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
Korporaal, S.J.A.; Sluis, R.J. van der; Eck, M. van; Hoekstra, M. 2021
The genetic disorder Down syndrome is associated with a decreased susceptibility for atherosclerotic cardiovascular disease. Hematological and immune abnormalities occur frequently in Down syndrome... Show moreThe genetic disorder Down syndrome is associated with a decreased susceptibility for atherosclerotic cardiovascular disease. Hematological and immune abnormalities occur frequently in Down syndrome patients. We evaluated, in a preclinical setting, the impact of a Down syndrome-like hematological/immune phenotype on atherosclerosis susceptibility. Hereto, hypercholesterolemic low-density lipoprotein receptor knockout mice were transplanted with bone marrow from either a trisomic Ts65Dn mouse or euploid wild-type control and subsequently fed a Western-type diet to induce the development of atherosclerotic lesions. T and B cell concentrations were markedly reduced in blood of Ts65Dn bone marrow recipients (p < 0.001). Expression levels of the pro-atherogenic scavenger receptor CD36 were respectively 37% and 59% lower (p < 0.001) in trisomic monocytes and macrophages. However, these combined effects did not translate into an altered atherosclerosis susceptibility. Notably, blood platelet numbers were elevated in Ts65Dn bone marrow recipients (+57%; p < 0.001), which was paralleled by higher platelet GPVI protein expression (+35%; p < 0.001) and an enhanced collagen-induced platelet activation (p < 0.001). In conclusion, we have shown that providing mice with a Down syndrome-like hematological profile does not change the susceptibility to atherosclerosis. Furthermore, our studies have uncovered a novel effect of the trisomy on platelet functionality that may be relevant in human clinical settings. Show less
One of the pathological hallmarks of Alzheimer's disease is amyloid‑β accumulation in the parenchymal brain tissue. Amyloid‑β is also found in the vessel wall of patients with cerebral amyloid... Show moreOne of the pathological hallmarks of Alzheimer's disease is amyloid‑β accumulation in the parenchymal brain tissue. Amyloid‑β is also found in the vessel wall of patients with cerebral amyloid angiopathy (CAA). These pathological accumulations of the amyloid‑β peptide are referred to as amyloidosis. Both patients with AD and CAA also commonly show cerebrovascular dysfunction. The aim of this thesis was to improve our understanding of the relation between cerebrovascular dysfunction and amyloidosis. To that end, cerebrovascular function measurements were designed and carried out in mouse models of cerebral amyloidosis. Chapter 2 and 3 show improvements of the current techniques to measure cerebrovascular function in mice. Surprisingly however, no difference was found in cerebrovascular function in two different models of amyloidosis, as shown in chapter 4 and 5. Possible explanations of the negative findings are further discussed in chapter 6. Despite the negative connotation of the outcome this thesis, this work is another small step towards a better understanding of the exact relationship between cerebrovascular dysfunction and amyloid‑β deposition in AD and CAA patients. Ultimately, this will help in the design of highly needed novel therapies for AD and CAA. Show less
Zaks, O.; Gaber, D.; Ben-Yaakov, K.; Sharvit-Bader, M.; Goz, A.; Rotfogel, Z.; ... ; Aharoni-Simon, M. 2021
Uveal melanoma (UM) and conjunctival melanoma (CM) are ocular malignancies that give rise to life-threatening metastases. Although local disease can often be treated successfully, it is often... Show moreUveal melanoma (UM) and conjunctival melanoma (CM) are ocular malignancies that give rise to life-threatening metastases. Although local disease can often be treated successfully, it is often associated with significant vision impairment and treatments are often not effective against metastatic disease. Novel treatment modalities that preserve vision may enable elimination of small tumors and may prevent subsequent metastatic spread.Very few mouse models of metastatic CM and UM are available for research and for development of novel therapies. One of the challenges is to follow tumor growth in-vivo and to determine the right size for treatment, mainly of the posterior, choroidal melanoma. Hence, the purpose of this study was to establish a simple, noninvasive imaging tool that will simplify visualization and tumor follow-up in mouse models of CM and UM.Tumors were induced by inoculation of murine B16LS9 cells into the sub-conjunctival or the choroidal space of a C57BL/6 mouse eye under a surgical microscope. Five to ten days following injection, tumor size was assessed by Phoenix MicronIVTM image-guided Optical Coherence Tomography (OCT) imaging, which included a realtime camera view and OCT scan of the conjunctiva and the retina. In addition, tumor size was evaluated by ultrasound and histopathological examination of eye sections.Tumor growth was observed 5-9 days following sub-conjunctival or sub-retinal injection of seven-thousand or seventy-thousand cells, respectively. A clear tumor mass was detected at these regions using the MicronIVTM imaging system camera and OCT scans. Histology of eye sections confirmed the presence of tumor tissue. OCT allowed an accurate measurement of tumor size in the UM model and a qualitative assessment of tumor size in the CM model. Moreover, OCT enabled assessing the success rate of the choroidal tumor induction and importantly, predicted final tumor size already on the day of cell inoculation.In conclusion, by using a simple, non-invasive imaging tool, we were able to follow intraocular tumor growth of both CM and UM, and to define, already at the time of cell inoculation, a grading scale to evaluate tumor size. This tool may be utilized for evaluation of new mouse models for CM and UM, as well as for testing new therapies for these diseases. Show less
