Atherosclerosis, the predominantly underlying pathology of cardiovascular events, is the consequence of lipid deposition in the arterial wall, mostly as consequence of high levels of serum... Show moreAtherosclerosis, the predominantly underlying pathology of cardiovascular events, is the consequence of lipid deposition in the arterial wall, mostly as consequence of high levels of serum cholesterol. Treatment of atherosclerosis is mainly focused at the reduction of cholesterol levels by lipid lowering medication, such as statins. Despite the use of statins and prophylactic treatments, such as a reduction in blood pressure and a reduction in risk factors to prevent atherosclerosis, cardiovascular disease is still the major cause of death in the Western world.1-3 As the struggle against atherosclerosis continues and its prevalence is increasing in the world, it is pivotal to find new targets for implementing new strategies against atherosclerosis. Inflammation is considered to be a major component in the process of atherosclerosis and is involved in initiation, progression and destabilization of the atherosclerotic lesion. Although the beneficial effects of statins on atherosclerosis may partly be ascribed to their anti-inflammatory properties, relatively little is known about the exact mechanism and contribution of different inflammatory cells and products in atherosclerosis. The aim of this thesis was to further elucidate the contribution of various components of the inflammatory response in atherosclerosis and thereby finding new intervention points to reduce the incidence and the consequences of atherosclerosis. 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
Scheuring van een vaatwandvernauwing (plaque) kan ertoe leiden dat materiaal vanuit deze plaques in direct contact komt met de bloedsomloop wat de bloedstolling kan activeren met als mogelijk... Show moreScheuring van een vaatwandvernauwing (plaque) kan ertoe leiden dat materiaal vanuit deze plaques in direct contact komt met de bloedsomloop wat de bloedstolling kan activeren met als mogelijk gevolg bloedvatafsluiting en hartinfarct. Vooral vetten zoals het lysofosfatidaat (LPA) in deze plaques zijn verantwoordelijk voor het in gang zetten van de bloedstolling. In dit project werd het mechanisme achter de LPA accumulatie in de plaque en de effecten van LPA op plaquestabiliteit onderzocht. Expressie analyse heeft aangetoond dat verschillende sleutelfactoren in de aanmaak, afbraak van LPA gedurende de plaqueontwikkeling toe- of afnemen en dat dit gepaard gaat met een netto ophoping van LPA in de plaque. Analyse van de vetsamenstelling van muizenplaques liet zien dat ook in ons muizenmodel voor hart- en vaatziekten LPA ophoping plaatsvindt en wel in dezelfde mate als in humane plaques. Bovendien hebben we een studie uitgevoerd naar het effect van LPA op atherosclerose in een muizenstam met een verhoogd cholesterol gehalte. Zowel toediening via de buikholte als lokale toediening van LPA ter plekke van de plaque blijkt te leiden tot een verslechtering van de plaquestabiliteit en zelfs tot bloedingen in de plaque, waarbij dit laatste mogelijk is terug te voeren op een verhoogde activering van mestcellen in het vaatweefsel. Dit blijkt ook uit het feit dat de effecten omkeerbaar zijn bij toediening van een remmer van mestcel activatie. Daarnaast werd een studie uitgevoerd naar de effecten van een op LPA gelijkend vetmolecuul, sfingosine 1-phosphate (S1P). Verhoging van de S1P concentratie door afwezigheid van een afbraakenzym voor S1P of toediening van een synthetische analoog van S1P, FTY720, bleek de plaqueontwikkeling aanzienlijk af te remmen door een selectieve onderdrukking van het immuunsysteem. Bij afwezigheid van het S1P afbraakenzym werd ook verlaging in cholesterolconcentratie in het bloed geconstateerd. Concluderend kunnen wij stellen dat LPA kan worden beschouwd als een overwegend pro-atherogeen, en S1P als een voornamelijk anti-atherogeen lipide. Verlaging van LPA en verhoging van S1P kan dus leiden tot een effectieve therapeutische benadering ter vermindering van instabiele atherosclerotische plaque vorming. Show less
