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Abstract The risk factors for atherosclerosis and coronary heart disease (CHD) are multiple and may interact with each other. Diet has a significant role among the main risk factors for atherosclerosis, as it regulates the levels of plasma lipids and lipoproteins, their oxidative modification or protection from oxidation, blood pressure, energy balance, and thrombogenesis. Nutrients can transfer their effects directly through plasma concentrations or modify the cell transduction or gene expression of important regulatory genes. The response to dietary modification varies between individuals. The plasma cholesterol response induced by dietary modification is at least partly regulated genetically and some of the variation is explained by other environmental factors. Apolipoprotein E (apo E) and apolipoprotein B (apo B) are the key regulatory proteins in cholesterol and lipoprotein metabolism. The genetic variation of apo E is associated with the plasma lipid levels and the CHD risk. The polymorphic variation of the apo B gene is also associated with increased plasma cholesterol and CHD risk. Obesity is associated with increased morbidity and mortality. Plasma lipid abnormalities, impaired glucose metabolism and increased blood pressure caused by obesity are the main reasons for increased CHD mortality among obese subjects. To study the magnitude of the response to dietary modification, genetically selected groups were investigated. Dietary modification had a significant impact on plasma total, LDL, and HDL cholesterol concentrations, and the individual response in plasma LDL cholesterol varied from 3 to 100%. The role of genetic variation in the apo E gene was not significant in the lipid response, but the blood pressure response was more distinct among subjects with the ε 4 allele than those with the ε 3 allele. The determination of apo B EcoRI and MspI gene polymorphisms revealed subjects with a greater response to diet, a finding which may have clinical importance in the future for the attempt to identify subjects for effective dietary counselling. The effect of caloric restriction on gene expression was studied in obese gallstone patients. Moderate weight reduction during caloric restriction was associated with reduced lipoprotein lipase gene expression, while the cholesteryl ester transfer protein gene expression remained unchanged. Some of the beneficial changes in plasma lipids and lipoproteins during and after weight reduction may be followed by altered transcription of their modifying genes. Meta-analysis is a modern and generally accepted method. Many clinical uncertainties can be solved by combining all the data available to a quantitative and objective analysis. However, the use of meta-analysis do not resolve the problem of the effect of publication bias.

The ATP-binding cassette transporter, sub-family A, member 1 (ABCA1) mediates the major pathway for cholesterol exit from non-hepatic cells and thereby controls the rate-limiting step in the biogenesis of high density lipoprotein (HDL) particles. In humans,ABCA1 deficiency results in Tangier disease, characterized by low levels of HDL cholesterol, cellular cholesterol accumulation and increased risk for atherosclerosis. More than 100 coding variants have been described in the ABCA1 gene. We attempted to understand how both naturally occurring and engineered mutations in ABCA1 impact its role in cholesterol transport in a variety of in vitro and in vivo systems. We attempted to correlate specific genetic variants in ABCA 1 with phenotypes in patients carrying the sevariants, and used an evolutionary approach to predict which specific variants in ABCA1would impact its function. We then turned to the study of tissue-specific genetic deletion of ABCA1 in mice to study its role in HDL biogenesis, atherosclerosis and glucose metabolism. We found that intestinal ABCA1 is an important site of HDL biogenesis and that activation of intestinal ABCA1 raises HDL levels in vivo. Hepatic ABCA1, which is a major site of HDL biogenesis, was shown to significantly contribute to susceptibility to atherosclerosis. Finally, we show that ABCA1 plays an unsuspected role in B-cell function and insulin secretion. These studies have contributed to our understanding of the impact of genetic variation in ABCA1 on diverse biological and pathological processes, and have identified novel aspects of ABCA 1 function in specific cell types. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

