Atherosclerosis, the pathophysiology of many cardiovascular diseases (CVD), is a chronic inflammatory process caused by the sustained accumulation of cholesterol, followed by endothelial dysfunction, and the resulting vascular inflammation. The established treatment for atherosclerosis, to date, involves the use of statins. These medications are hydroxymethylglutaryl coenzyme A reductase (HMG-CoA) inhibitors and lower the levels of by inhibiting HMG-CoA, a rate limiting step in the biosynthesis of cholesterol. Statin therapy varies in effectiveness based on dosage and individual differences, making effective treatment of patients challenging. More recently, extracellular vesicles (EVs) have emerged as a promising field in cardiovascular research. Once thought of as “platelet dust,” EVs are now recognized for their potential as therapeutic targets and tools. In this review, a comprehensive characterization of EVs is provided to explain how EVs are involved in normal physiological function and pathological processes of atherosclerosis. Evidence supports a model where EVs participate in the initiation and progression of atherosclerosis and may also be used as a delivery tool in disease therapy. Currently, cell-derived EVs can be therapeutic agents in animal models, an effective tool in gene therapy, or a drug delivery vehicle. Future experiments enhancing the therapeutic potential of EVs promise to deepen our understanding of EV-based therapy for atherosclerosis precision medicine.

The integration of biological and genetic data has established that diverse biological processes, involving multiple effectors, influence circulating levels of triglyceride and cholesterol. This diversity may underlie the genetic complexity of human dyslipidemias, including the common and highly atherogenic condition, Familial Combined Hyperlipidemia (FCHL). The aetiologies of FCHL are currently undetermined. In this thesis, a multi-pronged approach was employed to identify genes/variants contributing to the linkage observed between the chromosome 21q22.2-22.3 interval and lipid traits, in white-British FCHL families. Additionally GPIHBP1, which encodes glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1, was studied. GPIHBP1 represents a strong FCHL candidate gene due to its role in the lipolytic processing of triglyceride-rich lipoproteins. Combined genetic and gene expression analyses, focussed upon a refined 3.8Mb interval on chromosome 21q22.3 that was linked to lipid abnormalities in subsets of FCHL families, identified two genes (COL18A1 and PKNOX1 ) that warrant further investigation with regard to their contribution to FCHL. Promising results were also obtained for C21orf57, which resides just outside the 3.8Mb interval. Genetic association analyses in 1725 members of 239 FCHL families identified nominal association (P=0.0009) between a TSPEAR variant, rs34163868, and plasma triglyceride levels. Furthermore, transcript levels of CBS and TRPM2 were significantly altered by treatment with the PPAR-agonist bezafibrate in a rat hepatoma cell line, thus implicating these genes in triglyceride/fatty acid metabolism. In combined analysis of five independent cohorts, the minor allele of the GPIHBP1 variant, rs11538388 was protective against hypertriglyceridemia (P=2.98x10-4). The same allele was associated with decreased risk of coronary heart disease in the prospective Northwick Park Heart Study II (hazard ratio for carriers=0.76, P=0.0480) and delayed age of onset in the Southampton Atherosclerosis Study (odds ratio=0.76, P=0.0146). Collectively, these data demonstrate that the rs11538388 minor allele, or variant in linkage disequilibrium, is associated with more favourable processing of atherogenic lipoproteins.

FOXO3a is a pro-apoptotic transcription factor which shows increased activation in vascular smooth muscle cells (VSMCs) of advanced atherosclerotic plaques, specifically within the intimal layer. Since VSMC apoptosis plays a crucial role in the pathophysiology of atherosclerosis, we investigated the mechanisms underlying FOXO3a-mediated cell death in this particular cell type. We aimed to characterise a novel VSMC system (FOXO3aA3ERTM) and use these cells to validate MMP-13 and TIMP3 as new FOXO3a target genes. Also, we sought to determine the mechanisms of FOXO3aA3ERTM-mediated VSMC apoptosis, particularly regarding MMP-13 and TIMP3, potential MMP-13 substrates in the extracellular matrix and the precise apoptotic signalling involved. Furthermore, we aimed to investigate whether VSMC-specific activation of FOXO3aA3ERTM in mouse affects vascular remodelling during injury and whether this is reliant on MMP-13. Lastly, we aimed to address if endogenous FOXO3a upregulates MMP-13 in mouse and human VSMCs. Our laboratory has created a transgenic rat VSMC line which stably expresses an inducible FOXO3a mutant allele known as FOXO3aA3ERTM and previous microarray experiments identified matrix metalloproteinase 13 (MMP-13) as a potential novel FOXO3a target gene. Initially, we described several key features of the FOXO3aA3ERTM VSMCs used throughout this thesis, and subsequently demonstrated that MMP-13 is a bona fide target whose expression is rapidly upregulated upon FOXO3a activation, leading to markedly higher levels of protein, cleavage and proteolytic capacity. This induction of MMP-13 was responsible for the vast majority of FOXO3a-mediated apoptosis which was accompanied by prominent degradation of fibronectin, a glycoprotein found in the extracellular matrix. However, we could not identify a terminal apoptotic pathway. FOXO3a also downregulated the endogenous MMP inhibitor TIMP3, the recombinant protein of which reduced both MMP-13 proteolysis and FOXO3a-mediated apoptosis. Activation of FOXO3aA3ERTM in the VSMCs of medium and large arteries in mice resulted in heightened expression of MMP-13 in the vessel wall, which contributed to enhanced neointimal formation during carotid ligation. Finally, endogenous FOXO3a activation leads to increased MMP-13 expression in human VSMCs, but not mouse. Overall, we have shown that FOXO3a promotes VSMC apoptosis through MMP-13 both in vitro and in vivo, a novel pathway that has important implications for the pathogenesis and treatment of vascular disease.