Hart- en vaatziekten zijn, ondanks de toepassing van cholesterol verlagende medicijnen, nog steeds de meest voorkomende doodsoorzaak in de westerse wereld, en manifesteren zich onder andere door... Show moreHart- en vaatziekten zijn, ondanks de toepassing van cholesterol verlagende medicijnen, nog steeds de meest voorkomende doodsoorzaak in de westerse wereld, en manifesteren zich onder andere door hartinfarcten en hersenbloedingen. Cholesterol, en ook fosfolipiden zijn essentieel voor de opbouw van celmembranen, maar ook voor de synthese van bijvoorbeeld hormonen. Een evenwichtige cholesterol huishouding is dus niet alleen belangrijk voor het hele lichaam maar ook voor individuele cellen. Dit evenwicht wordt, in een gezonde situatie, in stand gehouden door een strak gereguleerde balans tussen de opname van cholesterol uit het voedsel en de novo cholesterol synthese. Het zogenaamde goede cholesterol, het HDL, dankt zijn beschermende werking aan het zogenaamde reverse cholesterol transport, het transport van cholesterol uit de periferie naar de lever, waar het kan worden verwerkt en afgevoerd via de gal naar de feaces. Dit proefschrift richt zich op de verschillende stappen van het reverse cholesterol transport. Concluderend: therapeutische modulatie van het __reverse cholesterol transport__ moet zich niet richten op __n specifiek gen/eiwit, maar er dient gestreefd te worden naar een simultane activatie van cholesterol transporteiwitten wat zou kunnen leiden tot een optimale bescherming tegen vaatvernauwing en dientengevolge hart- en vaatziekten. Show less
Blood-flow-induced shear stress plays an important role in cardiovascular development and disease. How endothelial cells sense shear stress remains to be elucidated. We postulated that the primary... Show moreBlood-flow-induced shear stress plays an important role in cardiovascular development and disease. How endothelial cells sense shear stress remains to be elucidated. We postulated that the primary cilium is a component of the endothelial shear sensor. This luminal cell protrusion contains microtubules and is connected to the microtubular cytoskeleton. We identified cilia on endothelial cells of the embryonic heart in areas of low or oscillatory shear stress. This shear-related distribution is reminiscent of the distribution of atherosclerotic lesions in the adult arterial system, as lesions develop at sites of low or oscillating shear (athero-prone flow). Ciliated endothelial cells are exclusively present at these atherosclerotic predilection sites in adult mice. Athero-prone (oscillatory) but not athero-protective (steady or pulsatile) flow induces ciliation of cultured endothelial cells. Moreover, the endothelial shear response is dependent on the microtubular cytoskeleton and primary cilia sensitise the endothelium for shear. Taken together, these data demonstrate that primary cilia are induced by athero-prone flow and that ciliated cells are more sensitive to shear stress. We conclude that the endothelial biosensor for shear stress is the microtubular cytoskeleton and that the attached primary cilium functions as a signal amplifier in areas subjected to athero-prone flow. Show less
In this thesis a novel technology is described to target adenovirus vectors. Adenovirus vectors are powerful tools to modulate gene expression. The use of these vectors however, is hampered by the... Show moreIn this thesis a novel technology is described to target adenovirus vectors. Adenovirus vectors are powerful tools to modulate gene expression. The use of these vectors however, is hampered by the fact that many for gene therapy interesting cell types do not, or only at low levels express the CAR receptor, necessary for infection. We developed a linker protein consisting of the virus-binding moiety of CAR genetically fused to the chicken protein avidin. Biotinylated ligands for cell specific receptors are bound to the linker protein via the avidin-biotin interaction. This now targeting protein is used to redirect adenovirus vectors to previously refractory cell types. Using this technology endothelial cell lines as well as primary endothelial cells can by infected at low MOI__s using an biotinylated cyclic RGD peptide. Primary bone marrow derived macrophages and macrophage cell lines are easily infected using a biotinylated dA6dG10 oligo nucleotide ligand. In vivo experiments showed a marked reduction of adenovirus mediated transgene expression by the liver, the organ responsible for virus uptake when unmodified adenovirus vectors are administered, after addition of several different ligands to the virus via the linker protein. Show less
