This thesis introduces the concept of "physics-based inverse design", working on the notion that the physical driving forces governing functionality are inherently encoded in independently... Show moreThis thesis introduces the concept of "physics-based inverse design", working on the notion that the physical driving forces governing functionality are inherently encoded in independently parameterized energy functions, which can be resolved through the use of inverse design strategies.The thesis describes the development of EVO-MD, a Python-based implementation of the physics-based inverse design concept. EVO-MD is capable of automatically setting-up, performing, and analyzing molecular dynamics simulations, allowing for the evolutionary optimization of complex and dynamic features in peptides. Examples of such applications include the optimization of lipid composition and curvature sensors, and the development of peptides with antiviral properties. 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
Cardiometabolic diseases including atherosclerotic cardiovascular disease (CVD) and non-alcoholic fatty liver disease (NAFLD) are the leading cause of preventable death worldwide.... Show moreCardiometabolic diseases including atherosclerotic cardiovascular disease (CVD) and non-alcoholic fatty liver disease (NAFLD) are the leading cause of preventable death worldwide. Hypercholesterolemia and inflammation are common major risk factors for atherosclerotic CVD as well as NAFLD. The studies described in this thesis aimed to get insight in strategies how to further improve cholesterol metabolism and inflammation, by exploring the therapeutic potential of brown fat activation and transcription factors involved in both processes. The results described in this thesis have increased our insight into regulation of cholesterol metabolism and inflammation by brown fat and nuclear receptors, respectively, and provided promising leads for innovative treatment of cardiometabolic diseases including brown fat activation, Δ24-dehydrocholesterol reductase inhibition, and farnesoid X receptor activation. 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
The aim of this thesis was to unravel a selection of a multitude of potential causal pathways that may underlie the association between excess body fat and cardiovascular disease, such as... Show moreThe aim of this thesis was to unravel a selection of a multitude of potential causal pathways that may underlie the association between excess body fat and cardiovascular disease, such as adipokines, inflammation, HDL-cholesterol and postprandial triglyceride response, and cholesteryl ester transfer protein (CETP). We showed that hs-CRP and GlycA as measures of inflammation, adiponectin, and leptin are not associated with clinical and subclinical cardiovascular disease in the general population. However, all may be relevant markers of disease risk. Also, postprandial triglyceride excursions, genetically-determined CETP and HDL-cholesterol, while not related with subclinical atherosclerosis in the general population, may be interesting targets to pursue in women and men separately, and in subgroups of individuals at high-cardiovascular risk. 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
Drug delivery across the skin is a challenging task because of the skin barrier. The skin barrier underlies in the outermost layer of the skin, the stratum corneum (SC). The lipids play a crucial... Show moreDrug delivery across the skin is a challenging task because of the skin barrier. The skin barrier underlies in the outermost layer of the skin, the stratum corneum (SC). The lipids play a crucial role in this barrier function. The focus of this PhD project was to elucidate the molecular structure of the lipid matrix present in the SC and to relate this structure with the barrier function. The lipid compositions selected for these studies were particularly chosen to understand the changes in barrier function in dry and diseased skin, i.e.,atopic eczema, Netherton syndrome compared to healthy skin. A variety of biophysical and analytical methods such as X-ray diffraction, neutron diffraction, infra-red spectroscopy, microscopy and LC/MS were combined to unravel the molecular structure. Diffusion studies and trans-epidermal water loss measurements were carried out to relate lipid organization with the lipid barrier. All the diffraction studies were performed in Grenoble, France at the ESRF (X-rays) and ILL (neutron). Neutron diffraction studies are in collaboration with King's College, University of London (Prof. J. Lawrence, Dr. D. Barlow). Show less
Current therapies for patients with atherosclerosis are targeted primarily on reducing blood cholesterol, but this fails to prevent a large number of cardiovascular events.95 In order to identify... Show moreCurrent therapies for patients with atherosclerosis are targeted primarily on reducing blood cholesterol, but this fails to prevent a large number of cardiovascular events.95 In order to identify new therapeutic strategies for treating atherosclerosis, it is important to study the interactions between the cells that make up the atherosclerotic lesion. This thesis describes the role of endothelial cells and macrophages during lesion formation, as well as the expression patterns of sterol sensors in vascular smooth muscle cells present in the lesion. These new insights contribute to our understanding of atherosclerosis open up new avenues of atherosclerosis research Show less
