A strong correlation exists between abdominal aortopathy and atherosclerosis. In thoracic aortopathy however, the prevalence of atherosclerosis and its role in the etiology of thoracic aortopathy... Show moreA strong correlation exists between abdominal aortopathy and atherosclerosis. In thoracic aortopathy however, the prevalence of atherosclerosis and its role in the etiology of thoracic aortopathy remained unknown. This thesis therefore studied the cardiovascular disease burden within this patient group. These results showed that the prevalence of atherosclerosis (i.e. cardiovascular disease burden), in contrast to abdominal aortopathy, is not increased within the thoracic aortopathy population. Show less
Acute cardiovascular syndromes, including myocardial infarction or stroke, are the principal cause of death in the Western society. The main underlying pathology of cardiovascular diseases is... Show moreAcute cardiovascular syndromes, including myocardial infarction or stroke, are the principal cause of death in the Western society. The main underlying pathology of cardiovascular diseases is atherosclerosis, which is caused by the accumulation of lipids and inflammatory cells in the vessel wall, in so-called atherosclerotic plaques. Current therapies mainly target the disturbed lipid homeostasis, but recent clinical trials have shown a clear benefit in treating patients with anti-inflammatory drugs. However, more specific targeting is required to avoid unwanted side effects. In this thesis, we have generated a detailed atlas of all the cells present in human atherosclerotic plaques using a novel state-of-the-art technique called single-cell RNA sequencing. This data set can be applied as a powerful tool to select potential drug targets with a functional relevance for atherosclerosis. We showed that the majority of the immune cells in the human atherosclerotic plaque consisted of T cells. Subsequently, we identified a pro-inflammatory population of T cells that likely responds to a plaque-derived antigen, suggesting that atherosclerosis has an autoimmune-like component. Finally, we have applied our single-cell atlas to define and validate targets to intervene with the recruitment and activation of mast cells and other immune cells in atherosclerosis. Show less
Atherosclerosis is a progressive disease resulting in the formation of an arterial plaque. Despite lipid lowering, recurrent cardiovascular events remain a risk. While atherosclerosis is primarily... Show moreAtherosclerosis is a progressive disease resulting in the formation of an arterial plaque. Despite lipid lowering, recurrent cardiovascular events remain a risk. While atherosclerosis is primarily lipid-driven, the immune system plays a critical role in the pathophysiology. Additional treatment could be achieved via immunomodulation. We aimed to identify potential biomarkers for monitoring of immunomodulatory drugs in future clinical trials and investigated pharmacological modulation of atherogenic pathways. We identified smokers and elderly healthy people as suitable groups for future clinical trials. We investigated the impact of sample aging on LPS responses, and optimized methodology for evaluation of LPS-driven neutrophil responses, in vitro and in vivo. As potential anti-atherogenic strategy, we evaluated the effect of pneumococcal vaccination on circulating oxLDL-IgM levels in man. The immunomodulatory impact of hydroxychloroquine, a drug with potential anti-atherogenic effects, was evaluated in healthy volunteers. A novel OX40L inhibitor was tested in healthy volunteers, since the OX40-OX40L axis may play a role in atherogenesis. OX40L inhibition was safe and effectively reduced T cell activity. Lastly, we showed that PD-1 agonism reduced atherosclerosis in Ldlr-/- mice. This thesis adds to the future development of effective and specific immunomodulatory treatments for atherosclerosis. Show less
This thesis investigated how coronary CT, a non-invasive imaging technique, can be used in clinical practice to better characterize coronary artery calcification and to improve risk stratification.... Show moreThis thesis investigated how coronary CT, a non-invasive imaging technique, can be used in clinical practice to better characterize coronary artery calcification and to improve risk stratification. We looked at sex-differences in the development of atherosclerosis and the differences in risks of a possible event. Development of atherosclerosis started 12 years later in women, but when both sexes have severe arteriosclerosis, women have a higher chance of an event (myocardial infarction and/or death). Furthermore, with serial coronary CT scans and detailed quantification of atherosclerosis, we analyzed which patients are more likely to have progression of atherosclerosis, despite the use of a statin (cholesterol lowering agent). Growth of plaque is associated with worse outcomes and these patients might benefit from a different and/or more aggressive treatment. Furthermore, measurement of the pericoronary adipose tissue has been postulated as a new biomarker to detect inflammation of the coronary arteries, which plays an important role in the formation and progression of atherosclerosis, and in this thesis we have attempted to establish reference values. Show less
