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
Non-alcoholic fatty liver disease (NAFLD) has rapidly become the most common cause of chronic liver disease, and its worldwide prevalence continues to increase in parallel of the obesity epidemic.... Show moreNon-alcoholic fatty liver disease (NAFLD) has rapidly become the most common cause of chronic liver disease, and its worldwide prevalence continues to increase in parallel of the obesity epidemic. NAFLD comprises a wide spectrum of liver damage ranging fat accumulation (steatosis) to steatosis with inflammation (non-alcoholic steatohepatitis, NASH), which can further progress to fibrosis. In particular patients with NASH have increased risk to develop other metabolic complications, such as cardiovascular disease.NAFLD is a complex disease, in which the origin and molecular mechanisms controlling the progression of simple steatosis to NASH remain poorly understood. Nevertheless, it is thought that inflammation is a critical component of NAFLD progression. This inflammation may be triggered by metabolic surplus (excess of energy or nutrients) and is also referred to as “metabolic inflammation”. White adipose tissue (WAT) is assumed to be largely involved in the development of metabolic inflammation. The studies described in this thesis contributed to the understanding of the role of WAT in the development of NAFLD and provide insight into the molecular processes that cause metabolic inflammation. Show less
The objective of this dissertation was to establish the value of QCT to further enhance the clinical applicability and accuracy of coronary CTA. The automatic characterization of coronary... Show more The objective of this dissertation was to establish the value of QCT to further enhance the clinical applicability and accuracy of coronary CTA. The automatic characterization of coronary atherosclerosis with QCT is feasible and correlates well with IVUS VH. Secondly, novel CTA risk score was created incorporating detailed information on the location, severity and composition of atherosclerosis as assessed with QCT. This CTA risk score allows accurate risk stratification of patients with suspected CAD. A drawback of coronary CTA is the fact that the hemodynamic significance of a lesion cannot be evaluated. In this thesis it was demonstrated that QCT provided better correlation with the presence of myocardial ischemia on SPECT MPI as compared to current visual assessment of coronary CTA. With regards to the specific setting of high risk diabetic patients without chest pain syndrome several conclusion can be derived from this thesis. First, if treated with optimal medical therapy, very few patients present with progression of myocardial ischemia. Second, the prognosis of these patients is good; the overall long-term event-rate is limited. Especially diabetic patients without CAD on coronary CTA have an excellent prognosis. Show less
The main objective of this thesis was to unravel relationships between obesity, insulin resistance, hyperglycemia, and atherosclerosis. It is well-established that patients with type 2... Show more The main objective of this thesis was to unravel relationships between obesity, insulin resistance, hyperglycemia, and atherosclerosis. It is well-established that patients with type 2 diabetes have a 2- to 3-fold increased risk of cardiovascular disease. We investigated whether insulin resistance and hyperglycemia are associated with atherosclerosis and incident cardiovascular disease before the onset of type 2 diabetes. Obesity can be considered as a common cause of both insulin resistance and atherosclerosis. Therefore, we investigated to what extent associations between insulin resistance, hyperglycemia and atherosclerosis were explained by body fat. We further aimed to study the specific role of visceral fat in the development of insulin resistance and atherosclerosis, and directly assessed abdominal subcutaneous and visceral adipose tissue depots. Show less