This PhD thesis contains the following chapters. The first part, containing chapter 2 and 3 mainly concerns model development. Chapter 2 describes the development of a mathematical modeling... Show moreThis PhD thesis contains the following chapters. The first part, containing chapter 2 and 3 mainly concerns model development. Chapter 2 describes the development of a mathematical modeling framework within which different diagnostic models based on lipoprotein profiles can be developed, and a first validation of a model in this framework using data from a stable__isotope flux study (78). Chapter 3 describes a further development of the model, and the development of a new lipoprotein model__based candidate diagnostic and its validation using a range of stable__isotope flux studies. The second part, containing chapter 4, 5, and 6, concerns model applications. Chapter 4 contains the first application of the model__based candidate diagnostic using HPLC__measured lipoprotein profiles, in the context of a nutritional intervention study conducted at TNO. Chapter 5 contains an exploration of lipoprotein profile data from a population study (the GOLDN study), and examines how subgroups of patients, identified via K__means clustering based on lipoprotein profiles, differ in their lipid response to a fenofibrate intervention. It also examines how the model__based candidate diagnostic behaves in these subgroups. Chapter 6 contains the study that shows that lipoprotein metabolism indicators may have predictive value for cardiovascular risk on top of classical risk markers. Chapter 7 contains a methodological reflection on the development of computational models for clinical diagnostic use based on clinical chemistry data. Finally, chapter 8 contains a general discussion of the research presented in this PhD thesis and suggestions for further research Show less
Late endosomal transport is disrupted in several diseases such as Niemann-Pick type C, ARC syndrome and Alzheimer__s disease. This thesis describes the regulation of late endosomal dynamics by... Show moreLate endosomal transport is disrupted in several diseases such as Niemann-Pick type C, ARC syndrome and Alzheimer__s disease. This thesis describes the regulation of late endosomal dynamics by cholesterol and multi-protein complexes. We find that cholesterol acts as a cellular tomtom that steers the direction of late endosomal transport by dynein-motors, and that this process is disrupted in Niemann-Pick disease. Furthermore cholesterol also regulates tethering via the multi-subunit HOPS complex. Homologues of this complex are mutated in ARC syndrome patients and the cellular consequences are described in this thesis. We find that the cholesterol sensing proteins ORP1L and MLN64 are major regulators of late endosomal transport, and that this transport is further regulated by the endoplasmatic reticulum. Show less
The aim of this thesis was to clarify which aspects of depression and anxiety are related to an increased metabolic risk, and which factors contribute to these associations. Taken together, our... Show moreThe aim of this thesis was to clarify which aspects of depression and anxiety are related to an increased metabolic risk, and which factors contribute to these associations. Taken together, our findings indicate that people with more severe symptoms of depression and anxiety are at particular risk of progressive dyslipidemia and (abdominal) obesity. The higher rates of smoking and systemic inflammation among people with depression or anxiety partially accounted for their adverse metabolic profile. Dysregulations of the autonomic nervous system partly explained why users of tricyclic antidepressants displayed an increased risk of dyslipidemia and (abdominal) obesity as well, and also of hypertension. These important findings shed light on useful avenues for future research, and on preventive and therapeutic insights and directions. Show less
