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 a major global burden and atherosclerosis is the main underlying pathological process. Despite better management of cholesterol levels, there remains a significant... Show moreCardiovascular disease is a major global burden and atherosclerosis is the main underlying pathological process. Despite better management of cholesterol levels, there remains a significant residual risk of developing atherosclerosis and cardiovascular events. Hence, novel pathways and targets should be identified to optimize atherosclerosis therapy. Despite dyslipidemia, the immune system is also heavily involved in the pathophysiology of atherosclerosis. Protective immune responses in the acute setting of increased cholesterol levels eventually turn into debilitating responses when the immune system is chronically stimulated. Hence, we aimed to identify new therapeutic targets to dampen the immune response in atherosclerosis. More specifically, we focused our efforts on modulating the B lymphocyte response, for which there was a scarcity of data. In this thesis we describe novel ways to modulate the B cell response in atherosclerosis. We have found that there are specific B cell subsets that have different effects on the progress of atherosclerosis. For instance, removal of TIM-1+ B cells resulted in increased atherosclerosis, while removal of BTLA+ follicular B cells reduced atherosclerosis. In conclusion, this thesis provides promising immunological targets for the treatment of atherosclerosis. 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
Atherosclerosis is the main underlying pathology of cardiovascular disease. Atherosclerosis is caused by an immune response which is directed against (modified) lipoproteins which accumulate in the... Show moreAtherosclerosis is the main underlying pathology of cardiovascular disease. Atherosclerosis is caused by an immune response which is directed against (modified) lipoproteins which accumulate in the vessel wall. Over time, this accumulation of lipids and immune cells induce morphological abnormalities in the vessel wall which cause the vessel lumen to narrow. This narrowing of the lumen (stenosis) causes ischemia in the downstream tissue. Prolonged ischemia causes myocardial ischemia and/or stroke. The research described in my thesis examines a well-recognized risk factor of atherosclerosis, being dyslipidemia, from an entirely new perspective. More specifically, it describes how dyslipidemia affects intrinsic metabolic processes in T cells, the conductors of the immune response characterizing atherosclerosis, and how this affects their function. My research has contributed to knowledge on the pathophysiology of atherosclerosis and might one day pave the way for the development of novel therapeutic approaches to treat cardiovascular disease. Show less
In this thesis, the importance of visceral obesity in the relation of obesity with cardiometabolic risk factors (chapter 2) was confirmed and it was shown that in individuals free of known... Show moreIn this thesis, the importance of visceral obesity in the relation of obesity with cardiometabolic risk factors (chapter 2) was confirmed and it was shown that in individuals free of known cardiovascular disease clustering of cardiometabolic risk factors is associated with changes in electrocardiographic parameters indicative of subclinical cardiovascular disease (chapter 3). The findings from chapter 3 also point to the importance of the prevention of these metabolic syndrome components, not only in obese, but also in non-obese individuals. Furthermore, both overall and abdominal adiposity were found to be associated with these deleterious changes in electrocardiographic parameters (chapter 4). Borderline Q-waves were associated with a negative cardiovascular risk profile and increased pulse wave velocity and intima-media thickness (chapter 5). Chapter 6 shows that several cardiovascular risk factors were associated with a wider spatial QRS-T angle, which reflects ventricular electrophysiological heterogeneity. Both carotid intima-media thickness, as measure of subclinical atherosclerosis, and pulse wave velocity, as measure of arterial stiffness, were associated with a wider spatial QRS-T angle. In chapter 7, improvement of electrocardiographic detection of left ventricular hypertrophy with conventional electrocardiographic criteria by taking into account body mass index and the spatial QRS-T angle is shown. Show less
Cardiovascular disease is the number one cause of death worldwide. The most important risk factor for developing this disease is high cholesterol levels in the blood. Other risk factors... Show moreCardiovascular disease is the number one cause of death worldwide. The most important risk factor for developing this disease is high cholesterol levels in the blood. Other risk factors contributing to cardiovascular disease can develop in individuals which are overweight. The clinical consequences of being overweight are clustered in the medical term: metabolic syndrome. Included in the metabolic syndrome are high blood pressure, dyslipidemia and glucose intolerance. At present, most cardiovascular disease patients are treated with statins which lower blood cholesterol levels. However, this treatment is not as effective in all patients and can cause some adverse drug reactions. Therefore, it is essential that novel therapeutic targets for the treatment of cardiovascular disease are identified. In this thesis, potential novel therapeutic targets in cardiovascular disease and metabolic syndrome are validated. In total, three potential targets were investigated: proteoglycan 4, protein arginine methyltransferase 3 and stabilin 1. Our studies showed the involvement of two of these targets in the development of cardiovascular disease and metabolic syndrome. Moreover, our results stress (1) that cardiovascular disease and metabolic syndrome are complex, multifactorial diseases with overlapping mechanisms and (2) that integration of research into both diseases can benefit therapeutic target identification and validation. Show less