Using a variety of relevant preclinical mouse models, this thesis not only showed several ways to improve antibody-based cancer immunotherapy but also demonstrates the importance of the choice of... Show moreUsing a variety of relevant preclinical mouse models, this thesis not only showed several ways to improve antibody-based cancer immunotherapy but also demonstrates the importance of the choice of the model for the translational relevance of the preclinical observations. Show less
Autosomal Dominant Polycystic Kidney Disease (ADPKD) progression involves a complex interaction of different molecular pathways, ultimately leading to cyst growth and loss of kidney function. The... Show moreAutosomal Dominant Polycystic Kidney Disease (ADPKD) progression involves a complex interaction of different molecular pathways, ultimately leading to cyst growth and loss of kidney function. The exact mechanism behind cyst formation is still not clearly understood. Moreover, we know some of the molecular pathways involved in cyst initiation and progression, but we do not know at which stage of the disease they play a role. In this thesis, we investigated the molecular pathways involved in renal injury-repair mechanisms and ADPKD. According to the currently available literature, injury-repair and ADPKD are two extremely intertwined mechanisms, which not only are characterised by activation of similar molecular pathways but are also able to influence each other. In fact, injury is able to accelerate cyst formation and progression, and cyst growth can cause injury to the surrounding tissue. Thus, the introduction of injury in the context of ADPKD can help to characterize the steps of disease progression, particularly in the early phases of cyst initiation, and direct future research to new possible therapeutic targets. Show less
Schwach, V.; Fernandes, M.G.; Maas, S.; Gerhardt, S.; Tsonaka, R.; Weerd, L. van der; ... ; Salvatori, D.C.F. 2020
Aims Cardiovascular diseases caused by loss of functional cardiomyocytes (CMs) are a major cause of mortality and morbidity worldwide due in part to the low regenerative capacity of the adult human... Show moreAims Cardiovascular diseases caused by loss of functional cardiomyocytes (CMs) are a major cause of mortality and morbidity worldwide due in part to the low regenerative capacity of the adult human heart. Human pluripotent stem cell (hPSC)-derived cardiovascular progenitor cells (CPCs) are a potential cell source for cardiac repair. The aim of this study was to examine the impact of extensive remuscularization and coincident revascularization on cardiac remodelling and function in a mouse model of myocardial infarction (MI) by transplanting doxycycline (DOX)-inducible (Tet-On-MYC) hPSC-derived CPCs in vivo and inducing proliferation and cardiovascular differentiation in a drug-regulated manner.Methods and results CPCs were injected firstly at a non-cardiac site in Matrigel suspension under the skin of immunocompromised mice to assess their commitment to the cardiovascular lineage and ability to self-renew or differentiate in vivo when instructed by systemically delivered factors including DOX and basic fibroblast growth factor (bFGF). CPCs in Matrigel were then injected intra-myocardially in mice subjected to MI to assess whether expandable CPCs could mediate cardiac repair. Transplanted CPCs expanded robustly both subcutis and in the myocardium using the same DOX/growth factor inducing regime. Upon withdrawal of these cell-renewal factors, CPCs differentiated with high efficiency at both sites into the major cardiac lineages including CMs, endothelial cells, and smooth muscle cells. After MI, engraftment of CPCs in the heart significantly reduced fibrosis in the infarcted area and prevented left ventricular remodelling, although cardiac function determined by magnetic resonance imaging was unaltered.Conclusion Replacement of large areas of muscle may be required to regenerate the heart of patients following MI. Our human/mouse model demonstrated that proliferating hPSC-CPCs could reduce infarct size and fibrosis resulting in formation of large grafts. Importantly, the results suggested that expanding transplanted cells in situ at the progenitor stage maybe be an effective alternative causing less tissue damage than injection of very large numbers of CMs. Show less
Geest, R. van der; Sluis, R.J. van der; Groen, A.K.; Eck, M. van; Hoekstra, M. 2019