The brain is increasingly recognized as the regulator of body homeostasis and as possible treatment target for cardiovascular disease. This thesis further reveals the role of the autonomic nervous... Show moreThe brain is increasingly recognized as the regulator of body homeostasis and as possible treatment target for cardiovascular disease. This thesis further reveals the role of the autonomic nervous system (ANS) in the control of lipid metabolism and inflammation, and identified pathological consequences of disturbed regulation. Part I focuses on regulation of lipid metabolism by the ANS, with special attention for brown adipose tissue (BAT) as an emerging pharmacological target for therapy. We describe novel targets that modulate BAT, both directly (e.g. CB1R) and via the brain (e.g. MC4R, GLP-1R) to show that BAT activation improves dyslipidemia, glucose tolerance and T2D and even atherosclerosis. In addition, we identified the biological clock as an important regulator of BAT function and showed the consequences of disturbed circadian rhythmicity for lipid metabolism. Part II of this thesis describes studies on the regulation of inflammation by the ANS, with focus on the anti-inflammatory reflex. During this reflex, binding of acetylcholine to _7nAChR and subsequent intracellular signaling results in transcriptional repression of pro-inflammatory genes. We investigated the effects of hematopoietic _7nAChR deficiency and the consequences of selective parasympathetic and sympathetic denervation of the spleen for this reflex, and for inflammation and atherosclerotic plaque development. 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
Cardiovascular disease (CVD) is the leading cause of death worldwide despite the successful development of several pharmaceutical interventions of which statin therapy is the dominating lipid... Show moreCardiovascular disease (CVD) is the leading cause of death worldwide despite the successful development of several pharmaceutical interventions of which statin therapy is the dominating lipid-lowering treatment option. Atherosclerosis, a chronic inflammatory disease of multifactorial origin, is a dominant contributor to the development of CVD. The research described in this thesis provides evidence for anti-atherogenic effects of several innovative pharmaceutical interventions that are currently being investigated in clinical trials, targeting hypertension and hypercholesterolemia, more specifically high low-density lipoprotein-cholesterol (LDL-C) and low high-density lipoprotein-cholesterol (HDL-C), as risk factors for CVD. Our results further support additional benefit of these treatment strategies in combination with statin treatment which is currently the __gold standard__ therapy for the treatment of CVD. Most of these lipid-modifying treatment strategies affect both LDL-C and HDL-C and we demonstrate that the beneficial effects of these treatment strategies predominantly derive from their non-HDL-C/LDL-C-lowering abilities. Nonetheless, results from preclinical studies and clinical trials support the notion that treatment strategies aimed at improving HDL function and raising apolipoprotein A-I may also inhibit the development of atherosclerosis and reduce the prevalence of CVD. 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
Cardiovascular risk assessment in patients with diabetes mellitus (DM) remains challenging. Risk scores to predict cardiovascular risk are widely used, but are developed in the general population... Show moreCardiovascular risk assessment in patients with diabetes mellitus (DM) remains challenging. Risk scores to predict cardiovascular risk are widely used, but are developed in the general population and tend to underestimate the cardiovascular risk of DM patients. Risk scores developed in diabetic populations to estimate cardiovascular risk have demonstrated good calibration and discriminations indices. However, external validation is still needed. A recent meta-analysis showed that the predictive ability of these scores developed in diabetic populations is not superior to those scores developed in general population. Accordingly, the additional use of other biomarkers or imaging tools seems a good alternative to better risk stratify diabetic patients. This thesis evaluates the application and performance of non-invasive cardiac imaging tests for cardiovascular risk assessment and management of DM patients. Identification of new markers of CAD derived from non-invasive cardiac imaging might result in a broader applicability of cardiovascular risk assessment. Non-invasive cardiac imaging tests might evaluate target organ damage as well as the presence, severity and extent of subclinical atherosclerosis preceding overt clinical CAD. Thus, high-risk patients for CAD can be identified and further decision making of each DM patient can be tailored in order to improve the clinical outcomes at long-term follow-up Show less