Cardiovascular disease remains the commonest cause of death worldwide. The majority of deaths are caused by atherosclerotic plaque rupture with resultant myocardial infarction or stroke, or rupture of abdominal aortic aneurysms. Conventional imaging modalities have consistently failed to identify atherosclerotic plaques or aneurysms with high-risk pathological features that are at highest risk of rupture or progression. The development of modern molecular imaging techniques targeted at these features could lead to the identification of such high-risk plaques and aneurysms in vivo and guide the development of novel treatment strategies. The aim of this thesis was to evaluate whether novel molecular modalities have a role in providing new insights into biological disease processes, and identify high-risk plaques and aneurysms. Using positron emission tomography-computed tomography (PET-CT), 18F-fluorodeoxyglucose and 18F-fluoride were utilised as markers of metabolic inflammation and active calcification. Cellular inflammation was assessed using ultrasmall superparamagnetic particles of iron oxide (USPIO) enhanced magnetic resonance imaging (MRI). In a prospective trial, 80 patients with myocardial infarction (n=40) and stable angina (n=40) underwent 18F-fluoride and 18F-fluorodeoxyglucose PET-CT, and invasive coronary angiography (Chapter 3). Intense 18F-fluoride uptake localised to recently ruptured plaque in patients with acute myocardial infarction. In patients with stable coronary artery disease, 18F-fluoride uptake identified coronary plaques with high-risk features on intravascular ultrasound. 18F-fluoride PET-CT is the first noninvasive imaging method to identify and localise ruptured and high-risk coronary plaques. Aortic vascular uptake of 18F- fluorodeoxyglucose was studied in patients with myocardial infarction and stable angina (Chapter 4). In a separate outcome of 1,003 patients enrolled in the Global Registry of Acute Coronary Events, we further evaluated whether infarct size predicted recurrent coronary events. Patients with myocardial infarction had higher remote atherosclerotic tracer uptake that correlated with the degree of myocardial necrosis, and exceeded that observed in patients with stable coronary disease. The outcome cohort demonstrated that patients with higher degree of myocardial necrosis had the highest risk of early recurrent myocardial infarction. This supports the hypothesis that acute myocardial infarction exacerbates systemic atherosclerotic inflammation and remote plaque destabilization: myocardial infarction begets myocardial infarction. In a prospective imaging cohort, the role inflammation and calcification was assessed in 63 patients with abdominal aortic aneurysms and 19 age and sex matched patients with atherosclerosis (Chapter 5). Compared to non-aneurysmal segments, enhanced inflammation and calcification was observed within the wall of aortic aneurysmal segments. In comparison to matched controls with atherosclerosis, the entire aorta in those with aortic aneurysm appears more highly inflamed, suggesting presence of a global aortopathy rather than a disease confined only to the abdominal region of the aorta. Aortic aneurysms have greater active inflammation and calcification than atherosclerotic controls suggesting a more intense, destructive and transmural pathological process. A subgroup of fifteen patients with aortic aneurysms underwent imaging with both PET-CT with 18F-fluorodeoxyglucose, and T2*- weighted MRI before and 24 h after administration of USPIO (Chapter 6). Whilst there was a moderate correlation between the two tracers, there were distinct differences in the pattern and distribution of uptake suggesting a differential detection of macrophage glycolytic and phagocytic activity respectively. These studies provide novel insights into vascular biological processes involved in the initiation, progression and rupture of atherosclerotic plaques and aortic aneurysms. Future longitudinal studies are needed to establish whether these techniques have a role in improving the clinical management and treatment of patients with coronary artery disease and aortic aneurysms.

: L’athérosclérose est une maladie inflammatoire chronique des grosses artères à localisation intimale. Elle est probablement la résultante d’une réaction inflammatoire mal contrôlée ayant pour but initial d’éliminer l’accumulation anormale de lipides au niveau de l’intima. Cette élimination est exercé par les monocytes/macrophages, dont l’infiltration et l’accumulation au niveau des lésions sont une étape cruciale de l’inflammation chronique locale provoquant en particulier la production de cytokines.Les mécanismes moléculaires responsables de cette accumulation monocytaire impliquent notamment les chimiokines et leurs récepteurs, acteurs clés de la mobilisation des leucocytes. Les souris génétiquement invalidées pour certaines chimiokines comme CCL2 et CX3CL1 ou pour leurs récepteurs respectifs sont partiellement protégées de l’athérosclérose. Par ailleurs, chez l’homme, des variations génétiques de CX3CR1 sont associées à une réduction du risque d’accidents cardiovasculaires. L’ensemble de ces résultats indiquent un rôle clé des chimiokines inflammatoires dans l’athérogenèse.L’objectif de cette thèse était de tester l’utilisation d’inhibiteurs des récepteurs de chimiokines comme outils thérapeutiques contre