs, and, ultimately, to help improve CVD risk prediction. While carotid, aortic, and peripheral arterial inflammation can be measured by 18F-fluorodeoxyglucose (FDG) PET-computed tomography (CT), as a glucose analog, high 18F-FDG signal spillover owing to physiological myocardial muscle metabolism prevents reliable coronary interpretation. Lack of cell specificity, and the influence of hypoxia on 18F-FDG uptake within macrophages and other plaque cells, are further limitations that drive the search for an alternative PET tracer for imaging inflammation in atherosclerosis. Up-regulation of the G-protein coupled receptor somatostatin receptor subtype-2 (SST2) occurs on the cell surface of activated macrophages. The central hypothesis tested in this thesis is that vascular SST2 PET imaging using 68Ga-DOTATATE might offer a more accurate marker of macrophage inflammation than 18F-FDG, with superior coronary imaging and therefore better power to discriminate high-risk vs. low-risk atherosclerotic lesions. Comprehensive molecular, histological and clinical evaluation of this experimental CVD imaging biomarker was undertaken, including a prospective clinical study with head-to-head comparison to 18F-FDG in coronary, carotid, and aortic vasculature. In vitro data showed that (i) target SSTR2 gene expression occurred in “pro-inflammatory” M1 macrophages, (ii) specific 68Ga-DOTATATE ligand binding to SST2 receptors occurred in CD68-positive macrophage-rich carotid plaque regions, and (iii) and carotid SSTR2 mRNA was highly correlated with both the pan-macrophage marker CD68 and in vivo 68Ga-DOTATATE PET signals. In clinical imaging, increased 68Ga-DOTATATE inflammatory signals correctly identified culprit vs. non-culprit arteries in patients with acute coronary syndrome and transient ischemic attack/stroke. 68Ga-DOTATATE also demonstrated good diagnostic accuracy for high-risk coronary CT features, and strong correlations with clinical CVD risk factors and 18F-FDG-defined vascular inflammation. While 18F-FDG also differentiated culprit vs. non-culprit carotid and high-risk coronary arteries, myocardial 18F-FDG overspill rendered coronary scans uninterpretable in most patients. In contrast, 68Ga-DOTATATE allowed unimpeded coronary interpretation in all patients. Findings of this thesis provide compelling evidence, from gene, to cell, to plaque, to patient, that SST2 PET imaging using 68Ga-DOTATATE provides a quantifiable marker of macrophage-related atherosclerotic inflammation and disease activity. Further studies are needed to establish whether 68Ga-DOTATATE PET can improve CVD risk prediction when added to current clinical methods, or offer a novel imaging platform to rapidly test the anti-inflammatory capacity of emerging atherosclerosis drugs. Broader translational applications of 68Ga-DOTATATE PET include possible use in diagnosis and therapeutic monitoring of vasculitis, endocarditis, myocarditis, and other manifestations of cardiovascular inflammation.

Atherosclerosis is characterised by the progressive growth of a plaque, where a fibrous cap covers a lipid-rich core. Rupture of this fibrous cap can lead to thrombosis and a heart attack or stroke. This dissertation considered the role of plaque microstructure in the mechanics and rupture risk of atherosclerotic plaques. Fibre structures in human atherosclerotic plaques were characterised through scanning electron microscopy, histology, and image processing. Local primary fibre orientation and the fibre dispersion were calculated. Plaque shoulder regions, where rupture is most frequent, had higher fibre dispersion and were more misaligned with the lumen wall. Comparative FE models were made from the plaque geometries and fibre structures found by image processing: one with evenly dispersed fibres (isotropic) and one with a preferred fibre orientation (anisotropic). The isotropic and anisotropic FE models predicted significantly different stresses. Stresses were often highest in the shoulder regions. Anisotropic stresses were calculated relative to fibres: axial stresses were highest, shear stresses were intermediate, and transverse stresses were low. Since the tissue is strongest in the fibre direction (axially), axial and shear failure modes should be considered. Material tests (uniaxial tension, trouser tear, and notched specimen) were used to characterise material properties in healthy porcine arteries. Fibre structures were evaluated by histology, multiphoton microscopy, and image processing. Samples were stiffer circumferentially and toughest against radial tears. Fractures progressed between fibres, rather than by breaking them. Material properties and fracture mechanisms were explained by fibre structures. In summary, atherosclerosis altered the fibre structure of the arteries, in ways that were mechanically significant and that explained clinical observations.