Atherosclerotic changes of the carotid artery are associated with elevated cardiovascular risk. Non-invasive imaging studies of the artery can provide information on the presence or absence of... Show moreAtherosclerotic changes of the carotid artery are associated with elevated cardiovascular risk. Non-invasive imaging studies of the artery can provide information on the presence or absence of abnormalities. Although the techniques are extensively used in clinical research their implementation in common practice is not widespread. In this thesis the potential benefits and challenges of carotid imaging in clinical practice are studied. Ultrasound and magnetic resonance imaging are the two modalities of interest. The findings suggest that ultrasound can be performed by the clinician in a routine outpatient setting. Clinicians are able to detect atherosclerotic plaques but not intima-media thickness. Plaques are highly prevalent in asymptomatic primary prevention patients. Magnetic resonance imaging is a new highly reproducible modality but requires further clinical validation. Its utility in individual patient risk assessment is unclear and ultrasound validity cannot be extrapolated to magnetic resonance. The use of a combination of the two imaging modalities may allow for estimation of the lamina adventitia in vivo. Finally, interpretation of the imaging parameters must be done in conjunction with all cardiovascular risk factors and treatment decision should not be based on imaging results alone. Show less
The most important risk factor for atherosclerotic cardiovascular disease is increased levels of LDL-C. Statins have long been the most potent LDL-C lowering drugs on the market. However,... Show moreThe most important risk factor for atherosclerotic cardiovascular disease is increased levels of LDL-C. Statins have long been the most potent LDL-C lowering drugs on the market. However, statin treatment is complicated by the fact that a considerable number of patients is unable to tolerate full therapeutic doses, or can be classified as statin low or non-responders. In >25% of patients at (very) high risk for cardiovascular disease, statin efficacy is too limited to achieve current guideline-mandated LDL-C target goals, and aggressive statin therapy decreases relative risk for ASCD by only 30-35%, leaving an unacceptable residual relative risk of 65-70% for life-threatening events. It is clear that on-treatment LDL-C levels and on-treatment measures of systemic inflammation are of equal importance in this residual risk. Both for residual cholesterol risk and residual inflammatory risk, effective drug therapy has been lacking for decades. In this thesis, we describe the first clinical studies with novel compounds based on increased LDL-C levels and inflammation (including the required methodology), and present the methodology that may be useful to develop future compounds based on dysfunctional endothelial barrier function resulting in subendothelial cholesterol accumulation and subsequent atheroma formation. Show less
Within this thesis, several diseases central in the field of cardiovascular disease will be outlined. First, the central dogma of molecular biology, RNA biology in general, RNA (alternative... Show moreWithin this thesis, several diseases central in the field of cardiovascular disease will be outlined. First, the central dogma of molecular biology, RNA biology in general, RNA (alternative)splicing and the role of RNA-binding proteins within these processes will be discussed to enhance the accessibility to non-molecular biologists. Subsequently, the current literature and insights into the RNA-binding protein Quaking will be outlined. Thereafter, a brief summary of the role of many distinct RNA-binding proteins (RBPs) in the cardiovascular system is provided, detailing their importance in the heart and cells of the blood vessels. This review provides some historical and biological perspectives, while simultaneously highlighting many recent advances in our understanding of RBP function in cardiovascular health and disease. By harnessing established and novel techniques, including RNA-sequencing, this thesis will describe the role of Quaking in vascular stenosis, atherosclerosis, inflammation and endothelial barrier function. Collectively, Quaking can be described as a genome-wide governor of RNA-processing that results in the proper translation into functional proteins. This thesis describes which RNA transcripts are under control of Quaking, which alternative transcripts are being generated through modulation by Quaking, while also describing the unique role for this protein in health and cardiovascular and renal disease. Show less
