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
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
Acute cardiovascular clinical events such as myocardial infarction and cerebral stroke represent the major cause of death in Western societies. These pathologies are primarily resulting from... Show moreAcute cardiovascular clinical events such as myocardial infarction and cerebral stroke represent the major cause of death in Western societies. These pathologies are primarily resulting from atherosclerosis, a progressive condition characterized by the accumulation of lipids, immune cells, and fibrous elements in large arteries. The pathogenesis of atherosclerosis involves complex interactions between a wide variety of cells, including monocytes, macrophages, neutrophils, and lymphocytes. It is essential to identify novel targets for therapeutic application in order to reduce the residual atherosclerotic cardiovascular disease risk in current and future patients. Recent studies have suggested that members of the protein arginine methyltransferase (PRMT) family can potentially serve as novel therapeutic targets for atherosclerosis because of their regulatory role in inflammation and metabolism. To validate the contribution of PRMTs in the progression of atherosclerosis, in the studies presented in this thesis we have investigated the effect of inhibition of PRMT functionality on atherosclerosis susceptibility in established atherosclerotic mouse models.To address the role of PRMTs in atherosclerosis, we therefore made use of specific PRMT inhibitors, i.e. TC-E 5003 for PRMT1 inhibition, TP-064 for PRMT4 inhibition, and GSK3326595 for PRMT5 inhibition, that thus far have primarily been applied in vivo in the context of cancer treatment. 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
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
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
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
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
Atherosclerosis is a chronic inflammatory disease in which lipids and cells of the immune system accumulate in the vessel wall. Clinical complications, such as a myocardial infarction or stroke may... Show moreAtherosclerosis is a chronic inflammatory disease in which lipids and cells of the immune system accumulate in the vessel wall. Clinical complications, such as a myocardial infarction or stroke may occur when advanced atherosclerotic lesions become unstable and rupture. In this thesis, the influence of the psychological stress response and stress-related neuropeptides on vascular inflammation and atherosclerotic lesion development has been investigated. We demonstrated that acute stress results in activation of a potent type of immune cell in the vessel wall, the mast cell, leading to increased inflammation and atherosclerotic plaque destabilization. Furthermore, we have shown that (peri)vascular mast cell activation leads to neutrophil recruitment, thus aggravating the local inflammatory response. In addition, we demonstrated increased expression of neuropeptide Y in advanced atherosclerotic lesions and that overexpression of this peptide results in increased lesion development. These insights emphasize a contributing role for psychological stress to atherosclerotic lesion development and as a risk factor for acute cardiovascular syndromes and opens up new avenues for possible future anti-inflammatory therapies to reduce the risk of cardiovascular disease. Show less
Atherosclerosis is the main underlying pathology of cardiovascular disease, the largest single cause of death in industrialized countries, and current treatment is still largely insufficient. In... Show moreAtherosclerosis is the main underlying pathology of cardiovascular disease, the largest single cause of death in industrialized countries, and current treatment is still largely insufficient. In recent years it has become evident that immune responses contribute to atherosclerosis. Therefore, during my PhD studies I focused on developing a therapy to induce and expand anti-inflammatory immune cells to reduce ongoing immune responses and atherosclerosis. I used the approach of cellular therapy and examined the effect of several different anti-inflammatory immune cells. For example, I made use of mesenchymal stem cells, which have previously been used to improve cardiac repair after myocardial infarction and were found to have anti-inflammatory properties. Additionally, I used drugs, e.g. inhibitors of protein degradation, and biologics, e.g. components of heat-killed bacteria, to directly increase the amount of anti-inflammatory immune cells. An interesting side-effect of some treatments was that they additionally reduced cholesterol levels. In summary, I have shown in pre-clinical models that immune cell-based therapies are promising for the treatment of atherosclerosis. As atherosclerosis is determined by both high cholesterol levels and inflammation reducing immune responses will greatly contribute to a better treatment of cardiovascular patients in the (near) future. Show less
Atherosclerosis is a chronic inflammatory disease, consisting of the buildup of lipids in the vessel wall. Advanced lesions may become unstable and rupture, leading to major cardiovascular... Show moreAtherosclerosis is a chronic inflammatory disease, consisting of the buildup of lipids in the vessel wall. Advanced lesions may become unstable and rupture, leading to major cardiovascular complications such as myocardial infarction or stroke. In this thesis, the role of the innate immune system in atherosclerosis has been investigated. We have shown that inhibition of complement component C5a results in reduced atherosclerotic lesion formation as well as reduced lesion destabilization. Also, we have provided evidence that activation of mast cells surrounding the atherosclerotic lesion results in increased accumulation of the neutrophil, thus aggravating the local inflammatory response. Moreover, we have investigated the effect of microRNA inhibition of atherosclerosis. MicroRNAs are short non-coding RNA strands with the ability to modulate the expression of multiple genes. With a unique Reversed Target Prediction we have identified microRNAs that are predicted to affect multiple atherosclerosis-related genes. We inhibited one of these predicted microRNAs: microRNA-494, and investigated its role in vivo. Interestingly, we observed a striking reduction in atherosclerotic lesion formation, as well as an increase in lesion stability. Show less
Many of the recognition molecules and mechanisms involved in immune responses have no bias towards external stimuli, but also sense and respond to pathological and physiological changes of non... Show moreMany of the recognition molecules and mechanisms involved in immune responses have no bias towards external stimuli, but also sense and respond to pathological and physiological changes of non infectious origin taking place within the body. Aiming at defining potential immuno-therapeutic strategies to treat human atherosclerosis, the focus of this work was the modulation of immune processes determinant of atherosclerosis lesion progression or cessation in mice, such as hematopoiesis, diapedesis and intravasation, leukocyte differentiation, cholesterol uptake apoptosis and cell survival. Modulation of these processes, by using bone marrow transplantation of hematopoietic stem cells with genetic deficiencies or over-expressing human or mouse engineered genes, demonstrated to alter the fate of atherosclerotic lesions at the balance between macrophage accumulation and lesion vulnerability versus resolution of inflammation and wound healing. This thesis demonstrates that processes responsible for the development and progression of atherosclerosis are dynamic and can be modulated to induce lesion stabilization and disease resolution. These results are promising for the development of novel therapeutics and challenge the current notion that atherosclerosis has a predetermined fate towards lesion vulnerability to rupture, which in humans results in thrombosis and clinical manifestations such myocardial infarction or stroke and sudden death. Show less
Vein graft surgery to treat occlusive arterial disease is a common applied procedure. Each year more than two million vein graft surgeries are performed worldwide. The major drawback of vein... Show moreVein graft surgery to treat occlusive arterial disease is a common applied procedure. Each year more than two million vein graft surgeries are performed worldwide. The major drawback of vein grafting is that within 10 years after vein graft surgery 50-60 % of the vein grafts suffer from patency loss due to thrombosis, intimal hyperplasia formation, accelerated atherosclerosis and rupture. Endogenous factors orchestrate the development and failure of vein grafts. Investigating the role of endogenous constituents on vein graft remodeling can enhance our basic knowledge of the involvement of these factors in vein graft remodeling. By interfering in the function of endogenous factors, as we showed in this thesis, vein graft remodeling can be negatively or positively influenced depending on the factor and strategy used. New therapeutic strategies can be developed based on this knowledge. In this thesis we investigate the role of innate immune components, complement system factors, toll like receptors, mast cells and NK cells and the role of Annexin A5 in vein graft remodeling. Furthermore we explored the role of plaque stability, plaque neovascularization and extracellular matrix remodeling in a hypercholersterolemic mouse vein graft model. Show less