Er is toenemend bewijs dat het niet noodzakelijk is om cholesterol en vetzuren in nuchtere toestand te meten, maar dat een niet-nuchtere meting in de meeste gevallen ook kan volstaan. De... Show moreEr is toenemend bewijs dat het niet noodzakelijk is om cholesterol en vetzuren in nuchtere toestand te meten, maar dat een niet-nuchtere meting in de meeste gevallen ook kan volstaan. De ontwikkeling van aderverkalking is complex en gaat veel verder dan alleen het cholesterolgehalte in het bloed. Aderverkalking, vet- en cholesterolstofwisseling en het immuunsysteem inclusief rode bloedcellen zijn nauw met elkaar verbonden. Hierbij lijkt het immuunsysteem zowel schadelijk als beschermend tegen de ontwikkeling van aderverkalking te kunnen werken. Vitamine D kan deels de schadelijke activatie van witte bloedcellen na vetinname voorkomen en heeft een gunstig effect op de elasticiteit van de slagaders. Rode bloedcellen kunnen juist een beschermend effect uitoefenen op de vaatwand door middel van transport van schadelijke cholesterolpakketjes. Dit transportmechanisme kan mogelijk plaatsvinden door middel van het niet-specifieke immuunsysteem met de complement receptor 1 danwel op basis van de ABO bloedgroep of een combinatie van beide Show less
In this dissertation clinical genetic investigations on migraine, related syndromes and comorbid conditions are described. The first migraine syndrome studied is Familial Hemiplegic Migraine (FHM),... Show moreIn this dissertation clinical genetic investigations on migraine, related syndromes and comorbid conditions are described. The first migraine syndrome studied is Familial Hemiplegic Migraine (FHM), a monogenic migraine variant. The clinical spectrum of FHM1-3 and the relation with closely related diseases such as Alternating hemiplegia of Chilhood, Early Seizures and Cerebral Edema after Trivial Head Trauma, epilepsy and episodic ataxia were investigated. The second monogenic migraine syndrome studied is Retinal Vasculopathy with Cerebral Leukodystrophy (later renamed CHARIOT), where common migraine is part of the clinical spectrum. The identification of TREX1 as the causal gene for RVCL is described. Investigation of the clinical spectrum showed retinal, cerebral and internal organ involvement, without an apparent genotype-phenotype correlation. Endothelial dysfunction of large arteries was shown in RVCL patients and is proposed as a possible disease mechanism. Lastly, migraine patients were identified in a Dutch genetic isolate and the relation with depression and atherosclerosis was assessed. For depression it was shown that shared genetic factors, at least partly, underlie the comorbidity with migraine, in particular migraine with aura. These studies improve our insight in genetic factors and pathofysiological mechanisms involved in migraine, which may ultimately contribute to better treatment options for migraine patients 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
Atherosclerosis, restenosis and cardiac remodeling after myocardial infarction can cause serious clinical problems that greatly contribute to both high morbidity and mortality. In all these... Show moreAtherosclerosis, restenosis and cardiac remodeling after myocardial infarction can cause serious clinical problems that greatly contribute to both high morbidity and mortality. In all these processes, the inflammatory responses caused by activation of the immune system play a very prominent role. This thesis elaborates on the role of specific components of the immune system and the therapeutic possibilities that lay hidden therein. This was done by focussing on the pathophysiological process in which Damage Associated Molecular Patterns (DAMPs) are released upon cell stress and cell death or other tissue damage, and may play an important role via different mechanisms. The data in this thesis illustrates specific involvement of DAMPs recognizing factors such as Toll-like Receptors in vascular remodeling and the therapeutic potential that lies within these findings. We show that endogenous activation of the immune s ystem plays an important role in the post-interventional vascular remodeling process. Multiple DAMPs such as HMGB1 are absent before intervention however they were found highly up regulated locally in the vessel wall after intervention indicating a specific relation with the intervention procedure. The presence and involvement of a variety of Toll Like Receptors in different models of vascular remodeling is interesting since these receptors are considered to be important recognizers of the DAMPs locally found in the vessel wall. Different intracellular signalling pathways and TLR accessory molecules seem to mediate the outcome of the specific DAMP-TLR interactions on vascular remodeling majorly. Protective effects of TLR3 and different outcomes of RP105 deficiency on vascular remodeling processes indicate the complexity of the underlying pathophysiological processes. These results can be partly explained by downstream TLR signalling and involvement of specific cell subtypes. The exploration of these underlying mechanisms offers new opportunities for biomarker selection and therapy development. Show less