Cardiovascular diseases are the leading cause of death worldwide, with atherosclerosis as most common underlying pathology. Atherosclerosis is characterized by arterial narrowing due to cholesterol... Show moreCardiovascular diseases are the leading cause of death worldwide, with atherosclerosis as most common underlying pathology. Atherosclerosis is characterized by arterial narrowing due to cholesterol and lipid accumulation. Despite available effective cholesterol lowering medication, considerable risk for recurrent vascular events remains. This residual risk is at least in part explained by high blood lipid levels. The research described in this thesis revealed novel therapeutic strategies that improve lipid metabolism and reduce atherosclerosis development in mice. Inhibition of the endocannabinoid system was found to be an effective strategy, as well as concomitant activation of two incretin hormone receptors, namely those for GIP and GLP1. For combined GIP/GLP1 receptor agonism we additionally showed strongly attenuated hepatic steatosis. We were also able to identify additional targets to attenuate hyperlipidemia by studying the mechanisms underlying the strong day-night rhythm of brown adipose tissue, which is a lipid combusting tissue. In this thesis, I also stress the importance of the choice in animal model when studying lipid-modifying interventions, and describe the development of the software tool RandoMice which can be used to improve the quality of preclinical studies by creating well-balanced experimental groups. Show less
Coronary computed tomography angiography allows detailed evaluation of the coronary atherosclerotic plaque, even before any cardiac symptoms are present. This thesis describes the important... Show moreCoronary computed tomography angiography allows detailed evaluation of the coronary atherosclerotic plaque, even before any cardiac symptoms are present. This thesis describes the important prognostic role that the burden, morphology, composition, and location of coronary plaque has, which allows to intervene at an early stage. Detection of subclinical coronary atherosclerosis allows early treatment and reduction of cardiovascular mortality and morbidity. Show less
Cardiometabolic health is tightly controlled by a complex network of organ communication. Dysfunction of these lines of communication is associated with the development of cardiometabolic diseases... Show moreCardiometabolic health is tightly controlled by a complex network of organ communication. Dysfunction of these lines of communication is associated with the development of cardiometabolic diseases, indicating inter-organ cross-talk as a therapeutic target. Herein, I explored the therapeutic potential of targeting inter-organ communication in cardiometabolic diseases, including obesity, atherosclerotic cardiovascular disease and non-alcoholic steatohepatitis, based on which I proposed novel therapies to tackle these diseases. On one hand, strategies can focus on regulating the gut microbiota-centered inter-organ cross-talk. We demonstrated that dietary interventions are efficient to modulate the gut microbiota composition and function, thereby regulating the gut microbial metabolite production. In particularly, we showed that dietary supplementation of butyrate, a gut microbial metabolite, and choline, a nutrient enriched in red meat, can beneficially modulate the gut microbiota to alleviate adiposity. On the other hand, therapies can also focus on liver-centered inter-organ cross-talk. We showed that improving hepatocyte mitochondrial function by γ hydroxybutyric acid not only improves liver metabolic function, but also reverses obesity and its associated metabolic diseases. Besides, cardiometabolic health can be improved by regulating systemic levels of hepatokines (e.g. FGF21). We showed that FGF21-based pharmacotherapies can regulate the cross-talk between the liver and adipose tissue to improve cardiometabolic diseases, especially fibrotic non-alcoholic steatohepatitis and atherosclerotic cardiovascular disease. Thus, the findings described in this thesis emphasize the importance of inter-organ cross-talk for cardiometabolic diseases, and have improved our knowledge on the mechanisms that underlie the risk in the ever-increasing population of individuals who suffer from cardiometabolic diseases. Show less
Cardiovascular diseases are still a major concern for the global health. The main underlying pathology of this disease is atherosclerosis which is characterized by the accumulation of lipids and... Show moreCardiovascular diseases are still a major concern for the global health. The main underlying pathology of this disease is atherosclerosis which is characterized by the accumulation of lipids and immune cells in the arterial wall leading to a chronic local inflammation and lesion formation. In this thesis, we aimed to (1) validate the use of zebrafish in cholesterol metabolism and atherosclerosis research, (2) study the role of certain classes of scavenger receptors in lipoprotein uptake and cholesterol-based functions, and (3) validated two immune-based potential targets for atherosclerosis. Show less