Cardiovascular diseases are among the most frequent causes of death in the world. The main underlying pathology of cardiovascular diseases is the development of atherosclerosis in the medium and... Show moreCardiovascular diseases are among the most frequent causes of death in the world. The main underlying pathology of cardiovascular diseases is the development of atherosclerosis in the medium and large-sized arteries. The role of several immune cell subsets has been identified in atherosclerosis and it has been established that the immune system contributes to initiation and progression of cardiovascular diseases. Atherosclerosis is thus currently described as a chronic inflammatory disease and both the innate and adaptive immune cells contribute to disease initiation and progression. This suggests that targeting the ongoing inflammatory response may limit atherogenesis. This thesis discusses several therapeutic immune targets to limit atherosclerotic lesion development. The results described show the interplay between lipids and the immune system and a number of anti-inflammatory strategies to reduce atherosclerotic lesion development. The studies show a clear advantage of reducing inflammation in atherosclerotic lesion development. Future (clinical) studies identifying new targets will lead to a better-personalized therapy reducing atherosclerosis. Show less
Cardiovascular disease (CVD) due to atherosclerosis is a major cause of death. In atherosclerotic lesions, two major types of macrophages can be distinguished: 1) pro-inflammatory M1-macrophages... Show moreCardiovascular disease (CVD) due to atherosclerosis is a major cause of death. In atherosclerotic lesions, two major types of macrophages can be distinguished: 1) pro-inflammatory M1-macrophages, which promote atherosclerosis and 2) anti-inflammatory M2-macrophages, which are regarded athero-protective. Hence, skewing of macrophages from an M1 to an M2-phenotype might be a promising alternative therapeutic strategy against CVD. In this PhD dissertation, using the bone marrow transplantation (BMT) technique, we examined several genes that were hypothesized to be promising drug targets to favorably modulate macrophage phenotype and function, including the classic M2 marker gene Arginase 1 (Arg1), the key regulators of M2-macrophage activation Akt2 (AKT Serine/Threonine Kinase 2) and MKP2 (MAP Kinase Phosphatase 2), and Usf1 (upstream stimulatory factor 1). Although we were successful in skewing macrophages into an M2-phenotype, this also led to accelerated foam cell formation, which counteracted the anti-inflammatory athero-protective effects of the M2-macrophage thereby limiting its therapeutic value. Overall this work underlines that modulation of macrophage polarization in atherosclerosis is feasible. However, more research is required to identify the key beneficial regulators and their overall effects in the complex microenvironment of atherosclerotic plaque before macrophage polarization can be exploited as an alternative therapeutic strategy against CVD. Show less
Cardiovascular syndromes are the major cause of death in Western societies. The main underlying pathology is atherosclerosis, a chronic disease affecting the arteries. During atherosclerosis... Show moreCardiovascular syndromes are the major cause of death in Western societies. The main underlying pathology is atherosclerosis, a chronic disease affecting the arteries. During atherosclerosis progression, LDL, or “bad” cholesterol, accumulates in the arterial wall, resulting in the formation of a lipid-rich atherosclerotic plaque. This event activates the immune system, which increases plaque inflammation. Mast cells are components of the immune system known for their role in allergy. However, it has been established that mast cells are also important in atherosclerosis. In this PhD dissertation, we explored the interaction of mast cells with other immune cells. We examined the interrelation between mast cells and T-lymphocytes and discovered that mast cells can function as antigen presenting cells in atherosclerosis and, enhance the development of an atherosclerotic plaque via a direct interaction. Nonetheless, mast cells can also act on the Natural Killer T-cells, resulting in a protective function against atherosclerosis. Importantly, we used a relatively novel technical approach to explore the characteristics of mast cells inside human atherosclerotic plaques. We found that mast cells are highly activated and thus possibly promote disease progression. In conclusion, mast cells possess both protective and harmful effects, acting as regulators of the immune response in atherosclerosis. Show less