Vegetable products, antioxidants help the body resistance in various pathological conditions, especially those associated with aging, cancer and cardiovascular diseases, and so are classified as agent quimioprotetores. The antioxidant properties of some phenolic compounds such as leucoantocianidina already have a proven action in respect of the reduction of cholesterol, however, to quantify the reduction of atherosclerosis were no reports. Thus, this study aims to examine the effectiveness of grape leucoantocianidina reduction in experimental atherosclerotic plaque in rabbits. We used New Zealand rabbits (n = 27) adults from 7 to 8 months of age divided into 3 groups: control group with 200 g feed water ad libitum (G1), group with feed 200g, 1.5 g cholesterol + 10 ml of yolk (G2), group with feed 200g, 1.5 g cholesterol + 10 ml of yolk and treated with 50mg/kg of leucoantocianidina grape (G3). All groups were fed during 100 days, aiming to replicate the model of induced atherosclerosis in New Zealand albino rabbits; assess the effects of grape leucoantocianidina for: total cholesterol, LDL, HDL, VLDL, total lipids, triglycerides; leucoantocianidina Quantifying the effects of grape on the arterial subendothelial layer using the Program IMAGE-PRO-PLUS 4.5. Blood samples for measurements of lipid profiles of animals occurred at 0, 33, 66 and 99 days. At the end of the experiment, the animals were euthanized. Segments of the aortic arch, carotid artery and femoral artery (right and left) were collected for histopathological analysis. As expected the G1 did not observe change, G2 found a severe thickening of the subendothelial layer, the group G3 was thickening of the subendothelial layer, however, a less severe form compared to G2. Under the microscope, foam cells were observed in the aortic arch and no changes in the arteries, femoral and carotid artery thickening and endothelial G2 significantly compared to the G1. Leucoantocianidina grape was able to reduce 4.35% of the thickening of the subendothelial layer, was able to significantly reduce hepatic steatosis (macrovesicular and microvesicular), as well as the size of xanthomas arising distributed in the body of animals. Leucoantocianidina The grape does not change, in this experimental model of atherosclerosis in rabbits, the levels of total cholesterol, LDL, VLDL, HDL, triglycerides and total lipids significantly. / Fundação de Amparo a Pesquisa do Estado de Alagoas / Produtos vegetais antioxidantes auxiliam o corpo na resistência as várias condições patológicas, especialmente aquelas associadas com o envelhecimento, câncer e as doenças cardiovasculares, e por isso são classificados como agentes quimioprotetores. As propriedades antioxidantes de alguns compostos fenólicos como a leucoantocianidina já tem ação comprovada no que diz respeito na redução do colesterol, no entanto, na quantificação da redução da aterosclerose não encontramos relatos. Desta forma, o presente trabalho tem por objetivo analisar efeito da leucoantocianidina de uva na redução de placa aterosclerótica experimental em coelhos. Foram utilizados coelhos da Nova Zelândia (n=27) adultos entre 7 a 8 meses de idade divididos em 3 grupos: grupo controle com ração 200 g e água ad libitum (G1); o grupo com ração 200g, 1,5 g de colesterol + 10mL de gema (G2); grupo com ração 200g, 1,5 g de colesterol + 10mL de gema e tratado com 50mg/kg da leucoantocianidina de uva (G3). Todos os grupos foram alimentados durante o período de 100 dias, tendo como objetivo reproduzir o modelo de indução de aterosclerose em coelhos albinos Nova Zelândia; Verificar os efeitos da leucoantocianidina de uva sobre o: colesterol total, LDL, HDL, VLDL, lipídios totais, triglicerídeos; Quantificar os efeitos da leucoantocianidina de uva sobre a camada subendotelial arterial utilizando o Programa IMAGE-PRO-PLUS 4.5. A coleta de sangue para as dosagens do perfil lipídico dos animais aconteceram nos momentos 0, 33, 66 e 99 dias. Ao término do período experimental, os animais foram submetidos à eutanásia. Segmentos do arco aórtico, da artéria carótida e artéria femoral (direitas e esquerdas) foram coletados para análise histopatológica. Como esperado o grupo G1 não observou alteração, o grupo G2 verificamos um espessamento severo da camada subendotelial, o grupo G3 houve espessamento da camada subendoletial, no entanto, numa forma menos grave comparado com o grupo G2. Ao microscópio, foram observadas células espumosas no arco aórtico e sem alterações nas artérias: femoral e carótida e espessamento de endotélio no grupo G2 de forma significante comparando-os ao G1. A leucoantocianidina de uva foi capaz de reduzir 4,35 % do espessamento da camada subendotelial, foi capaz de reduzir forma significante a esteatose hepática (macrovesicular e microvesicular), bem como o tamanho dos xantomas que surgiram distribuídos nos corpo dos animais. A leucoantocianidina de uva não modifica, no presente modelo experimental de aterosclerose em coelhos, os níveis de colesterol total, LDL, VLDL, HDL, triglicerídeos e lipídeos totais de forma significante.