The first genetic CAD risk locus to be identified by genome-wide association studies, single nucleotide polymorphisms (SNPs) at 9p21.3 predispose to increased risk of CAD. By bioinformatics scan analysis of the 9p21.3 locus; we interrogated the 59 linked SNPs over the 53,202bp to identify putative transcription factor-binding consensus sequences. We hypothesize that some genetic polymorphisms at the 9p21.3 locus are functional and will disrupt specific regulatory sequences within enhancers. Here, I investigated how polymorphisms affect TEAD-dependent regulation at the 9p21.3 locus, and also how polymorphisms affect GATA factor-dependent regulation at the 9p21.3 locus, using cultured HEK293 and primary human aortic smooth muscle cells (HAoSMCs) to transfect the pGL3-promoter plasmid constructs containing the reference or risk variant sequences (rs10611656, rs4977757, rs10757269, rs9632885). We showed by luciferase reporter assay that the risk allele of the SNPs disrupt activation by various TEAD transcription factors. We also performed electrophoretic mobility shift assay (EMSA) to test for allele-specific transcription factor binding that affect the family of TEAD transcription factors and the GATA factors. EMSA showed binding of TEAD3 and TEAD4, and differential binding for both GATA genotypes, and luciferase reporter assay confirmed that TEAD3 and TEAD4 activate the non-risk but not the risk allele, and for GATA factors no significant activation was shown. Our investigations lead us to conclude that rs10811656 and rs4977757 are functional and disrupt specific TEAD regulatory sequences within enhancers

Endothelial dysfunction and/or injury is pivotal to the development of cardiovascular and inflammatory pathology. Endothelial dysfunction and/or injury has been described in Human Immunodeficiency Virus (HIV) infection. Elaboration of circulating markers of endothelial activation, such as soluble adhesion molecules and procoagulant proteins, occurs in HIV infection. Certain endothelial cells, such as those lining liver sinusoids, human umbilical vein endothelial cells, bone marrow stromal endothelial cells or brain microvascular endothelial cells, have been shown to be variably permissive for HIV infection. Entry of virus into endothelial cells may occur via CD4 antigen or galactosyl-ceramide receptors. Other mechanisms of entry including chemokine receptors have been proposed. Nevertheless, endothelial activation may also occur in HIV infection either by cytokines secreted in response to mononuclear or adventitial cell activation by virus or else by the effects of the secreted HIV-associated proteins, gp 120 (envelope glycoprotein) and Tat (transactivator of viral replication) on endothelium. Enhanced adhesiveness of endothelial cells, endothelial cell proliferation and apoptosis as well as activation of cytokine secretion have all been demonstrated. Synergy between select inflammatory cytokines and viral proteins in inducing endothelial injury has been shown. In HIV infection, dysfunctional or injured endothelial cells potentiate tissue injury, inflammation and remodeling, and accelerate the development of cardiovascular disease.

Macrophages are central players during atherosclerosis. Especially, macrophage cholesterol efflux, which promote the removal of free cholesterol from foam cells, are crucial to prevent lipid accumulation and reverse atherogenesis. microRNAs (miRNAs) are important regulators of various pathways involved in atherosclerosis. During inflammation, macrophages secrete extracellular vesicles (EVs) carrying miRNAs to communicate signals to nearby cells. However, the role of macrophage-derived EVs in atherogenesis is not known. In the first study, we find that EVs derived from cholesterol-loaded macrophages can inhibit macrophage migration in vitro and in vivo. This effect appears to be mediated by the transfer of several miRNAs, including miR-146a, to recipient macrophages where they repress the expression of specific pro-migratory target genes Igf2bp1 and HuR. Our studies suggest that EV-derived miRNAs secreted from atherogenic macrophages may accelerate the development of atherosclerosis by decreasing cell migration and promoting macrophage entrapment in the vessel wall. Understanding macrophage communication via EVs provided the rationale for the design of nanoparticles (NPs) that mimic macrophage EVs to deliver beneficial miRNAs to the atherosclerotic plaque. While cationic lipid/polymer-based NPs have been employed as systemic delivery vehicles of siRNA, none of these have been used to deliver miRNAs to macrophages in vivo. In the second study, we developed a chitosan NP platform for effective delivery of miRNAs to alter macrophage function in vivo. We showed that our NPs made using a cross-linked chitosan polymer can protect as well as transfer miR-33 to naïve macrophages and regulate the expression of its target gene (Abca1) as well as cholesterol efflux in vitro and in vivo. Finally, almost all miRNAs that have been characterized are efflux-repressing miRNA, thereby accelerating atherosclerosis. miR-223 is one of a few miRNAs whose overexpression can promote cholesterol efflux, modulate the inflammatory response, and thus, be antiatherogenic. However, its contribution to the pathogenesis of atherosclerosis in vivo and the mechanism underlying its effects has not been thoroughly characterized. We herein find that miR-223 is capable of suppressing plaque development via modulating cholesterol efflux and inflammatory responses, thus may serve as a potential therapeutic to reduce atherosclerosis. These effects of miR-223 appear to be dependent on the inhibition of its target gene, the transcription factor Sp3. Overall, this thesis highlights the importance of both endogenous and extracellular miRNAs in controlling different aspects of atherogenic response.