In this thesis several aspects of metabolic syndrome are addressed. The focus involves questions concerning the genetics of obesity, TG and cholesterol and hyperglycemia. Since we hypothesized that... Show moreIn this thesis several aspects of metabolic syndrome are addressed. The focus involves questions concerning the genetics of obesity, TG and cholesterol and hyperglycemia. Since we hypothesized that obesity is the most important trigger of metabolic impairment, the MetS definition in this thesis was chosen to include the obesity measure waist circumference as an essential component. In the study described in chapter 2, the heritability of the metabolic syndrome was addressed and compared to the heritability of its individual components. Since the individual components of MetS were shown to be more heritable than MetS itself, the studies described in chapter 3 and 4 focused on the genetics of the individual MetS component plasma TG. For this purpose, a candidate gene approach was employed using HTG patients and healthy controls. The involvement of a series of candidate genes was confirmed. The study described in chapter 5 followed a similar approach to that used in the studies described in chapter 3 and 4. Several candidate genes were studied in patients suffering from hyperlipoproteinemia (HLP) type III, which is characterized by elevated levels of total plasma cholesterol and plasma TG. HLP type III is characterized by APOE2 homozygosity. Contributing genetic factors in the (metabolically stressed) APOE2/2 environment were confirmed. Plasma adiponectin, an adipose tissue secreted hormone (adipokine), has been suggested to be a biomarker for MetS. In chapter 6 we describe a study which particularly aimed to determine the effect of menopause on the discriminating accuracy of adiponectin to predict MetS. Especially low levels of plasma adiponectin in postmenopausal women were found to be a risk for MetS. However, the discriminating accuracy of adiponectin for the presence of MetS was exceeded by BMI in men and pre __and post menopausal women. Since plasma adiponectin levels are very well correlated with MetS components or related traits, the study described in chapter 7 addressed the question whether these correlations are caused by a genetic overlap (genetic correlation). The genetic correlation was mono-laterally validated with regard to the adiponectin gene (ADIPOQ). Chapter 8 describes a study towards finding novel loci associated with adiponectin or loci that are possibly involved in the genetic overlap between adiponectin and MetS components or related traits. This study followed a genome-wide association (GWA) approach. The results of this GWA were used in a joined analysis with two other cohorts in a meta-analysis. In addition, a selected proportion of SNPs was submitted for replication in several cohorts. Chapter 9 provides a general discussion by reviewing all previous chapters in the thesis. Furthermore, chapter 9 includes suggestions and proposals for future analyses towards unraveling genetic and environmental factors involved in the expression and manifestation of metabolic risk factors. Show less
The studies described in this thesis show that inflammation and CETP are both important factors in lipid metabolism and atherosclerosis. In the first part of this thesis we showed that high dietary... Show moreThe studies described in this thesis show that inflammation and CETP are both important factors in lipid metabolism and atherosclerosis. In the first part of this thesis we showed that high dietary cholesterol can induce hepatic inflammation via disturbed cholesterol homeostasis and ER stress, revealing new targets for the treatment of metabolic inflammation. Next, we demonstrated that intervention in both systemic and vascular inflammation can reduce atherosclerosis progression and/ or induce regression, highlighting the importance of developing drugs targeting the inflammatory component of atherosclerotic disease. In the second part of this thesis we showed that CETP inhibition per se may be anti-atherogenic, but that combination therapy of the CETP inhibitor torcetrapib with atorvastatin may have obscured its atheroprotective effect. Furthermore, we showed that the VLDL-increasing effect of CETP largely explains its atherogenic effect, at least in APOE*3-Leiden.CETP mice, and that CETP inhibition may negatively affect lesion stability. Our data suggest that CETP inhibition may not be the most optimal strategy to increase HDL-C levels and thereby reduce atherosclerosis. We anticipate that strategies improving HDL functionality, rather than raising the HDL level, are more likely to effectively reduce CVD. Show less
Atherosclerosis is a multifactorial disease of the large arteries and the leading cause of morbidity and mortality in industrialized countries. There is ample evidence that hypercholesterolemia (i... Show moreAtherosclerosis is a multifactorial disease of the large arteries and the leading cause of morbidity and mortality in industrialized countries. There is ample evidence that hypercholesterolemia (i.e. elevated plasma levels apo-B-containing lipoproteins) is a major causative factor in atherogenesis. It is equally clear that atherogenesis has an inflammatory component which is thought to drive the progression of the disease. However, while the lipid component in atherosclerosis development is relatively well-understood, the origin and exact contribution of the inflammatory component remains largely unknown. The aim of this thesis is to further define and delineate the contribution of the inflammatory component to the atherosclerotic process and to elucidate the link between cholesterol and inflammation in atherosclerotic lesion formation and progression. The studies of this thesis show that, besides plasma cholesterol, inflammation contributes to a substantial extent to atherogenesis. Intervention strategies directed at lowering apoB-containing lipoproteins and reducing inflammation may therefore be more effective than current lipid-lowering strategies. Direct experimental evidence for this assumption mainly comes from animal experiments as described in this thesis. Human intervention studies are necessary to evaluate whether these findings can also be translated to the human situation. Show less