The worldwide prevalence of obesity is steadily increasing. Obesity leads to insulin resistance and atherosclerosis, which are the pathologies underlying type 2 diabetes and cardiovascular disease,... Show moreThe worldwide prevalence of obesity is steadily increasing. Obesity leads to insulin resistance and atherosclerosis, which are the pathologies underlying type 2 diabetes and cardiovascular disease, respectively. Inflammation is an important factor connecting obesity to these disorders, but the exact mechanisms connecting obesity, the immune system, type 2 diabetes and cardiovascular disease are still under investigation. The research described in this thesis was performed 1) to gain more insight into the role of the immune system in obesity, dyslipidemia, insulin resistance and atherosclerosis, 2) to study whether inflammation contributes to the disadvantageous metabolic phenotype of a human population with a particularly high risk to develop type 2 diabetes and cardiovascular disease, and 3) to study the therapeutic potential of decreasing inflammation by pharmacological strategies to reduce obesity and improve glucose and lipid metabolism in pre-clinical models. The studies described in this thesis have increased our understanding of the role of inflammation in adipose tissue function and lipid metabolism during the development of type 2 diabetes and cardiovascular disease. Moreover, novel potential therapeutic strategies were identified to combat obesity, metabolic inflammation and associated metabolic disorders, such as treatment with interferons, salsalate and GPR120 agonists. Show less
Carotid atherosclerosis, a disease in which plaque builds up inside the vessel wall, is a major cause of ischemic stroke. Traditionally, atherosclerosis risk stratification is heavily based on... Show moreCarotid atherosclerosis, a disease in which plaque builds up inside the vessel wall, is a major cause of ischemic stroke. Traditionally, atherosclerosis risk stratification is heavily based on the percentage of stenosis. However, a growing body of evidence suggests that luminal stenosis may not be the only cause of symptoms but the plaque composition may be more likely to impact the disease outcome. High-resolution vessel wall magnetic resonance imaging (VWMRI) is one of the most promising modalities for visualizing and evaluating carotid atherosclerotic plaque. The quantitative assessment of carotid atherosclerotic disease requires vessel wall segmentation and plaque classification, which is generally performed by manual delineations. However, manual contour tracing is labor-intensive, time-consuming and subject to inter-observer and inter-scan variability, which makes manual image analysis impractical for studies where large volume of data needs to be processed. Therefore, the main goal of this thesis is to: 1) develop approaches to automatically, robustly and reproducibly segment the carotid vessel wall and classify the atherosclerotic plaque from multi-spectral VWMRI; 2) validate the developed methods with reference standard; 3) extract the imaging biomarkers that can assist carotid artery disease evaluation. Show less
This thesis describes the role of 14q32 microRNAs in vascular remodelling. The 14q32 microRNA cluster contains 54 microRNAs in humans and is highly conserved in mammals. In part I of this thesis,... Show moreThis thesis describes the role of 14q32 microRNAs in vascular remodelling. The 14q32 microRNA cluster contains 54 microRNAs in humans and is highly conserved in mammals. In part I of this thesis, we describe the role of 14q32 microRNAs in several processes of vascular remodelling. We have shown that inhibition of several 14q32 microRNAs, miR-329, miR-494 and miR-495, results in increased neovascularisation after hindlimb ischemia in mice. In addition, inhibition of the same microRNAs reduced atherosclerotic plaque formation and restenosis in experimental mouse models under hypercholesterolemic conditions. In part II of this thesis, we zoom in to the post-transcriptional regulation of 14q32 microRNAs through RNA binding proteins. The third and last part of this thesis studies the expression of microRNAs in subcutaneous adipose tissue of critical limb ischemia patients and discusses the potential use of microRNAs as biomarker to predict the risk of amputation in these patients. In conclusion, this thesis provides novel insights in the role of 14q32 microRNAs in processes of vascular remodelling. Experimental studies have identified 14q32 microRNAs as potential therapeutic targets for treatment and prevention of atherosclerosis, restenosis and peripheral arterial disease. Show less
The aim of this thesis was to better understand the cellular origin of cholesteryl ester transfer protein (CETP) and to investigate the effects of the CETP inhibitor anacetrapib on the... Show moreThe aim of this thesis was to better understand the cellular origin of cholesteryl ester transfer protein (CETP) and to investigate the effects of the CETP inhibitor anacetrapib on the development of atherosclerosis. First, we investigated the specific characteristics of hepatic macrophages that express CETP. Our data clearly indicated that in the liver, CETP is exclusively expressed by F4/80+Ly6C-Clec4f+Vsig4+ macrophages that represent resident, rather than immature macrophages. Next, we showed that the HDL response to the inflammatory stimulus lipopolysaccharide is mediated by hepatic macrophages via down regulation of CETP expression in the liver that causes an increase in the level of HDL-C. In the second part of this thesis, we examined the effects and mechanism of pharmacological inhibition of CETP by anacetrapib on the development of atherosclerosis. We concluded that anacetrapib mainly decreases atherosclerotic lesion development via a reduction of non-HDL-C. Finally, we concluded that anacetrapib reduces (V)LDL-C by increasing hepatic remnant clearance via two mechanisms: 1) inhibition of CETP activity, resulting in remodelled VLDL particles that are more susceptible to hepatic clearance, and 2) a CETP-independent reduction in plasma PCSK9 level that has the potential to increase LDL receptor-mediated hepatic remnant clearance. Show less