l’athérosclérose. Dans ce but, notre laboratoire a développé une molécule aux propriétés antagonistes du récepteur CX3CR1, marqueur utilisé pour la caractérisation phénotypique des monocytes. Nos travaux sur deux modèles murins d’athérosclérose mettent en évidence que le blocage de CX3CR1 par notre antagoniste réduit la taille des plaques d’athérosclérose formées sans modifier leur composition cellulaire ni le taux de cholestérol plasmatique circulant. Cette diminution est corrélée à une diminution du nombre d’une sous-population monocytaire circulante spécifique, ainsi qu’à une diminution de leurs propriétés d’adhérence et de survie. D’un point de vue curatif, l’antagoniste de CX3CR1 est capable de limiter la progression des plaques d’athérosclérose sans la prévenir totalement.L’utilisation d’un outil ciblant spécifiquement le récepteur CX3CR1 nous à permis d’une part de mieux comprendre le rôle de ce dernier dans les processus de monocytose et d’athérogenèse et d’autres part d’évaluer la faisabilité d’approches thérapeutiques visant à limiter le nombre de monocytes infiltrant les lésions d’athérosclérose. Les perspectives de ces travaux consistent d’une part à approfondir encore le rôle de CX3CR1 dans la mobilisation monocytaire, notamment au niveau de la moelle osseuse, et d’autre à utiliser l’antagoniste testé en association avec d’autres drogues ciblant les récepteurs de chimiokines impliqués dans l’athérogenèse, tels que CCR2 et CCR5. / Atherosclerosis account for nearly 30% of death in industrialized countries. It is a chronic inflammatory disease of the large arteries intima. It has been suggested that it is the result of an uncontrolled inflammatory reaction secondary to an abnormal accumulation of lipids in the intima. The lipid clearance is performed by monocytes / macrophages, Their infiltration and accumulation in atherosclerotic lesions is a critical step of a local chronic inflammation associated with an increased production of cytokines. The molecular mechanisms of the generation of atherosclerotic lesions involve monocytes, chemokines and their receptors which are key players controlling leukocytes mobilization. Mice genetically invalidated for chemokines such as CCL2 and/or CX3CL1 or their respective receptors are partially protected from atherosclerosis. Furthermore, in humans, genetic polymorphisms of CX3CR1 are associated with a reduced risk of cardiovascular events. Taken together, these results highlight a key role for inflammatory chemokines in atherogenesis. The aim of this thesis was to investigate wether inhibitors of chemokine receptors could play a role as therapeutic tools against atherosclerosis. To this end, our laboratory had developed an antagonist of CX3CR1, a crucial phenotypic and functional marker of monocytes. Our work, on two murine models of atherosclerosis, demonstrates that blocking CX3CR1 by our antagonist reduces the size of atherosclerotic lesions. This decrease is correlated with a lower number of circulating inflammatory monocytes, as well as a decrease in their adhesion and survival properties. Therefore, CX3CR1 antagonist coud be able to limit the progression of atherosclerotic plaques. Targeting CX3CR1 allowed us to understand the role of this receptor in the pathophysiology of atherogenesis by its effects on circulating inflammatory monocytes and to evaluate the feasibility of the use of this antagonist as a therapeutic tool to reduce atherosclerotic lesions. Perspectives of this work are firstly to deepen the role of CX3CR1 in monocyte mobilization, especially from the bone marrow, and secondly to test this antagonist in combination with others drugs targeting chemokine receptors involved in atherogenesis, such as CCR2 and CCR5 in order to better control the evolution of atherosclerotic lesions.

No description available.

Li Tin Wai Olive. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 191-217). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.II / ABSTRACT --- p.III / 中文摘要 --- p.IX / PUBLICATIONS --- p.XIV / TABLE OF CONTENTS --- p.XV / LIST OF ABBREVIATIONS --- p.XXI / LIST OF FIGURES AND TABLES --- p.XXIII / Chapter CHAPTER 1. --- INTRODUCTION --- p.1 / Chapter CHAPTER 2. --- LITERATURE REVIEW --- p.7 / Chapter 2.1. --- Cardiovascular disease --- p.7 / Chapter 2.1.1. --- Introduction --- p.7 / Chapter 2.1.2. --- Atherosclerosis --- p.7 / Chapter 2.1.3. --- Cellular and molecular deregulation in early atherosclerosis --- p.10 / Chapter 2.1.3.1. --- Introduction --- p.10 / Chapter 2.1.3.2. --- Endothelial dysfunction --- p.11 / Chapter 2.1.3.3. --- Nitric oxide --- p.12 / Chapter 2.1.3.4. --- Adhesion molecules and the early events of atherogenesis --- p.13 / Chapter 2.1.3.4.1. --- Introduction --- p.13 / Chapter 2.1.3.4.2. --- Intracellular adhesion molecule-1 --- p.15 / Chapter 2.1.3.4.3. --- Nuclear factor kappa B --- p.18 / Chapter 2.1.3.5. --- Summary --- p.19 / Chapter 2.2. --- Nitric oxide in molecular vascular biology --- p.20 / Chapter 2.2.1. --- Introduction --- p.20 / Chapter 2.2.2. --- Nitric oxide synthase --- p.21 / Chapter 2.2.2.1. --- Introduction --- p.21 / Chapter 2.2.2.2. --- Endothelial nitric oxide synthase --- p.24 / Chapter 2.2.2.3. --- Inducible nitric oxide synthase --- p.25 / Chapter 2.2.2.4. --- Nitric oxide concentration dependent effector pathways --- p.26 / Chapter 2.2.3. --- Nitric oxide and its regulation in vascular events --- p.28 / Chapter 