The tenn Acute Coronary Syndrome (ACS) encompasses a spectrum of patients who present with chest discomfort or other symptoms caused by myocardial ischemia or infarction. Since critical or acute care nurses care for such patients, they should have a thorough knowledge of ACS pathophysiology and current treatments for ACS The purpose of this research study is to explore and describe the knowledge level that the critical care nurses in a state hospital in East Texas feel they have regarding ACS. This study was quantitative, descriptive and contextual in design, in which a sample survey was performed, using a questionnaire based on a literature study. The response of most ofthe critical care nurses tested was that they felt they had insufficient knowledge. An in-service training session has been proposed to help improve the nurses' knowledge and expertise on ACS. / Health Studies / M.A. (Nursing Science)

abstract: Background. Effects of lifestyle interventions on early biomarkers of oxidative stress and CVD risk in youth with prediabetes are unknown. Objective. To evaluate the effects of a lifestyle intervention to prevent type 2 diabetes among obese prediabetic Latino adolescents on oxidized lipoproteins. Design: In a quasi-experimental design, 35 adolescents (51.4% male, age 15.5(1.0) y, body mass index (BMI) percentile 98.5(1.2), and glucose 2 hours after an oral glucose tolerance test-OGTT 141.2(12.2) mg/dL) participated in a 12-week intervention that included weekly exercise (three 60 min-sessions) and nutrition education (one 60 min-session). Outcomes measured at baseline and post-intervention were: fasting oxidized LDL and oxidized HDL (oxLDL and oxHDL) as oxidative stress variables; dietary intake of fresh fruit and vegetable (F&V) and fitness (VO2max) as behavioral variables; weight, BMI, body fat, and waist circumference as anthropometric variables; fasting glucose and insulin, 2hour glucose and insulin after an OGTT, insulin resistance (HOMA-IR), and lipid panel (triglycerides, total cholesterol, VLDL-c, LDL-c, HDL-c, and Non-HDL) as cardiometabolic variables. Results. Comparing baseline to post-intervention, significant decreases in oxLDL concentration were shown (51.0(14.0) and 48.7(12.8) U/L, p=0.022); however, the intervention did not decrease oxHDL (395.2(94.6) and 416.1(98.4) ng/mL, p=0.944). F&V dietary intake (116.4(97.0) and 165.8(91.0) g/d, p=0.025) and VO2max (29.7(5.0) and 31.6(4.7) ml*kg-1*min-1, p<0.001) significantly increased. Within-subjects correlations between changes in F&V intake and oxidized lipoproteins, adjusted for VO2max changes, were non-significant (R=-0.15, p=0.52 for oxLDL; R=0.22, p=0.25 for oxHDL). Anthropometric variables were significantly reduced (weight -1.3% p=0.042; BMI -2.2% and BMI percentile -0.4%, p=0.001; body fat -6.6% and waist circumference -1.8%, p=0.025). Cardiometabolic variables significantly improved, including reductions in glucose 2hour (-19.3% p<0.001), fasting insulin (-12.9% p=0.008), insulin 2hour (-53.5% p<0.001), and HOMA-IR (-12.5% p=0.015), with 23 participants (66%) that reverted toward a normal glucose tolerance status. Most lipid panel significantly changed (triglycerides -10.2% p=0.032; total cholesterol -5.4% p=0.002; VLDL-c -10.4% p=0.029; HDL-c -3.2% p=0.022; and Non-HDL -5.5% p=0.0007). Conclusion. The intervention resulted in differential effects on oxidized lipoproteins and significant improvements in behavioral, anthropometric and cardiometabolic variables, reducing the high metabolic risk of obese prediabetic kids. / Dissertation/Thesis / Doctoral Dissertation Exercise and Nutritional Sciences 2017

The role of inducible nitric oxide synthase (iNOS) and/or nitric oxide (NO) in atherosclerosis remains elusive. Several researchers argued whether iNOS and/or NO are pathogenic or cardio protective. The pathogenesis of atherosclerosis is complex and includes mechanisms associated with inducible nitric oxide synthase (iNOS). We have demonstrated that the expression and function of iNOS may be selectively down regulated by pro-oxidants such as antimycin A and diethyl maleate (DEM). To further explore the underlying mechanisms associated with these effects we have investigated whether antimycin A and/or DEM modulated the activation of key cellular signalling molecules associated with the induction of iNOS. Expression of p38 mitogen activated kinase (MAPK) and Akt were induced by exposure to lipopolysaccharide (LPS) and interferon-gamma (IFN-γ). Oxidative stress (OS) was induced using antimycin A, DEM and hydrogen peroxide (H2O2). All three OS inducers caused a significant generation of free radicals whereas only antimycin A and DEM generated superoxide radical (O2-). Also nitrite production and iNOS expression may be down regulated, in part; by pro-oxidants generating O2- but not hydroxyl radicals (OH-). Antimycin A and DEM concentration dependently inhibited the phosphorylation of p38 MAPK and Akt and this was restored when the cells were pre-treated with Atorvastatin whereas H2O2 was without any significant effect. Taken together, the data suggest novel actions for both pro-oxidants and atorvastatin which may have important implications in coronary artery disease where suppression of iNOS may be deleterious and maintaining its expression may be cardio-protective.