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21418号 / 医博第4408号 / 京都大学大学院医学研究科医学専攻 / (主査)教授 木村 剛, 教授 髙橋 良輔, 教授 竹内 理 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Rationale. Cigarette smoking is a well-known risk factor for cardiovascular disease, including arterial diseases such as atherosclerosis and abdominal aortic aneurysm. However, our understanding of how exposure to cigarette smoke impacts arterial disease pathogenesis is not well known. Consequently, this doctoral thesis focuses on understanding the development of atherosclerosis and aortic aneurysm in the context of exposure to cigarette smoke. In particular, since monocytes and macrophage are key immune cells implicated in arterial pathology, this work concentrates on understanding the impact of cigarette smoke exposure on the function and kinetics of monocytes and arterial macrophages. Main Findings. Using a mouse model that combines two clinically relevant risk factors, hyperlipidemia and cigarette smoke, we showed that smoke exposure increases atherosclerosis and induces the spontaneous formation, progression, and rupture of abdominal aneurysms. We also provide experimental evidence that atherosclerosis strongly associates with regions of elastin damage and arterial dilation, suggesting atherogenesis may directly contribute to abdominal aneurysm formation. Given the importance of macrophages in arterial disease, we investigated arterial macrophage heterogeneity and function following exposure to cigarette smoke. We report that cigarette smoke exposure increased the abundance of arterial monocytes and macrophages, whereas heterogeneity was primarily driven by hypercholesterolemia in aneurysmal tissue. Specifically, hypercholesterolemia is associated with an increase in macrophage populations with putative functions in inflammation and tissue remodelling including Trem2 foamy macrophages, inflammatory macrophages, and interferon-inducible macrophages. Moreover, we demonstrated that arterial macrophages play a critical role in elastin fragmentation within the arterial wall of smoke exposed mice. Finally, we investigated the impact of cigarette smoke on kinetic factors that can contribute to arterial macrophage accumulation. We found that, despite increased development of arterial disease, exposure to cigarette smoke is associated with an overall suppression of circulating monocytes and pro-inflammatory cytokines. Using a parabiosis model, we show monocyte recruitment is significantly increased and is likely a key factor contributing to accumulation of arterial macrophages following exposure to cigarette smoke. We also present evidence suggesting that endothelial dysfunction, related to a loss of endothelial nitric oxide synthase, contributes to increased arterial monocyte recruitment following exposure to cigarette smoke. Conclusions and Significance. Overall, we provide evidence that atherosclerosis likely contributes to abdominal aneurysm pathology in a model of cigarette smoke-induced aneurysm formation. We further provide insight into how tobacco smoke promotes arterial disease development through increased local accumulation of arterial macrophages despite suppressed monopoiesis and systemic inflammation. We identify monocyte recruitment and endothelial dysfunction as key factors contributing to the increased accumulation of arterial macrophages, with no overall differences in macrophage heterogeneity, following smoke exposure. In addition to providing insight into the increased risk of arterial disease following exposure to cigarette smoke, this study also provides experimental evidence that atherogenesis can contribute to abdominal aneurysm pathology. Overall, this thesis furthers our understanding of arterial disease pathogenesis and can provide a foundation for further mechanistic or therapeutic focused research aimed at reducing the burden of cardiovascular disease. / Thesis / Doctor of Philosophy (PhD) / Diseases that affect the heart and major blood vessels are one of the leading causes of illness and death worldwide. Atherosclerosis is one such disease caused by the buildup of fatty deposits in the walls of major blood vessels called arteries. This buildup can eventually block the artery and lead to a heart attack or stroke. Abdominal aortic aneurysms are another type of disease that affects arteries. In this case, the walls of the artery grow weak and begin to balloon out until the artery eventually breaks causing severe internal bleeding and death. One of the most important cells involved in the development of atherosclerosis and aneurysms is the macrophage, a type of white blood cell that is an important part of the immune system and found in diseased arteries. Although we know that cigarette smoking is one of the most significant risk factors for developing atherosclerosis and abdominal aneurysms, we do not fully understand why. Therefore, the goal of this thesis project was to investigate how cigarette smoke affects the development of arterial disease with a focus on understanding how it impacts the movement and function of macrophages. Using a mouse model, we found that the development of atherosclerosis and aneurysm are likely related, and also identified ways that exposure to cigarette smoke increases the numbers of macrophages in arteries. This work advances our understanding of how arterial diseases may be related and provides insight into how smoking can increase the risk of developing arterial disease.