Endocytosis can be conceptually broken down into two stages: The formation of the nascent vesicle containing the ingested proteins and lipids, and its subsequent maturation into a degradative... Show moreEndocytosis can be conceptually broken down into two stages: The formation of the nascent vesicle containing the ingested proteins and lipids, and its subsequent maturation into a degradative compartment. During this maturation process proteins can be recycled back to the plasma membrane to prevent degradation. The vesicular membrane and the luminal contents undergo considerable remodeling to transform this compartment into a degradative one. This stops signal transduction by degrading the receptors within the vesicle and also generates antigens for presentation on MHC class II molecules in order to elicit an adaptive immune response. The engulfment of large particles and microorganisms (e.g. pathogens) is referred to as phagocytosis. The vesicle created by phagocytosis is called the phagosome. Phagosome maturation is considered to involve a complex sequence of fusion and fission events with the sub compartments of the endocytic pathway. In many ways, phagosome maturation recapitulates the progression of cargo along the endocytic pathway. In this thesis we have described molecular mechanisms that regulate the endocytic route and how pathogens explore this endocytic route to ensure survival. Show less
This thesis contributes to a better understanding of the roles of apoCI, LPL, and CETP in lipoprotein metabolism. Our data illustrate that the activity of LPL, and thereby the level of plasma TG,... Show moreThis thesis contributes to a better understanding of the roles of apoCI, LPL, and CETP in lipoprotein metabolism. Our data illustrate that the activity of LPL, and thereby the level of plasma TG, is crucially determined by the relative abundance of apolipoproteins. In addition, we showed that LPL is an important determinant in remnant-particle clearance in the absence of the three main apoE-recognizing receptors. Finally, we demonstrated that CETP presents a pro-atherogenic factor in mice resembling a human lipid distribution over lipoproteins and that atorvastatin and fenofibrate treatment influence HDL-metabolism via inhibition of CETP, which may thus add to their therapeutic benefit. Since there were initial concerns that inhibition of CETP would reduce the flux of cholesteryl esters from the periphery back to the liver, thereby possibly increasing the risk for atherosclerosis, it is of interest that we found that fenofibrate-mediated inhibition of CETP did not hamper the total flux of HDL-cholesteryl esters. This holds promise for therapies based on CETP inhibition. Show less
The metabolic syndrome is an increasing problem in our Western society. Many of the features of the metabolic syndrome, like obesity, insulin resistance, dyslipidemia, and hepatic steatosis are... Show moreThe metabolic syndrome is an increasing problem in our Western society. Many of the features of the metabolic syndrome, like obesity, insulin resistance, dyslipidemia, and hepatic steatosis are established risk factors for cardiovascular disease. Growing evidence supports the important role of body free fatty acid handling and/or body distribution of triglycerides in the pathogenesis of the metabolic syndrome-associated problems. We used several different approaches to study the development of obesity, insulin resistance, dyslipidemia, and liver steatosis. In chapter 2 we found that absence of apoC3, a natural LPL inhibitor, enhances FA uptake from plasma triglycerides in adipose tissue leading to increased susceptibility to diet-induced obesity, followed by more severe development of insulin resistance. Therefore, we have shown that regulation of body distribution of triglycerides, in a LPL-dependent process, plays an important role in obesity development. In chapter 3 we found that acute inhibition of the β-oxidation of FA indeed increases hepatic lipid content, but neither stimulates hepatic VLDL secretion nor reduces insulin sensitivity. In chapter 4 we showed that the combination of proteomics with relevant physiological parameters in a sensitive animal model, is a powerful tool, which will aid in identifying workingmechanisms of various dietary FA. In chapter 5 we found that sphingolipids protect the liver from fat and cholesterol-induced steatosis. Since sphingolipids are nutritional compounds present in several daily foods, such as milk and meat, addition of sphingolipids to the diet may decrease traditional cardiovascular risk factors, such as plasma cholesterol and triglycerides. Show less