Various modalities are available in the diagnostic and prognostic evaluation of patients presenting with known or suspected coronary artery disease (CAD). A rapidly expanding technique is... Show moreVarious modalities are available in the diagnostic and prognostic evaluation of patients presenting with known or suspected coronary artery disease (CAD). A rapidly expanding technique is noninvasive coronary angiography with Multi-Slice Computed Tomography (MSCT), which allows accurate detection of significant stenoses. The main value of the technique lies in the noninvasive exclusion of CAD in patients with intermediate pre-test likelihood. Although imaging in populations such as patients with previous stent placement appears to be more challenging, promising results have been obtained in these populations as well. However, it remains important to realize that the presence of coronary atherosclerosis with luminal obstruction does not invariably imply the presence of ischemia. Accordingly, a noninvasive angiographic imaging technique as MSCT cannot be used to predict the hemodynamical importance of lesions. In patients with borderline stenosis, therefore, functional testing (which can be performed by nuclear imaging, stress echocardiography or MRI) will remain necessary to determine management. Nonetheless, detection of CAD at a far earlier stage than functional imaging is an important advantage of MSCT. Initial investigations suggest that MSCT may distinguish different plaque characteristics between various presentations. Potentially, this information could be useful for risk stratification. Finally, additional non-coronary information can be derived as well. LV function can be evaluated with high accuracy while also information on the cardiac venous system can be obtained. Show less
One of the most important characteristics of atherosclerosis is the chronic inflammatory response in which T cells and NKT cells are very important. In this thesis several methods to modulate the... Show moreOne of the most important characteristics of atherosclerosis is the chronic inflammatory response in which T cells and NKT cells are very important. In this thesis several methods to modulate the activity of these T and NKT cells in atherosclerosis are described. The induction of regulatory T cells (as mentioned in chapters 2 and 3) positively influences the immune response in atherosclerosis. In addition, treatment with dendritic cells (DCs) loaded with oxidized LDL (oxLDL) resulted in the activation of oxLDL-specific T cells and in an increase in oxLDL-specific antibodies. These IgG antibodies have a protecting effect on atherosclerosis (chapter 4). Furthermore, the complex role of NKT cells in the immunology of atherosclerosis is described (chapters 5, 6 and 7). NKT cells can play a protective role in atherosclerosis when they are 1) activated directly by _-GalCer or when they are 2) activated by OCH loaded on DCs. The studies described in this thesis led to a better insight in the role of the immune system in high-fat diet induced atherosclerosis, and they also create a possible therapeutic protocol which can probably be applied to reduce the incidence of cardiovascular diseases in humans. Show less
With the use of combinatorial phage display, solid phase peptide synthesis and a multidiscipline of molecular and cellular assays in vascular biology, the research described in this thesis has... Show moreWith the use of combinatorial phage display, solid phase peptide synthesis and a multidiscipline of molecular and cellular assays in vascular biology, the research described in this thesis has resulted in the identification of two novel peptides targeting to SR-AI and CD40 respectively which hold promise as targeted contrast agents for the diagnosis of atherosclerosis symptom. In addition, a peptide named VIVIT and its derivatives had been discovered and synthesized which constitute a more selective and less toxic drug candidate than currently used immunosuppressant cyclosporine A or FK506, leading to new generation immunosuppressants and therapeutics for autoimmune diseases such as rheumatoid arthritis or allograft transplantation and cardiovascular disorders including atherosclerosis, restenosis and cardiac hypertrophy. Show less
The main cause of cardiovascular disease (CVD) is atherosclerosis. Several genes that affect atherosclerosis development have been identified by the use of genetically modified mice (i.e.... Show moreThe main cause of cardiovascular disease (CVD) is atherosclerosis. Several genes that affect atherosclerosis development have been identified by the use of genetically modified mice (i.e. transgenic and knock-out mouse models). Many of these genes exert their role in atherosclerosis development as a result of effects on lipoprotein metabolism and inflammation. Transgenic mouse models have also been proven to be suitable for evaluating the mechanisms underlying the anti-atherosclerotic action of experimental drugs aimed to reduce atherogenic lipoprotein levels. However, thus far no suitable animal model was present to evaluate the mechanism of action of anti-atherosclerotic effect of HDL-raising therapeutic strategies. In this thesis, we further explored the role of apolipoprotein CI (apoCI) and cholesteryl ester transfer protein (CETP) in lipoprotein metabolism, inflammation, and atherosclerosis. Furthermore, we developed a mouse model that will be suitable for testing potential high-density-lipoprotein (HDL) raising therapies as a novel strategy to treat CVD. Show less