In this thesis, we have addressed two key objectives: 1) to gain more insight in various pathophysiological aspects of cardiometabolic diseases including in the disease proneSouth Asian population,... Show moreIn this thesis, we have addressed two key objectives: 1) to gain more insight in various pathophysiological aspects of cardiometabolic diseases including in the disease proneSouth Asian population, and 2) to study the physiological effects of cold exposure and identify a novel pharmacological approach to directly target BAT. Show less
Cardiovascular disease (CVD) is a major cause of death worldwide. The underlying cause of most CVD is atherosclerosis. Atherosclerosis is characterized by progressive plaque build-up in the... Show moreCardiovascular disease (CVD) is a major cause of death worldwide. The underlying cause of most CVD is atherosclerosis. Atherosclerosis is characterized by progressive plaque build-up in the arterial wall.Noncoding RNAs (ncRNAs) are RNAs that are not translated into protein. This thesis focuses on two types: microRNAs and small nucleolar RNAs (snoRNAs). MicroRNAs inhibit the production of proteins and act on multiple proteins simultaneously. In CVD, many different proteins are involved. Changing expression of one microRNA can therefore have a major impact.Numerous snoRNAs have been associated with diseases, including CVD. The function of half of the human C/D box snoRNAs, however, is unknown.The first aim of this thesis is to investigate inhibition of microRNA-494-3p in advanced atherosclerosis. The second aim is to elucidate the function of SNORD113-6, a snoRNA that is involved in CVD.The thesis shows that inhibition of microRNA-494-3p halts plaque progression and increases stability of advanced plaques. This reduces the risk of e.g. a myocardial infarction.Furthermore, SNORD113-6 influences the function of fibroblasts, scar cells, and thus plays a role in maintaining function of our blood vessels.These insights may open up new therapeutic possibilities in future treatment of CVD. Show less
The research described in this thesis shows that hypercholesterolemia, a well-established risk factor for atherosclerosis, can impact skin lipid pool and barrier function already at young age. In... Show moreThe research described in this thesis shows that hypercholesterolemia, a well-established risk factor for atherosclerosis, can impact skin lipid pool and barrier function already at young age. In the field of atherosclerosis, we showed that the small peptide Lyp-1 can be used as a targeting molecule in liposomal formulations to deliver liver X receptor agonist to plaque resident foam cells/lipid-rich macrophages. Elucidation of the mechanisms underlying the intercommunication between plasma lipids and skin lipids may also bring valuable opportunities to prevent and treat dermatological pathologies in dyslipidemic patients; perhaps in combination with anti-atherogenic therapies. Thus, by deepening our knowledge we may improve our advice to the patients and ultimately improve their quality of life. Show less
This thesis aimed to provide evidence that supports a central role for NGCs in CVD by studying the expression, regulation and function of neuronal guidance cues (NGCs) in endothelial cells and... Show moreThis thesis aimed to provide evidence that supports a central role for NGCs in CVD by studying the expression, regulation and function of neuronal guidance cues (NGCs) in endothelial cells and monocytes, the 2 cells types that play main role in development of atherosclerosis. The findings laid the foundation for future research of NGCs as novel tar- gets for intervention of atherosclerosis. Show less
Cholesterol influences many pathways, including serving as precursor for adrenal steroidogenesis. Imbalance of cholesterol levels has been implicated in several diseases including cardiovascular... Show moreCholesterol influences many pathways, including serving as precursor for adrenal steroidogenesis. Imbalance of cholesterol levels has been implicated in several diseases including cardiovascular diseases and its underlying pathology, atherosclerosis. Here we concentrate on the role of a) lipid metabolism, especially high-density lipoprotein (HDL), in the development and regression of atherosclerosis and b) apolipoprotein E in adrenal glucocorticoid (GC) synthesis. We showed the importance of HDL size and functionality on atherosclerotic lesion formation in scavenger receptor-BI (SR-BI) knockout mice. Normalisation of the enlarged HDL particle size phenotype in these mice, trough depletion of phospholipid transfer protein, decreased atherosclerotic susceptibility and, contrary, development of a metabolic syndrome like phenotype. Furthermore, we studied the importance of HDL during regression of existing lesions in hypercholesterolemic apolipoprotein-E (ApoE) knockout mice. Normalizing the hypercholesterolemia resulted in regression of lesions and additional HDL depletion impaired the regression.The specific contribution of lipoprotein fractions to steroidogenesis is unknown. We lowered the (very) large-density lipoprotein fraction in ApoE-KO mice, resulting in a decreased GC output. ApoE is also produced within the adrenal where its local role is unclear. By transplanting an ApoE KO adrenal into an adrenalectomized wild-type mouse we revealed that local ApoE does not impact GC synthesis. Show less