2.2.3.1. --- Introduction --- p.28 / Chapter 2.2.3.2. --- Regulation of vascular tone --- p.30 / Chapter 2.2.3.3. --- "Regulation of platelet adhesion, activation and aggregation" --- p.32 / Chapter 2.2.3.4. --- Regulation of endothelial adhesiveness and leukocyte adhesion - Anti-adhesive effect of nitric oxide --- p.32 / Chapter 2.2.3.5. --- "Regulation of vascular smooth muscle growth, migration and proliferation" --- p.33 / Chapter 2.2.3.6. --- Antioxidative effect of nitric oxide --- p.34 / Chapter 2.2.3.7. --- Regulation of endothelial apoptosis --- p.35 / Chapter 2.2.3.8. --- Nitric oxide and its relationship with other risk factors --- p.36 / Chapter 2.3. --- "Menopause, cardiovascular diseases and Traditional Chinese Medicine" --- p.37 / Chapter 2.3.1. --- Traditional Chinese Medicine and menopause --- p.37 / Chapter 2.3.2. --- Dang Gui Buxue Tang --- p.38 / Chapter 2.3.3. --- Danggui --- p.39 / Chapter 2.3.3.1. --- Botanic origins --- p.39 / Chapter 2.3.3.2. --- Usage --- p.39 / Chapter 2.3.4. --- Huangqi --- p.40 / Chapter 2.3.4.1. --- Botanic origins --- p.40 / Chapter 2.3.4.2. --- Usage --- p.40 / Chapter 2.3.5. --- Modern scientific research --- p.41 / Chapter 2.3.5.1. --- General cardioprotective role --- p.41 / Chapter 2.3.5.2. --- Vascular tone modulation --- p.42 / Chapter 2.3.5.3. --- Haemostasis --- p.42 / Chapter 2.3.5.4. --- Endothelial cell --- p.43 / Chapter 2.3.5.4.1. --- Nitric oxide pathway --- p.43 / Chapter 2.3.5.4.1.1. --- Direct alteration of nitric oxide secretion --- p.43 / Chapter 2.3.5.4.1.2. --- Alteration of Nitric oxide synthase expression or activity --- p.43 / Chapter 2.3.5.4.2. --- Alteration of adhesion molecule expression --- p.44 / Chapter 2.3.5.4.3. --- Alteration of adhesion molecule expression as an effect of nitric oxide secretion --- p.45 / Chapter 2.3.5.5. --- Antioxidant effect --- p.45 / Chapter 2.3.5.6. --- Estrogenicity of DBT --- p.46 / Chapter 2.4. --- Research plan --- p.47 / Chapter 2.4.1. --- Formulation of research hypotheses --- p.47 / Chapter 2.4.1.1. --- Hypotheses --- p.50 / Chapter 2.4.2. --- Plan of study --- p.50 / Chapter 2.4.2.1. --- Dang Gui Buxue Tang extraction and standardization of content --- p.50 / Chapter 2.4.2.2. --- Cell model development --- p.52 / Chapter 2.4.2.3. --- Experimental studies --- p.54 / Chapter 2.4.2.4. --- eNOS activity determination - the nitric oxide metabolite assay --- p.56 / Chapter 2.4.2.5. --- Endotoxin contamination in DBT --- p.57 / Chapter 2.4.3. --- Sample size and statistical analysis --- p.59 / Chapter CHAPTER 3. --- MATERIALS AND METHODS --- p.62 / Chapter 3.1. --- Dang Gui Buxue Tang extraction and content standardization --- p.62 / Chapter 3.1.1. --- Plant materials --- p.62 / Chapter 3.1.2. --- DBT authentication --- p.62 / Chapter 3.1.3. --- DBT processing prior to extraction --- p.63 / Chapter 3.1.4. --- DBT extraction --- p.63 / Chapter 3.1.5. --- Quantitative standardization of DBT markers by High Pressure Liquid Chromatography --- p.66 / Chapter 3.1.5.1. --- DBT markers: standard preparation --- p.66 / Chapter 3.1.5.2. --- Sample preparation --- p.67 / Chapter 3.1.5.3. --- Quantitative analysis of DBT constituents by HPLC --- p.67 / Chapter 3.1.6. --- DBT polysaccharide standardization --- p.68 / Chapter 3.1.6.1. --- Glucose standard preparation --- p.68 / Chapter 3.1.6.2. --- Sample preparation --- p.68 / Chapter 3.1.6.3. --- Quantitative determination of polysaccharide by Phenol-Sulfuric acid colorimetric assay --- p.68 / Chapter 3.1.7. --- DBT endotoxin contamination determination --- p.69 / Chapter 3.1.7.1. --- "Positive, negative and inhibition controls" --- p.69 / Chapter 3.1.7.2. --- Qualitative determination of sample endotoxin --- p.70 / Chapter 3.2. --- Cell culture --- p.70 / Chapter 3.2.1. --- Characterization of cultured cells --- p.72 / Chapter 3.2.2. --- Passage --- p.73 / Chapter 3.3. --- DBT treatment --- p.73 / Chapter 3.3.1. --- Solvent system of DBT treatment --- p.73 / Chapter 3.3.2. --- Dosage and duration of DBT treatment --- p.74 / Chapter 3.3.3. --- Positive and negative controls --- p.74 / Chapter 3.4. --- MTT-based cytotoxicity assay --- p.75 / Chapter 3.5. --- Reverse transcriptase- polymerase chain reaction --- p.76 / Chapter 3.5.1. --- Sample preparation --- p.76 / Chapter 3.5.1.1. --- Total RNA isolation --- p.76 / Chapter 3.5.1.2. --- DNase treatment --- p.77 / Chapter 3.5.1.3. --- RNAethanol precipitation --- p.78 / Chapter 3.5.1.4. --- Complementary DNA synthesis --- p.78 / Chapter 3.5.2. --- Polymerase chain reaction --- p.79 / Chapter 3.5.2.1. --- Polymerase chain reaction conditions --- p.79 / Chapter 3.5.2.2. --- Primers --- p.79 / Chapter 3.5.3. --- Visualization of the PCR products --- p.81 / Chapter 3.5.3.1. --- Gel electrophoresis --- p.81 / Chapter 3.5.3.2. --- Gel Doc software --- p.82 / Chapter 3.5.3.3. --- Densitometry --- p.82 / Chapter 3.5.4. --- Real time RT-PCR --- p.82 / Chapter 3.6. --- Quantitative Immunocytochemical studies --- p.84 / Chapter 3.6.1. --- Coverslip preparation --- p.84 / Chapter 3.6.2. --- Sample preparation --- p.84 / Chapter 3.6.3. --- Immunocytochemical staining preparation --- p.85 / Chapter 3.6.3.1. --- "Immunocytochemical staining for vWF, α-actin, iNOS, ICAM-1, NF-kB using DAKO catalyzed signal amplification (CSA) system (anti-mouse)" --- p.86 / Chapter 3.6.3.2. --- Immunocytochemical staining for eNOS using Santa Cruz immunoCruz staining system (anti-goat) --- p.87 / Chapter 3.6.4. --- Counterstaining and mounting --- p.88 / Chapter 3.6.5. --- Result interpretation --- p.89 / Chapter 3.6.5.1. --- Microscopy and digital image capture --- p.89 / Chapter 3.6.5.2. --- Determination of Image (file) Energy --- p.89 / Chapter 3.7. --- Total Nitrite/Nitrate quantitative colorimetric assay --- p.90 / Chapter 3.7.1. --- Sample preparation --- p.90 / Chapter 3.7.2. --- Total Nitrite/Nitrate quantitative colorimetric assay --- p.91 / Chapter CHAPTER 4. --- RESULTS --- p.93 / Chapter 4.1. --- Dang Gui Buxue Tang extraction and standardization of content --- p.93 / Chapter 4.1.1. --- DBT extraction - general data --- p.93 / Chapter 4.1.2. --- DBT polysaccharide standardization --- p.97 / Chapter 4.1.3. --- DBT marker standardization --- p.101 / Chapter 4.1.4. --- DBT endotoxin contamination determination --- p.104 / Chapter 4.2. --- Cell model development --- p.108 / Chapter 4.2.1. --- Endothelial morphology --- p.108 / Chapter 4.2.2. --- Immunocytochemistiy --- p.108 / Chapter 4.2.3. --- MTT cytotoxicity assay --- p.110 / Chapter 4.3. --- Study 1 --- p.112 / Chapter 4.3.1. --- Immunocytochemistry (Hypothesis 1) --- p.112 / Chapter 4.3.2. --- RT-PCR (Hypothesis 1) --- p.117 / Chapter 4.3.3. --- Real time RT-PCR (Hypothesis 1) --- p.121 / Chapter 4.3.4. --- Immunocytochemistry (Hypothesis 2) --- p.125 / Chapter 4.3.5. --- RT-PCR (Hypothesis 2) --- p.128 / Chapter 4.3.6. --- Total Nitrite/Nitrate quantitative colorimetric assay --- p.131 / Chapter 4.4. --- Study 2 --- p.133 / Chapter 4.4.1. --- Immunocytochemistry --- p.133 / Chapter 4.5. --- Study 3 --- p.138 / Chapter 4.5.1. --- Immunocytochemistry --- p.138 / Chapter 4.5.2. --- RT-PCR --- p.141 / Chapter 4.5.3. --- Real time RT-PCR --- p.145 / Chapter 4.6. --- Endotoxin contamination in DBT --- p.149 / Chapter 4.6.1. --- Effects of endotoxin on eNOS --- p.149 / Chapter 4.6.2. --- Immunocytochemistry on immunostained endothelial cells --- p.151 / Chapter 4.6.3. --- Effects of endotoxin on iNOS --- p.153 / Chapter 4.6.4. --- Effect of endotoxin on NF-kB --- p.157 / Chapter 4.6.5. --- Effects of endotoxin on ICAM-1 --- p.160 / Chapter CHAPTER 5. --- DISCUSSION --- p.165 / Chapter 5.1. --- DBT extraction and standardization of content --- p.165 / Chapter 5.1.1. --- Optimal DBT extraction conditions --- p.165 / Chapter 5.1.2. --- Evidence to support formulae usage --- p.166 / Chapter 5.1.3. --- Limitation of the methodology used --- p.166 / Chapter 5.2. --- Cell model development --- p.167 / Chapter 5.2.1. --- Choice of DBT concentration range in the study --- p.167 / Chapter 5.2.1.1. --- Choice of concentration range in consideration of endotoxin contamination --- p.167 / Chapter 5.2.1.2. --- Choice of concentration range in consideration of DBT's cytotoxicity effects --- p.168 / Chapter 5.2.1.3. --- Choice of concentration range in consideration of prevous studies --- p.168 / Chapter 5.3. --- Study 1 --- p.169 / Chapter 5.3.1. --- Action of DBT on eNOS expression --- p.169 / Chapter 5.3.2. --- Action of DBT on iNOS expression --- p.170 / Chapter 5.3.3. --- Action of DBT on Nitric oxide metabolite assay --- p.171 / Chapter 5.3.3.1. --- Result interpretation with rejected hypothesis 2 --- p.171 / Chapter 5.3.3.2. --- Assay limitations and improvements --- p.171 / Chapter 5.4. --- Study 2 --- p.172 / Chapter 5.4.1. --- Action of DBT on NF-kB expression --- p.172 / Chapter 5.4.2. --- Assay limitations and improvements --- p.173 / Chapter 5.5. --- Study 3 --- p.174 / Chapter 5.5.1. --- Action of DBT on ICAM-1 expression --- p.174 / Chapter 5.6. --- Endotoxin contamination in DBT --- p.175 / Chapter 5.6.1. --- Action of endotoxin contamination in DBT on various markers --- p.175 / Chapter 5.6:2. --- Experimental limitation --- p.176 / Chapter 5.6.3. --- Endotoxin removal --- p.177 / Chapter 5.6.3.1. --- Introduction --- p.177 / Chapter 5.6.3.2. --- Endotoxin removal methodologies suitable for herbal use --- p.179 / Chapter 5.7. --- Action of DBT on angiogenesis stimulation --- p.181 / Chapter 5.7.1. --- Evidence for DBT's proangiogenic effects from various studies --- p.181 / Chapter 5.7.2. --- Influence of endotoxin contamination on angiogenesis stimulation --- p.182 / Chapter 5.7.3. --- Assay limitations and future developments --- p.183 / Chapter CHAPTER 6. --- GENERAL DISCUSSION AND SUMMARY --- p.186 / Chapter CHAPTER 7. --- REFERENCES --- p.191

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / As células tronco são caracterizadas pela sua capacidade de se diferenciar em várias linhagens de células e exibir um pontente efeito parácrino. O objetivo deste trabalho foi avaliar o efeito da terapia com células da medula óssea (BMCs) na glicose sanguínea, no metabolismo lipídico e remodelamento da parede da aorta em um modelo experimental para aterosclerose. Camundongos C57BL/6 foram alimentados com uma dieta controle (grupo CO) ou uma dieta aterogênica (grupo AT - 60% gordura). Após 16 semanas, o grupo AT foi dividido em quatro sub grupos: grupo AT 14 dias e o grupo AT 21 dias receberam uma injeção de PBS na veia caudal e mortos 14 e 21 dias após respectivamente; grupo AT-BMC 14 dias e AT-BMC 21 dias que receberam uma injeção com BMCs na veia caudal e mortos 14 e 21 dias após, respectivamente. O grupo CO foi sacrificado juntamente com outros grupos. O transplante BMCs reduziu os niveis de glicose, triglicerídeos e colesterol total no sangue. Não houve