Accurate visualization and quantification of atherosclerosis in a non-invasive manner by means of MR is, nowadays, of high importance, not only regarding morphology but also composition of the... Show moreAccurate visualization and quantification of atherosclerosis in a non-invasive manner by means of MR is, nowadays, of high importance, not only regarding morphology but also composition of the atherosclerotic plaques. MR has proven to be capable of detecting early, subclinical vulnerable plaque. However, when the analysis of atherosclerosis is based on visual interpretation of the images or manually delineated structures, the outcome is often not reliable. The main objective of this thesis was to investigate novel image processing techniques to automatically quantify markers of the severity of atherosclerosis in a reproducible manner, by automatically outlining the boundaries of the blood vessel wall, lumen and plaque burden. Different algorithms have been developed and applied to different vascular beds (aorta and carotid arteries) proving to be versatile and powerful tools, that provide quantitative reproducible parameters. These new techniques have been validated in limited populations, proving to be accurate and reproducible. They are, therefore, suitable to be further adapted and to be employed in the clinical vascular research. This assists the physician in making a diagnosis and identifying high-risk patients, who may benefit from treatment. 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
This thesis centers on the mechanisms of estrogen action and the effects on the development of atherosclerosis. We have focused on the liver as central organ in lipid and glucose metabolism and the... Show moreThis thesis centers on the mechanisms of estrogen action and the effects on the development of atherosclerosis. We have focused on the liver as central organ in lipid and glucose metabolism and the vessel wall as the actual site where the injury occurs. To gain insight in tissue-specific actions of estrogens, we have spent considerable effort to develop tools for liver and blood vessel specific modulation of the estrogen receptor (ER) signaling cascade. The generation, characterization and application of these tools in vitro and in vivo will be described in the different chapters of this thesis. Show less
The presence of calcium deposits in the vessel wall is indicative of advanced atherosclerosis, and the extent of coronary calcification has been found to add prognostic significance to conventional... Show moreThe presence of calcium deposits in the vessel wall is indicative of advanced atherosclerosis, and the extent of coronary calcification has been found to add prognostic significance to conventional risk factors of coronary artery disease. However, the mechanisms underlying vascular calcification are still obscure. The major objective of the work described in the first part of this thesis was to elucidate the mechanisms involved in atherosclerotic calcification. To study the process of VSMC calcification we developed and characterized an in vitro model of neonatal rat VSMC calcification. To investigate whether pharmacotherapy may affect vascular calcifications, we have studied the effect of a calcium antagonist (amlodipine) and a statin (atorvastatin) and their combination on this process. Inflammation is an important mechanism in the atherosclerotic process, and prospective and cross-sectional clinical and epidemiological studies have shown that CRP is consistently associated with CVD. The causality of CRP in atherosclerosis is discussed. To enable the study of the effect of CRP on atherosclerosis development in vivo, ApoE*3-Leiden/hCRP transgenic mice were generated and studied. The effects of a calcium antagonist (amlodipine), administered either alone or in combination with a statin (atorvastatin), on early atherosclerosis development in ApoE*3-Leiden/hCRP was investigated. Show less