This thesis focuses on using liposomes in two different treatment strategies; vaccination (or immunotherapy) and delivery of a small molecule, and in two different disease models; cancer and... Show moreThis thesis focuses on using liposomes in two different treatment strategies; vaccination (or immunotherapy) and delivery of a small molecule, and in two different disease models; cancer and atherosclerosis. For each of these treatment strategies, the liposomal formulation was tailored to obtain the desired therapeutic effect. Chapter 2 reviews some of the most important physicochemical properties (size, shape, and rigidity) that determine the immunological effects of liposomes and other nanoparticles. In chapter 3 we present a detailed study on the effect of the rigidity of anionic liposomes, as measured by atomic force microscopy, on antigen-specific regulatory T-cell (Treg) responses. In chapter 4, we show that our optimized anionic liposomes can induce potent antigen-specific Treg responses, and can be used to delay atherosclerosis progression in a mouse model. Chapter 5 also focuses on liposomal treatment of atherosclerosis, but here targeted liposomes were prepared to successfully deliver a small molecule to foam cells in atherosclerotic plaques. In Chapter 6, we used cationic liposomes in combination with an adjuvant for cancer immunotherapy in mice. Finally, we summarize the overall findings in chapter 7 and discuss perspectives of using liposomes for vaccination and targeted drug delivery. Show less
Atherosclerosis is the most important underlying process that drives cardiovascular disease, and is characterized by an accumulation of cholesterol which triggers an inflammatory response in the... Show moreAtherosclerosis is the most important underlying process that drives cardiovascular disease, and is characterized by an accumulation of cholesterol which triggers an inflammatory response in the vessel wall. This results in the recruitment of many types of inflammatory cells towards the plaques that form in the vessel wall, among which are CD8+ T-cells. In this thesis, the role of CD8+ T-cells in the advanced stages of lesion development has been investigated, as this is the most clinically relevant stage of the disease. This thesis demonstrates that CD8+ T-cells exert a protective function. We show that the absence of CD8+ T-cells in a mouse model results in less stable atherosclerotic lesions with increased numbers of inflammatory cells. In a subsequent study, we show that CD8+ T-cells express an enzyme that inhibits the inflammatory process. We also show that injecting a specific subset of CD8+ T-cells is protective against the development of atherosclerotic lesions in mice. Importantly, we show that this data can be translated to atherosclerosis development in humans, as we demonstrate similar results using patient material obtained from endarterectomy surgery. Finally, we show that developing therapies directed towards activating CD8+ T-cells may be of value to inhibit the immune response, and thus reduce the risk of cardiovascular disease. Show less
The thesis discussed several strategies that may contribute to further CVD risk reduction in the future. We described two novel lipid-lowering strategies, we unraveled (part of) the etiology of the... Show moreThe thesis discussed several strategies that may contribute to further CVD risk reduction in the future. We described two novel lipid-lowering strategies, we unraveled (part of) the etiology of the cardiovascular safety issues of TKIs that are used for the treatment of CML, and we investigated the dose effects of PFOA on lipoprotein metabolism. Looking forward, we developed a novel mouse model that can be used for the study of diabetic macrovascular complications, and we evaluated the potential of OSM as novel target in CVD. Show less
Cardiovascular diseases are the primary cause of death in the world with atherosclerosis as primary underlying cause. Atherosclerosis is characterized by cholesterol accumulation in the vessel wall... Show moreCardiovascular diseases are the primary cause of death in the world with atherosclerosis as primary underlying cause. Atherosclerosis is characterized by cholesterol accumulation in the vessel wall and inflammation of the vessel wall of medium to large size arteries. Both cholesterol accumulation and inflammation are pathogenic in the context of atherosclerosis. Current treatment regimens are tailored to reduce cholesterol levels in the blood. However, even a successful lowering of cholesterol is in many patients not sufficient to prevent a major cardiovascular event due to unresolved inflammation. Therefore, the