diferença significativa em relação à massa corporal entre os grupos transplantados e não transplantados, sendo todos diferentes do grupo CO. Não houve diferença significativa na curva glicemica entre os grupos AT 14 dias, AT-BMC 14 dias e AT 21 dias e estes diferentes do grupo CO e do grupo AT-BMC 21 dias. O Qa (1/mm2) foi quantitativamente reduzido no grupo AT 14 dias e AT 21 dias quando comparado ao grupo CO. Este Qa se mostrou elevado no grupo AT-BMC 21 dias quando comparado a todos os grupos. O aumento da expessura da parede da aorta foi observado em todos os grupos aterogênicos, entretanto o aumento da espessura foi significativamente menor no grupo AT-BMC 21 dias em relação ao grupo AT 14 dias e AT 21 dias. A percentagem de fibras elásticas se apresentou significativamente maior no grupo AT 21 dias quando comparado ao CO e AT-BMC 21 dias. Não houve diferença significativa entre o grupo CO e AT-BMC 21 dias. Vacúolos na túnica média, delaminação e o adelgaçamento das lamelas elásticas foram observados nos grupos AT-14 dias e AT-21 dias. O menor número destes foi visualizado no grupo AT-BMC 14 dias e AT-BMC 21 dias. A imunomarcação para alfa actina de músculo liso (α-SMA) e fator de crescimento vascular e endotelial (VEGF) mostrou menor marcação em grupos transplantados com BMCs. A marcação para antígeno nuclear de proliferação celular (PCNA) mostrou-se mais expressiva no grupo AT-BMC 21 dias grupo. Marcação para CD105, CD133 e CD68 foi observada nos grupos AT 14 dias e AT 21 dias. Estas marcações não foram observadas nos grupos AT-BMC 14 dias e AT-BMC 21 dias. Nas eletromicrografias observamos o remodelamento benéfico no grupo AT-BMC14 dias e AT-BMC 21 dias, com a organização estrutural similar ao grupo CO. Vesículas de pinocitose, projeção da célula muscular lisa e a delaminação da lamina elástica interna são observados nos grupos AT 14 dias e AT 21 dias. Célula endotelial preservada, com lamina elástica interna de contorno regular e contínua é observada no grupo CO e nos grupos AT-BMC 14 dias e AT-BMC 21 dias. Como conclusão, os nossos resultados reforçam o conceito de que, em um modelo aterosclerótico utilizando camundongos e dieta aterogênica, a injeção de BMCs melhora os níveis de glicose, metabolismo lipídico e ocasiona um remodelamento benéfico na parede da aorta. / Stem cells are characterized by their ability to differentiate into multiple cell lineages and display the paracrine effect. The aim of this work was to evaluate the effect of therapy with bone marrow cells (BMCs) on blood glucose, lipid metabolism and aortic wall remodeling in mice through the administration of a high fat diet and subsequent BMCs transplantation. C57BL/6 mice were fed a control diet (CO group) or an atherogenic diet (AT group). After 16 weeks, the AT group was divided into four groups: an AT 14 days group and AT 21 days group, that were given an injection of vehicle and sacrificed at 14 and 21 days after, respectively; AT-BMC 14 days group and AT-BMC 21 days group that was given an injection of BMCs and sacrificed at 14 and 21 days after. The CO group was sacrificed along with other groups. The BMCs transplant had reduced blood glucose, triglycerides and total cholesterol. There was no significant difference in relation to body mass between the transplanted groups and non-transplanted groups, with all are different to CO group. There was no significant difference in the glycemic curve between AT 14 days group, AT-BMC 14 days group and AT 21 days group and these are different to CO and the AT-BMC 21 days group. The Qa (1 / mm2) was quantitatively reduced in the AT 14 days group and AT 21 days group when compared to the CO group. This Qa proved high in AT-BMC 21 days BMC compared to all groups. The increased thickness of the aortic wall was observed in all atherogenic groups, but was significantly smaller in group AT-BMC 21 days compared to AT 14 days group and AT 21 days group. The percentage of elastic fibers was significantly higher in the AT 21 days group when compared to the CO and AT-BMC 21 days. There was no significant difference between the CO and AT-BMC 21 days. Vacuoles in the media tunic, delamination and the thinning of the elastic lamellae were observed in AT 14 days group and AT 21 days group. The smallest number of these apresentation were displayed on the AT-BMC 14 days group and and AT-BMC 21 days. The immunostaining for α-SMA and VEGF showed lower in AT-BMC 14 days group and AT-BMC 21 days group. The markup for PCNA appears to be greater in the AT-BMC 21 days group. Marking to CD105, CD133 and CD68 were observed in AT 14 days group and AT 21 days group. These markings were not observed in AT-BMC 14 days group and AT-BMC 21 days group. In electron micrographs observed the beneficial remodeling in AT-BMC 14 day group and AT-BMC 21 days, with the structural organization was similar to the CO group. Vesicles of pinocytosis, projection of smooth muscle cell and delamination of the internal elastic lamina are seen in groups AT 14 days group and AT 21 days group. Endothelial cell preserved, regular and continuous contour in internal elastic lamelae is observed in the CO group, AT-BMC 14 days group and AT-BMC 21 days group. In conclusion, our results support the concept that an atherosclerotic model using mice and atherogenic diet, the injection of BMCs improve glucose, lipid metabolism and causes a beneficial remodeling of the aortic wall.