Chronic glucocorticoid overexposure predisposes to the development of atherosclerotic cardiovascular disease in humans. Cholestatic liver disease is associated with increased plasma glucocorticoid... Show moreChronic glucocorticoid overexposure predisposes to the development of atherosclerotic cardiovascular disease in humans. Cholestatic liver disease is associated with increased plasma glucocorticoid levels. Here we determined, in a preclinical setting, whether the chronic presence of cholestatic liver disease also induces a concomitant negative impact on atherosclerosis susceptibility. Hereto, regular chow diet-fed atherosclerosis-susceptible hypercholesterolemic apolipoprotein E (APOE) knockout mice were treated with the bile duct toxicant alpha‑naphthylisothiocyanate (ANIT) for 8 weeks. ANIT exposure induced the development of fibrotic cholestatic liver disease as evident from collagen deposits and compensatory bile duct hyperproliferation within the liver and the rise in plasma levels of bilirubin (+60%; P<0.01) and bile acids (10-fold higher; P<0.01). Adrenal weights (+22%; P<0.01) and plasma corticosterone levels (+72%; P<0.01) were increased in ANIT-treated mice. In contrast, atherosclerosis susceptibility was not increased in response to ANIT feeding, despite the concomitant increase in plasma free cholesterol (+30%; P<0.01) and cholesteryl ester (+42%; P<0.001) levels. The ANIT-induced hypercorticosteronemia coincided with marked immunosuppression as judged from the 50% reduction (P<0.001) in circulating lymphocyte numbers. However, hepatic glucocorticoid signaling was not enhanced after ANIT treatment. It thus appears that the immunosuppressive effect of glucocorticoids is uncoupled from their metabolic effect under cholestatic disease conditions. In conclusion, we have shown that cholestatic liver disease-associated endogenous glucocorticoid overexposure does not increase atherosclerosis susceptibility in APOE knockout mice. Our studies provide novel preclinical evidence for the observations that the hypercholesterolemia seen in cholestatic human subjects does not translate into a higher risk for atherosclerotic cardiovascular disease. Show less
Hoekstra, M.; Nahon, J.E.; Jong, L.M. de; Kröner, M.J.; Leeuw, L.R. de; Eck, M. van 2019
The nuclear receptor liver X receptor (LXR) impacts on cholesterol metabolism as well as hepatic lipogenesis via transcriptional regulation. It is proposed that inhibition of the protein arginine... Show moreThe nuclear receptor liver X receptor (LXR) impacts on cholesterol metabolism as well as hepatic lipogenesis via transcriptional regulation. It is proposed that inhibition of the protein arginine methyltransferase 3 (PRMT3) uncouples these two transcriptional pathways in vivo by acting as a specific lipogenic coactivator of LXR. Here we validated the hypothesis that treatment with the allosteric PRMT3 inhibitor SGC707 will diminish the hepatic steatosis extent, while leaving global cholesterol metabolism, important in cholesterol-driven pathologies like atherosclerosis, untouched. For this purpose, 12-week old hyperlipidemic apolipoprotein E knockout mice were fed a Western-type diet for six weeks to induce both hepatic steatosis and atherosclerosis. The mice received 3 intraperitoneal injections with SGC707 or solvent control per week. Mice chronically treated with SGC707 developed less severe hepatic steatosis as exemplified by the 51% reduced (P < 0.05) liver triglyceride levels. In contrast, the extent of in vivo macrophage foam cell formation and aortic root atherosclerosis was not affected by SGC707 treatment. Interestingly, SGC707-treated mice gained 94% less body weight (P < 0.05), which was paralleled by changes in white adipose tissue morphology, i.e. reduction in adipocyte size and browning. In conclusion, we have shown that through PRMT3 inhibitor treatment specific functions of LXR involved in respectively the development of fatty liver disease and atherosclerosis can be uncoupled, resulting in an overall diminished hepatic steatosis extent without a negative impact on atherosclerosis susceptibility. As such, our studies highlight that PRMT3 inhibition may constitute a novel therapeutic approach to limit the development of fatty liver disease in humans. Show less
In this thesis, the role of the liver and lungs in atherosclerosis development were studied. The liver plays an important role in lipid metabolism and inflammation, the two main processes involved... Show moreIn this thesis, the role of the liver and lungs in atherosclerosis development were studied. The liver plays an important role in lipid metabolism and inflammation, the two main processes involved in atherogenesis. We show that continuous enhanced inflammation in hepatocytes increased the hepatic production of VLDL and aggravated atherosclerosis development in hyperlipidemic APOE*3-Leiden (E3L) mice as compared to control E3L mice. Poor lung function, most commonly caused by chronic obstructive pulmonary disease (COPD), is a risk factor for atherosclerosis development. To this end, we investigated whether elastase-induced alveolar wall destruction, a model for COPD, would worsen atherosclerosis development in E3L mice. No difference in atherosclerotic lesion size was observed between mice after elastase or vehicle instillation, indicating that alveolar destruction per se is not responsible for the increased risk for atherosclerosis in COPD patients. Furthermore, we studied the anti-atherosclerotic effects of resveratrol which can be found in red wine and Asian medicinal herbs. Hyperlipidemic E3L.CETP mice were fed a diet without (control) or with resveratrol, atorvastatin, or both. Resveratrol protected against atherosclerosis development, but did not add to the anti-atherogenic effects of atorvastatin. Finally, the clinical implications and future perspectives of these results are discussed. Show less