immune system provides an interesting therapeutic target for the treatment of atherosclerosis. In this thesis we have explored the effect on atherosclerosis of several immunomodulatory strategies in pre-clinical models.As cholesterol is not soluble in water, cholesterol is transported in the bloodstream in particles called lipoproteins. The low-density lipoprotein (LDL) carries the highest concentration of cholesterol and accumulates in the vessel wall where a pathogenic specific immune response against LDL is instigated. In this thesis we have used several strategies to modulate the specific immune response against LDL, inducing LDL-specific regulatory T cells, antibodies, and cytotoxic T cells. Through immunoproteasomal inhibition we assessed the effect of general immune inhibition on atherosclerosis. Show less
This thesis explores different avenues to develop insurmountable antagonists for CC Chemokine Receptors, such as CCR1, CCR2 and CCR5. These receptors, which belong to the large family of G protein... Show moreThis thesis explores different avenues to develop insurmountable antagonists for CC Chemokine Receptors, such as CCR1, CCR2 and CCR5. These receptors, which belong to the large family of G protein-coupled receptors (GPCRs), are implicated in a variety of inflammatory and immune diseases, including atherosclerosis, rheumatoid arthritis and cancer. Thus, numerous drug candidates have been developed over the years to target them. Despite promising preclinical data, most of these candidates have failed in clinical trials due to lack of efficacy, making necessary the development of novel tools and concepts to better study and target these receptors. Thus, throughout this thesis we have explored different mechanisms to achieve insurmountable inhibition, which include intracellular allosteric modulation, covalent inhibition and long residence time. Moreover, the crystal structure presented in this thesis provides a new template for the rational design of future antagonists. Finally, with the identification of several selective or multitarget intracellular ligands for CCR1, CCR2 and CCR5, we are expanding the toolbox to further modulate chemokine receptors. Overall, the results of this thesis may contribute to the development of novel chemokine receptor antagonists, and GPCRs in general, with improved in vivo efficacy. Show less
Cardiovascular disease is the leading cause of death worldwide. The primary underlying pathology of cardiovascular disease is atherosclerosis. Atherosclerosis is a chronic, multifactorial disease... Show moreCardiovascular disease is the leading cause of death worldwide. The primary underlying pathology of cardiovascular disease is atherosclerosis. Atherosclerosis is a chronic, multifactorial disease in which lipid accumulates in the arterial wall, leading to a local inflammatory reaction and atherosclerotic plaque formation. Atherosclerotic disease develops largely asymptomatic over a lifetime. However, plaque rupture or erosion can cause the formation of a superimposed thrombus, blocking the flow of blood, and cause acute cardiovascular events such as myocardial infarction or ischemic stroke. Defects in cholesterol metabolism and hypercholesterolemia, which are major risk factors for atherosclerosis, have been shown to affect hematopoiesis, immune cell production and platelet counts and reactivity. Therefore, bone marrow cholesterol handling is an interesting target in the battle against cardiovascular disease, and acute cardiovascular events in particular. This thesis describes novel interactions between cholesterol metabolism and the production of immune cells and platelets, and its effects on atherosclerosis and atherothrombosis development. Show less
Cardiometabolic disease such as obesity, type 2 diabetes, and atherosclerosis, are a leading cause of morbidity and mortality in the Western world. Two important risk factors for the development of... Show moreCardiometabolic disease such as obesity, type 2 diabetes, and atherosclerosis, are a leading cause of morbidity and mortality in the Western world. Two important risk factors for the development of cardiometabolic disease are hyperlipidemia and inflammation. Recently, evidence strongly indicates a role for the gut microbiota in the development of cardiometabolic disease. Therapeutic approaches are therefore aimed at modifying the gut microbiota composition and function to beneficially affect the development of cardiometabolic disease and its underlying risk factors. A potential candidate to modify gut microbiota composition are indigestible carbohydrates, or prebiotics. In this thesis, we aimed to understand the interplay between various indigestible carbohydrates, gut microbiota composition and function, and the development of obesity, type 2 diabetes, and atherosclerosis. Together, the studies described in this thesis increased our knowledge on the potential of various indigestible carbohydrates in the modulation of the gut microbiota to affect the development of cardiometabolic disease, suggesting a promising strategy to further pursue with some caution. Show less