Individuals with diabetes mellitus often develop complications that traditionally have been separated into microvascular pathologies, such as retinopathy, nephropathy and neuropathy, or macrovascular pathologies, including cardiovascular disease. Increasing evidence suggests that these micro- and macro-vascular complications may be linked. Our objective is to determine if direct effects of hyperglycemia on a microvascular bed that supplies cells in large arteries, the vasa vasorum, promotes diabetes-associated accelerated atherosclerosis. Normoglycemic apolipoprotein-E deficient (ApoE-/-) mice showed continuous atherosclerosis progression throughout the study that was directly correlated to increased vasa vasorum density with time. Hyperglycemic ApoE-/- Ins2+/Akita mice and streptozotocin-injected (STZ) ApoE-/- mice also demonstrated progressive plaque growth over time, but had accelerated atherosclerosis at 15 weeks of age compared to normoglycemic controls. The increased atherosclerosis in hyperglycemic mice correlated with impaired angiogenesis at 10 and 15 weeks of age. These mice showed increased expression for a marker of hypoxia in the atherosclerotic lesions yet decreased expression of vascular endothelial growth factor (VEGF), suggesting disruption of hypoxia-mediated angiogenesis. Cell culture experiments suggested that alternative splicing of an antiangiogenic form of VEGF in macrophages as well as post-translational modifications of macrophages and smooth muscle cells may contribute to reduced VEGF expression and decreased vasa vasorum neovascularization. After 25 weeks of age, vasa vasorum expansion plateaued in normoglycemic mice but continued to increase in hyperglycemic ApoE-/- STZ-injected mice. The increase in vasa vasorum neovascularization correlates to increases in plasma cholesterol. We have shown that hyperglycemia alters the microvascular structure of the vasa vasorum in two distinct mouse models of diabetes. Initially, elevations in glucose correlate to a significant reduction in lesion vascularization that results in increased lesional hypoxia that may promote the development and progression of atherosclerosis. At later time points there appears to be a burst of neovascularization that correlate with increases in cholesterol. / Thesis / Master of Science (MSc) / People with diabetes have elevated glucose levels that affect the vessels that distribute blood in our body. This puts them at higher risk of developing cardiovascular disease and having heart attacks and strokes. One set of vessels, known as the vasa vasorum, delivers blood to the walls of larger vessels. The primary goals of this study are 1) to determine if diabetes affects the vasa vasorum and, 2) to determine if changes to the vasa vasorum increase a person’s risk of developing cardiovascular disease. The results of this study show that diabetes in mice decreases the number of vasa vasorum vessels. The decrease in vasa vasorum blood vessels appears to influence the larger blood vessels they supply which promotes an environment that is more prone to cardiovascular disease. This information could be used in the future to develop drugs that target the vasa vasorum and possibly decrease cardiovascular events.

Atherosclerosis is a multifactorial inflammatory disease of the arterial wall and its clinical manifestations, including myocardial infarction and stroke, are the leading causes of death in western societies. Recent data has suggested that disruption of protein homeostasis in a cell's endoplasmic reticulum (ER), a condition known as ER stress, is associated with the progression of atherosclerosis. Furthermore, signaling by the serine/threonine kinase glycogen synthase kinase (GSK)-3α/β mediates pro-atherogenic processes. This thesis examines the role of ER stress and GSK3α/β signaling in atherosclerosis. Initially, three apolipoprotein-E deficient (ApoE-/-) mouse models of accelerated atherosclerosis were established. Relative to ApoE-/- mice fed a chow diet, pro-atherogenic conditions promoted hepatic steatosis, atherosclerosis, ER stress and GSK3β activity. A subset of mice from each group were given the GSK3α/β inhibitor valproate. Valproate supplementation suppressed hepatic steatosis, atherosclerosis and GSK3β activity in each mouse model without altering ER stress levels. This study revealed a role for ER stress and GSK3α/β in multiple murine models of atherosclerosis. Next, we investigated ER stress and GSK3α/β signaling in macrophage foam cell formation. In macrophages, ER stress induced GSK3α/β activity in a protein kinase R-like endoplasmic reticulum kinase (PERK) dependent manor. GSK3α/β inhibition attenuated ER stress induced lipid accumulation and the expression of distal components of the PERK pathway. Overexpression of constitutively active GSK3β induced foam cell formation. In mice, valproate supplementation attenuated PERK signaling in peritoneal macrophages and macrophages within atherosclerotic lesions. Together, these results point to GSK3α/β being a downstream component of the PERK pathway and that PERK-GSK3α/β signaling mediates ER stress induced foam cell formation. Lastly, we investigated the tissue and homolog specific functions of GSK3α and GSK3β in atherosclerosis. In high fat diet (HFD) fed low-density lipoprotein receptor deficient (LDLR-/-) mice, deletion of GSK3α or GSK3β in hepatocytes did not alter liver lipid content or atherosclerosis. Myeloid cell deletion of GSK3α, but not GSK3β, attenuated HFD induced atherosclerosis. Mechanistically, deletion of GSK3α in macrophages promotes the anti-atherogenic M2 macrophage phenotype by modulating signal transducer and activator of transcription (STAT)-3 and STAT6 phosphorylation and activation. Together, the data presented in this thesis suggest; 1) GSK3α/β inhibition attenuates atherosclerosis in multiple mouse models, 2) PERK-GSK3α/β signaling regulates macrophage foam cell formation and 3) myeloid cell GSK3α mediates atherosclerosis and macrophage phenotype. / Thesis / Doctor of Philosophy (PhD)