The research in this thesis is aimed at the elucidation of the role of the glucocorticoid receptor (GR) in hippocampal neuroplasticity and functioning. To achieve this, we have developed a novel... Show moreThe research in this thesis is aimed at the elucidation of the role of the glucocorticoid receptor (GR) in hippocampal neuroplasticity and functioning. To achieve this, we have developed a novel method to specifically knockdown GR in a discrete cell population of the mouse brain. In this thesis I report silencing of GR expression selectively in a population of neuronal progenitors and immature neurons of the dentate gyrus, using RNA-interference (RNAi) delivered by a lentiviral vector. Characterization of these cells resulted in the discovery that GR knockdown causes a striking modulation of hippocampal neurogenesis and remodelling of hippocampal circuitry. Functional studies further revealed consequences of GR knockdown for contextual memory performance and behavioural coping strategies during stressful conditions. The results demonstrate the feasibility to apply RNAi in discrete cell populations for study of the action mechanism of glucocorticoids underlying control of neuroplasticity and behaviour. Show less
Activation of the complement system provides an important mechanism of defense of an organism against invading pathogens. In the healthy individual this defense is finely regulated to prevent... Show moreActivation of the complement system provides an important mechanism of defense of an organism against invading pathogens. In the healthy individual this defense is finely regulated to prevent attack of the complement system against cells and tissues of the host, however in abnormal situations this regulation can be out of balance. In the present thesis we look at a mouse model where the classical pathway of complement, in conjunction with anti-C1q autoantibodies, is shown to be involved in the development of renal disease (Chapter 2). In Chapter 3, a novel mouse model of complement-mediated glomerulonephritis is described. This model seems to be dependent on the alternative pathway of complement activation as well as on Fc receptors, and provides a novel tool to dissect the contribution of different effector systems in renal inflammation.Then the natural complement-inhibitory properties of the defensin Human Neutrophil Peptide-1 and the extracellular matrix molecules decorin and biglycan are investigated, which are presented to play a role in the regulation of complement in vitro, which hopefully can be extended to in vivo situations of health and disease in the near future (Chapter 4 & 5). The thesis is concluded with a general discussion in Chapter 6. Show less
In a murine model for rheumatoid arthritis, we wished to investigated whether it was possible to skew the immune response with a cellular vaccin to protect the mice against the induction and/or... Show moreIn a murine model for rheumatoid arthritis, we wished to investigated whether it was possible to skew the immune response with a cellular vaccin to protect the mice against the induction and/or progeression of arthritis. the model that was used for this purpose was Collagen-Induced Arthritis (CIA). As dendritic cells (DCs) are the main antigen-presenting cells and key players in setting immune responses and connecting innate witth adaptive immunity, it is favorable to use these cells to manipulate the immune system to circumvent autoimmunity, in this case CIA. Because CIA is still implicated as a Th1-mediated disease, the aim was to skew the immune system towards a more Th2-like phenotype or to induce a T cell with a regulatory capacity. Therefore, several ways to stimulate DCs and subsequently the evolving T cell response were selected, to analyze whether Th2 cells or regulatory T cells were activated, resulting in the inhibition of arthritis. Show less
This thesis examines different risk factors, in relation to restenosis after Percutaneous coronary interventions (PCI), with its main focus on genetic markers. Restenosis is the main drawback of... Show moreThis thesis examines different risk factors, in relation to restenosis after Percutaneous coronary interventions (PCI), with its main focus on genetic markers. Restenosis is the main drawback of PCI. Genetic variance poses an opportunity to enhance stratification of individuals who will be more prone to develop restenosis. Restenosis is a multifactorial process, therefore only limited part of the number of candidate genes that are potentially involved in restenosis can be described. Since the inflammatory reaction is known to be highly important in restenosis, our study has its main focus on inflammatory markers. To examine various candidate genes and their polymorphisms we made use of the GENetic DEterminants of Restenosis (GENDER) study, a multicenter follow-up study, including 3,104 consecutive patients, who were successfully treated with PCI. In the different chapters we describe the study population and the clinical and genetic factors investigated. Furthermore, we made use of a mouse model to improve our understanding of restenosis. Our results have contributed to a better understanding of the restenotic process, they could provide novel therapeutic targets as well as contribute to development of improved risk stratification of patients who are scheduled for elective PCI, thereby creating the opportunity to individualize treatment in the future. Show less