The main underlying mechanism resulting in cardiovascular complications is the formation of atherosclerotic lesions in arteries. The progression of the lesion is hallmarked by a chronic... Show moreThe main underlying mechanism resulting in cardiovascular complications is the formation of atherosclerotic lesions in arteries. The progression of the lesion is hallmarked by a chronic inflammatory immune response in the arterial wall. Activated T helper cells differentiate into subsets, each producing a specific set of cytokines. T helper cell subsets contribute to the formation of the lesions, with a pro-atherogenic role for T helper 1 and a protective role for regulatory T cells. Cytokines of the IL-12 family play a crucial role in de differentiation of T helper cells. In this thesis I studied the role of IL-12 family members IL-27, IL-30 and IL-35 in atherosclerotic lesion formation. Furthermore the contribution of IL-37, part of the IL-1 family, was studied. In addition to modifications in lesion size and composition, I focus on the effects of the cytokines on T cell activation and differentiation. 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
The studies described in this thesis have contributed to the discovery of CETP as a biomarker for the hepatic macrophage content, a hallmark of NASH for which no non-invasive diagnostic method is... Show moreThe studies described in this thesis have contributed to the discovery of CETP as a biomarker for the hepatic macrophage content, a hallmark of NASH for which no non-invasive diagnostic method is currently available, and discovery of novel therapeutic modalities for atherosclerosis and NASH. First of all, we gained more insight into the true cellular origin of CETP (i.e. the liver macrophage), and the mechanisms underlying the CETP-lowering effects of HDL-raising agents (i.e. by reducing the hepatic macrophage content). We extrapolated the association between the reduction of hepatic lipid content and plasma CETP concentration upon lipid-lowering interventions from mice to humans. Furthermore, we demonstrated the role of CETP in discrepant effects of rHDL on VLDL metabolism between mice and humans, and reported a species difference in the central regulation of hepatic VLDL metabolism by NPY between mice and rats, which underscores a general concern in animal research in view of extrapolating findings from specific animal studies to explain observations done in humans. Additionally, we demonstrated that CORT has long-lasting beneficial effects on atherosclerosis development suggesting a possibility for therapeutic application of anti-inflammatory agents in CVD. Finally, we described GLP-1 receptor agonism as a novel strategy to improve lipid metabolism and hepatic inflammation, which may result in novel strategies to treat both atherosclerosis and NASH. Show less
In this thesis we reported our investigations of the relationship between soil-transmitted helminths (STH) and a number of outcomes, in particular malaria, insulin resistance (a marker for type-2... Show moreIn this thesis we reported our investigations of the relationship between soil-transmitted helminths (STH) and a number of outcomes, in particular malaria, insulin resistance (a marker for type-2 diabetes (T2D)) and atherosclerosis (a marker for cardiovascular diseases (CVD)) on Flores island, Indonesia. In the study on Flores Island, the use of albendazole as a single dose at three monthly intervals decreased helminth infections significantly. However, this intensive deworming could not eliminate helminth infections. Despite no effect on malaria parasitemia and clinical symptoms was found, we noted that in vitro immune responses were improved after albendazole treatment and significant increases in malaria-specific and mitogen-induced tumor necrosis factor and interferon _ cytokine production were observed. We also reported that helminth infections are associated with improved insulin sensitivity and lower risk factors for CVD. A possible approach to confirm our results will be a long-term, well-powered, placebo controlled (adequate) anthelminthic trials to investigate asymptomatic malaria (in area where clinical malaria is highly prevalent); as well as to study whether alleviation of helminthic pressure is inversely correlated with anti-inflammation, lipid levels and insulin sensitivity, and therefore leads to an accelerated development of T2D and CVD. Show less