Made available in DSpace on 2014-06-11T19:31:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2004Bitstream added on 2014-06-13T18:41:27Z : No. of bitstreams: 1 hueb_jc_dr_botfm.pdf: 402586 bytes, checksum: 0363aade5dace29841c7485277d2b0f8 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Placa de aterosclerose em aorta torácica é uma importante causa de acidente vascular cerebral (AVC) e ataque isquêmico transitório (AIT). Sua gênese estaria relacionada com migração, para a circulação cerebral, de trombos e cristais de colesterol que se desprenderiam de placas complexas, localizadas na aorta torácica proximal. Existem várias semelhanças entre a fisiopatologia do desenvolvimento da placa de aterosclerose e a remodelação miocárdica. Por causa disso, formulou-se a hipótese de que a avaliação de pacientes com AVC e AIT, por meio do ecocardiograma transtorácico ((ETT), pode identificar características associadas com risco aumentado de placa de aterosclerose em aorta. Os objetivos desse estudo foram: 1) avaliar a incidência de placa de aterosclerose em aorta torácica de pacientes com história de AVC e AIT prévios, por meio do ecocardiograma transesofágico (ETE); 2) avaliar se existe associação entre a presença dessas placas e sinais de remodelação ventricular, observados por meio do ETT; e, finalmente, 3) analisar os níveis séricos de proteína C reativa de alta sensibilidade (PCRas), nesses pacientes... / Atherosclerosis plaque in the thoracic aorta is an important cause of acute cerebrovascular events. It would be caused by migration of thrombi and cholesterol cristals released from complex plaques, located at the proximalis thoracic aorta, to the cerebral circulation. Because there are several similarities between the physiopathology of atherosclerosis plaque development and myocardial remodeling. We hypothesized that patients with cerebrovascular events, and atherosclerosis plaque have cardiac morpho-functional alterations. The objectives of the present study were: 1) to evaluate the incidence of thoracic aorta artherosclerosis plaques in patients with a previous cerebrovascular events history, by transesophageal echocardiogram (TEE); 2) to evaluate if there is an association between the presence of plaques and signs of ventricular remodeling, observed by means of transthoracic echocardiogram; and, 3) to analyze the high sensitivity C-reactive protein (hs-CRP) seric levels, in those patients. One hundred and sixteen patients (79 male) with a previous... (Complete abstract click electronic address below)

O Metotrexato (MTX) é um fármaco utilizado como anti-inflamatório no tratamento da artrite reumatóide (AR). O risco de doença cardiovascular em pacientes com AR é menor quando tratados com MTX. Apesar dessa evidência, há poucos relatos da utilização de MTX para o tratamento da aterosclerose. Foi desenvolvida em nosso laboratório uma nanopartícula rica em colesterol (LDE), a qual é reconhecida pelos receptores da lipoproteína de baixa densidade (LDLr) após injeção na corrente sangüínea. A LDE concentra-se em células com hiperexpressão de LDLr, em processos proliferativos como a aterosclerose. Dessa maneira, a LDE pode ser utilizada como veículo para o direcionamento de fármacos contra essas células. A molécula de MTX foi latenciada e a modificação do fármaco aumentou a sua incorporação à LDE. A proposta desse estudo foi avaliar a eficácia de MTX associado à LDE (LDE-MTX) no tratamento da aterosclerose em coelhos além de investigar o efeito desse complexo na expressão de genes inflamatórios que participam do processo aterogênico. Para realização do estudo foram utilizados quatro grupos de 10 coelhos (raça New Zealand) cada, sendo que todos foram submetidos a uma dieta rica em colesterol por 8 semanas. Após as primeiras 4 semanas de dieta, os grupos foram tratados com LDE-MTX (grupo LDE-MTX), MTX comercial (grupo MTX comercial), LDE (grupo controle LDE) ou solução salina (grupo controle Salina), via endovenosa por 4 semanas. O grupo LDE-MTX não apresentou toxicidade ao longo do tratamento de acordo com os parâmetros utilizados, enquanto que o grupo MTX comercial apresentou uma queda acentuada de eritrócitos ao final do tratamento (p<0,001). A análise morfométrica macroscópica mostrou que os grupos LDE-MTX e MTX comercial reduziram as lesões ateroscleróticas quando comparados ao grupo controle Salina (66 e 76%, respectivamente) (p<0,001). Por microscopia, a camada íntima do arco aórtico e torácico foi reduzida nos grupos LDE-MTX (67% e 75%) e MTX comercial (81% e 92%, respectivamente) quando comparados com os mesmos fragmentos do grupo controle Salina (p<0,05). A presença de macrófagos na camada íntima dos grupos LDE-MTX e MTX comercial foi reduzida