Cette thèse porte sur la sélection d’anticorps humains et la découverte de nouveaux biomarqueurs en vue d’un diagnostic et d’une thérapie de l’athérosclérose. Le premier volet concerne, dans un premier temps, la sélection d’anticorps humains dans un contexte physiopathologique chez un modèle animal de l’athérosclérose. La sélection in vivo a permis de sélectionner des anticorps sur large pannel de cibles dans leur conformation native et sous l’influence de leur microenvironnement. Suite à cela, afin d’accéder à la spécificité des clones, une méthode innovante de criblage a été développée, la cytométrie de flux à moyen débit. Cette technique, contrairement à l’ELISA couramment utilisé pour la caractérisation d’anticorps ou de peptides, ne nécessite pas de connaitre l’antigène et permet également de travailler à des concentrations de protéines moindres. Ainsi, deux cents anticorps ont pu être sélectionnés. Les tests d’affinité et de caractérisation de ces anticorps ont, par la suite, mené à l’identification d’une nouvelle cible de la plaque d’athérome, la galectine 3, protéine impliquée dans l’activation des macrophages ainsi que la rétention des LDL oxydées par ces dernières. Le deuxième volet concerne une seconde approche de criblage in silico des anticorps issus de la sélection in vivo, notamment grâce à l’utilisation de techniques de séquençage haut débit (Pacific Biosciences). Les résultats de cette étude pilote ont permis demettre en exergue plusieurs points : (1) la possibilité d’obtenir des séquences >1000bpcorrespondant à des scFv après analyse par IMGT/HighV-QUEST (database des immunoglobulines internationales) et (2) la possibilité d’identifier des séquences de clones préférentiellement enrichies après une sélection in vivo. En conclusion et perspectives, ces deux méthodes de criblage complémentaires nous donnent accès à de nouvelles voies d’obtention d’anticorps humains pour (1) le couplage à des nanoparticules pour le diagnostic par IRM de la plaque d’athérome ainsi que (2) apporter une thérapie in situ au site inflammatoire. / This thesis is based on the selection of human antibodies and identification of newbiomarkers to develop a diagnosis and a therapy of atherosclerosis. The part one treats, first,the selection of human antibodies in a pathophysiological context in an animal model ofatherosclerosis. To access not only to a large panel of targets but also proteins in their nativeconformation and influenced by their microenvironment, an in vivo selection was performed.In a second time, an innovative high throughput flow cytometry screening was implemented.Contrary to the use of ELISA screening which needs to have an identified target, the flowcytometry doesn’t have this limitation and uses a lower quantity of proteins than ELISAassays. By this screening, two thousand clones were selected. Characterization and specificitytests of these antibodies have yielded to the discovery of galectin 3 has a new biomarker. Thisprotein is implicated in macrophage activation, binding of oxLDL by macrophages to formfoam cells. The second part is about a second screening approach of the antibodies issuingfrom the in vivo selection by using Next Generation Sequencing methods (PacificBiosciences). The results of this primary study allowed for highlighingt two points: (1) thefeasibility to obtain long reads >1000bp identified as scFv after analysis by IMGT/HighVQUEST(international database of immunglobulins) and (2) the feasibility to identify hits ofclones preferentially enriched during the in vivo selection. In conclusion and perspectives,these two complementary methods give the opportunity to obtain human antibodies for (1) thecoupling to nanoparticles for diagnosis by MRI of atheroma plaque and (2) serve as ligandsfor in situ delivery of drugs.