In this thesis we focus on atherosclerosis as the main cause of cardiovascular disease. Since inflammation and cell death are important processes in the onset and progression of atherosclerosis, we... Show moreIn this thesis we focus on atherosclerosis as the main cause of cardiovascular disease. Since inflammation and cell death are important processes in the onset and progression of atherosclerosis, we investigate the role of several genes involved in inflammation and cell death in the vessel wall and their effect on atherosclerosis. We use several ways to modulate gene expression. Examples from different chapters are whole body deletion of TNF (2), local gene targeting of Fas Ligand to the cap of the plaque (3), conditional gene targeting of mdm2, thereby upregulating p53 (4), and beta-galactosidase (5), and pharmacological targeting of PPARs (6). In this thesis we use various mouse models of atherosclerosis, such as the apoE deficient mouse, the "humanized" apoE3*Leiden mouse and accelerated atherosclerosis induced by collar placement. Show less
Aderverkalking (atherosclerose) is een ziekte waarbij door verdikking van de vaatwand vernauwing van slagaderen optreedt. Door deze vernauwing is het mogelijk dat er te weinig zuurstofrijk bloed de... Show moreAderverkalking (atherosclerose) is een ziekte waarbij door verdikking van de vaatwand vernauwing van slagaderen optreedt. Door deze vernauwing is het mogelijk dat er te weinig zuurstofrijk bloed de organen bereikt. In het geval van atherosclerose in slagaderen die hart of hersen van bloed moeten verzien leidt de vernauwing niet zelden tot de dood. Het feit dat atherosclerose de belangrijkste onderliggende oorzaak is van wereldwijde sterfte onderstreept het maatschappelijk belang van ontwikkeling van een adequate therapie tegen atherosclerose. In dit proefschrift werden nieuwe vaccinatietechnieken aangewend om het proces van atherosclerose tegen te gaan. Door de tolerantie voor lichaamseigen stoffen of cellen te doorbreken met deze vaccinatietechnieken bleek het mogelijk te zijn atherosclerose in muizen sterk af te remmen. Het wegnemen van de functie van ontstekingsbevorderende eiwitten (interleukinen 12 en 17) met deze vaccinatietechieken leidde tot een 70-90% afname in atherosclerose. Ook het met vaccinatie specifiek opruimen van cellen die betrokkken zijn bij atherosclerose-geassocieerde processen, zoals nieuwvaatvorming, bleek een geschikte methode te zijn om atherosclerose tegen te gaan. Het voordeel van de nieuwe vaccinatietechnieken die bij dit onderzoek zijn gebruikt is gelegen in het feit dat met enkele injecties voor lange tijd (ten minste 24 weken) bescherming tegen atherosclerose bewerkstelligd kan worden. Andere voordelen van deze vaccinaties zijn het gebrek aan afweerreacties tegen lichaamsvreemde medicijnen en de lage productiekosten van dergelijke vaccins. De combinatie van de sterke afname in atherosclerose en de bijkomende voordelen van de vaccinatietechnieken leidde in dit proefschrift tot de conclusie dat vaccinatie een belangrijke bijdrage kan leveren aan de ontwikkeling van therapieën tegen hart- en vaatziekten in mensen. Show less
The work described in this thesis was aimed at identifying the role of cell cycle and apoptosis genes in atherosclerosis. Atherosclerosis is the primary cause of cardiovascular disease, a disorder... Show moreThe work described in this thesis was aimed at identifying the role of cell cycle and apoptosis genes in atherosclerosis. Atherosclerosis is the primary cause of cardiovascular disease, a disorder occurring in the large and medium-sized arteries of the body. Although in the beginning 90s promising lipid lowering therapies predicted a strong reduction in cardiovascular deaths, in westernized societies it is still the underlying cause of about 40% of all deaths, indicating that treatment of atherosclerosis goes beyond lipid lowering solely. In addition to lipids, continuous cell growth (cell cycle) and cell death (i.e. apoptosis and necrosis) processes play a central role in the development of atherosclerosis. To investigate the role of several cell cycle and apoptosis genes (i.e. p53, Rb and Mdm2) in atherosclerosis we generated and characterized several mouse models based on site-specific recombinase (SSR) technology. The studies described in this thesis show that next to therapies aiming at lifestyle interventions, lipid therapies and regulation of inflammation, targeting cell cycle and apoptosis genes on lesional or cellular level might prove the most effective way to reduce the burden of atherosclerosis. Show less