em 59% e 57% (p<0,001) no arco aórtico e 37% e 38% na região da aorta torácica (p=0,016) em relação ao grupo controle Salina, respectivamente. A porcentagem de MMP-9 no arco aórtico foi reduzida em 48% em ambos os grupos tratados (p=0,0003), enquanto que na aorta torácica LDE-MTX e MTX comercial diminuíram 54% e 66% (p<0,0001) respectivamente, em relação ao grupo controle Salina. Na região da aorta abdominal, a redução de MMP-9 também foi observada nos grupos LDE-MTX (68%) e MTX comercial (70%) (p=0,016). Quando se comparou ao grupo controle LDE, os dois grupos tratados tiveram porcentagens semelhantes de redução em todas as análises morfométricas. Nos estudos de expressão gênica in vivo, 5 genes inflamatórios se mostraram hipoexpressos (TNF-&#945;, MCP-1, IL-1&#946;, IL-18 e MMP-12) e um gene (IL-10) apresentou aumento de expressão no arco aórtico de coelhos tratados com LDE-MTX em relação ao grupo controle. No experimento in vitro, 5 dos 10 genes (TNF-&#945;, VAP-1, IL-1&#946;, CXCL2 e TLR2) avaliados tiveram redução da expressão e 1 (TGF-&#946;1) se mostrou hiperexpresso na linhagem de endotélio humana (HUVEC) tratada com as duas formulações de MTX. Os resultados indicam que tanto a LDE-MTX quanto MTX comercial reduzem acentuadamente as lesões ateroscleróticas em coelhos e parecem minimizar a resposta inflamatória na doença aterosclerótica. Contudo, LDE-MTX apresentou uma tolerabilidade maior ao tratamento, pois não apresentou toxicidade hematológica em comparação com MTX comercial. / Methotrexate (MTX) is the most frequently used drug for rheumatoid arthritis treatment. The incidence of vascular disease in these patients is lower when treated with MTX. However, few studies have been done using MTX for atherosclerosis treatment. In previous studies, we showed that, after injection into blood stream, a cholesterol-rich nanoparticle (LDE) binds to low density lipoprotein receptors (LDLr) and concentrates in tissues with intense cell proliferation such as atherosclerosis. LDE may thus carry drugs directed against those tissues reducing the toxicity of chemotherapeutic agents. For stable association with LDE, a lipophylic methotrexate derivative was used. The purpose of this study was to test MTX associated to LDE (LDE-MTX) in rabbits with atherosclerosis and investigate their anti-inflammatory effects on inflammatory mediators. Atherosclerosis was induced in rabbits by cholesterol rich diet during eight weeks. After 4 weeks from the introduction of the atherogenic diet, 4 groups of 10 animals were treated with LDE-MTX (LDE-MTX group), commercial MTX (commercial MTX Group), LDE (LDE control group) and saline solulion (Saline control group). MTX dose in both preparations was 4mg/kg/week during 4 additional weeks. LDE-MTX group showed superior tolerability with pronouncedly lesser hematologic toxicity in comparison to commercial MTX (p< 0.001). By morphometric analysis, both LDE-MTX and commercial MTX treatment groups showed a pronounced reduction of lesion area compared with Saline control group (66-76% respectively) (p<0001). By microscopy, intimal width at aortic arch and thoracic segments was reduced by 67% and 75% in LDE-MTX group compared to Saline control group, respectively (p<0.05). Commercial MTX group showed a reduction of 81% and 92% at aortic arch and thoracic segments compared to Saline control group, respectively (p<0.05). Presence of macrophages in intima layer at aortic arch was reduced by 59% and 57% (p<0.001) in LDE-MTX and commercial MTX groups while at thoracic segments was diminished by 37% and 38% (p=0.016) compared to Saline control group, respectively. MMP-9 percentage was diminished by 48% in both treated groups at aortic arch (p=0.0003) while at thoracic segment, LDE-MTX and commercial MTX reduced by 54% and 66% (p<0.0001) compared to Saline control group, respectively. Furthermore, MMP-9 percentage also diminished at abdominal segment in LDE-MTX group (68%) and commercial MTX (70%) when compared to Saline control group (p=0,016). When compared to LDE control group, both treated groups had similar percentage reduction in all morphometric analysis. In vivo studies, the expression of 5 inflammatory genes was downregulated (TNF-&#945;, MCP-1, IL-1&#946;, IL-18 and MMP-12) and 1 was upregulated (IL-10) when rabbits with induced atherosclerosis were treated with LDE-MTX. Besides, 5 inflammatory genes were downregulated (TNF-&#945;, VAP-1, IL-1&#946;, CXCL2 e TLR2) and 1 was upregulated (TGF-&#946;1) when human endothelial cell line (HUVEC) was treated with both MTX preparations. Therefore, MTX can be an effective drug for atherosclerosis treatment and associated to LDE, side effects of this chemotherapeutic agent can be minimized.