The aim of this thesis was to develop novel treatment strategies for different types of eye melanoma utilizing zebrafish models. We first establish orthotopic and ectopic xenograft models for uveal... Show moreThe aim of this thesis was to develop novel treatment strategies for different types of eye melanoma utilizing zebrafish models. We first establish orthotopic and ectopic xenograft models for uveal and conjunctival melanoma by engraftment of the immortalized cells derived from these tumors into zebrafish embryos. Next, we expanded these models with spheroids and zebrafish patient-derived xenografts for pre-clinical, personalized screening of anti-uveal melanoma drug responses. We demonstrated that these models can be harnessed to explore the in vivo interactions of the tumor cells with blood vessels and macrophages leading to angiogenic response. We finally apply the conjunctival melanoma model to clarify the inhibitory effects of ginsenosides and correlate their structures with potential antitumoral mechanisms. Show less
Ocular melanoma is a rare disease that originates from melanocytes in the eye. It is the most prevalent primary ocular malignancy in adults, and has a high metastatic rate. Two important questions... Show moreOcular melanoma is a rare disease that originates from melanocytes in the eye. It is the most prevalent primary ocular malignancy in adults, and has a high metastatic rate. Two important questions for good patient care are: 1) How to differentiate between (benign) nevi, and (malignant) melanoma?, and 2) How to treat this tumor best, particularly in cases with metastases?This thesis addresses two types of ocular melanoma: melanoma of the internal parts of the eye (uveal melanoma) and melanoma of the mucous membrane covering the eye (conjunctival melanoma). This thesis combines patient-related projects with projects from the lab.With new imaging techniques we demonstrate that oxygen values differ in eyes with melanoma compared to other eyes including those with a nevus. We use OCT-angiography to depict tumour vessels non-invasively in conjunctival and iris lesions. These two techniques may be used in the future to differentiate lesions, and to monitor patients after treatment.With studies in the lab we show that new drugs (immunotherapy) that are recently used in cutaneous melanoma, can also be used to treat conjunctival melanoma. We show that vascular growth in uveal melanoma is related to other (genetic and immunologic) characteristics, providing new clues for therapy. Show less
In dit proefschrift begin ik met een algemene inleiding in Hoofdstuk 1 om kort de relevantie van EC-gedrag in vasculaire morfogenese en in angiogenese te presenteren. Bovendien bespreek ik hoe de... Show moreIn dit proefschrift begin ik met een algemene inleiding in Hoofdstuk 1 om kort de relevantie van EC-gedrag in vasculaire morfogenese en in angiogenese te presenteren. Bovendien bespreek ik hoe de EC-functie ingewikkeld wordt gereguleerd door positieve en negatieve factoren, en hoe hun functie kan worden gemanipuleerd voor therapeutische winst bij kanker en hart- en vaatziekten. In Hoofdstuk 2 bespreken we in detail de rol van de TGF-β-signaleringsroute in EndMT en bespreken we de bijdrage van dit proces aan de ontwikkeling van ziekten, evenals de mogelijke toepassingen ervan in weefselmanipulatie. In Hoofdstuk 3 onthullen we gedetailleerde werkprotocollen om TGF-β-geïnduceerde EndMT te onderzoeken en hoe de betrokkenheid van EndMT-effectoren te beoordelen met behulp van CRISPR/Cas9-genediting. In Hoofdstuk 4 hebben we de functie van EndMT transcriptiefactoren onderzocht en hun werkingsmechanisme opgehelderd. We ontdekten dat de EndMT-transcriptiefactoren (TF's) SNAIL en SLUG cruciaal zijn voor EndMT in endotheelcellen van muizen en dat de ID-eiwitten hun functie in EndMT compenseren. In Hoofdstuk 5 geven we een technisch overzicht van embryonale zebravis-xenotransplantaattesten om TGF-β-familiesignalering in de progressie van borstkanker bij de mens te onderzoeken, waaronder intravasatie/extravasatie van tumorcellen en tumorangiogenese. In Hoofdstuk 6 identificeren en onderzoeken we twee nieuwe BMP type I receptor macrocyclische kinaseremmers met therapeutisch potentieel om angiogenese in normale en tumorvatvorming bij zebravissen te normaliseren. In Hoofdstuk 7 vat ik alle studies in het proefschrift samen en geef ik enkele toekomstperspectieven met betrekking tot onze resultaten. Show less
During my PhD we have investigated different approaches to block intraplaque angiogenesis in atherosclerosis. Intraplaque angiogenesis is a physiological response to the increased oxygen demand in... Show moreDuring my PhD we have investigated different approaches to block intraplaque angiogenesis in atherosclerosis. Intraplaque angiogenesis is a physiological response to the increased oxygen demand in the plaque but also has adverse effects by facilitating intraplaque hemorrhage and influx of inflammatory mediators, resulting in plaque instability and consequent rupture. To study this phenomenon we used in vitro assays as well as the accelerated atherosclerosis vein graft model in ApoE3*Leiden mice, a unique model in which the formed plaque shows characteristics that highly resemble human atherosclerotic lesions, including intraplaque angiogenesis and hemorrhage and a high inflammatory cell content. We focused on different approaches to restore plaque stability via improving intraplaque oxygen levels as well as via blocking different growth factors signaling. Moreover we studied the effects of our treatments on the interaction between angiogenesis and inflammation both in vitro and in vivo. Show less
Cardiovascular diseases (CVDs) remain the leading cause of death worldwide, and thus, novel therapies are required. CVDs generally result in local shortages in the blood supply, known as ischemia.... Show moreCardiovascular diseases (CVDs) remain the leading cause of death worldwide, and thus, novel therapies are required. CVDs generally result in local shortages in the blood supply, known as ischemia. Neovascularization is the body's innate response mechanism that stimulates the restoration of blood flow to ischemic tissues. During the last decade, microRNAs have emerged as critical regulators of both CVD and neovascularization. Recent studies demonstrated that microRNAs are altered in many ways; however, whether these microRNA modifications could be physiologically relevant remained unclear. We examined whether specific microRNAs with a known cardiovascular function are subject to particular microRNA-alterations and if they could be relevant in cardiovascular disease. Our experiments demonstrated that the level of specific microRNA alterations, including isomiR formation, adenosine-to-inosine editing, and N6-adenosine methylation, changed in response to cardiovascular pathology. Many of these alterations changed the microRNAs function, which had a direct effect on processes like neovascularization. For example, microRNA adenosine-to-inosine editing increased after ischemia in both mice and humans and promoted neovascularization. These findings suggest that microRNA modifications can potentially be harnessed as a biomarker for cardiovascular disease, or even a novel therapeutic target. Show less
The endothelial glycocalyx (EG) is critically involved in vascular integrity and homeostasis, where it regulates endothelial cell mechanotransduction, vascular permeability, coagulation and... Show moreThe endothelial glycocalyx (EG) is critically involved in vascular integrity and homeostasis, where it regulates endothelial cell mechanotransduction, vascular permeability, coagulation and inflammation. Loss of the glomerular EG component, hyaluronan, results in albuminuria and vascular destabilisation. Glomerular loss of hyaluronan has a profound effect on endothelial stability, and similar effects were observed in tumor vessels of metastatic melanomas and in muscular tissue of diabetic patients with critical limb ischemia. Endothelial hyaluronan biosynthesis is critically determined by the endothelial metabolic state which glycolysis is a determining factor of endothelial hyaluronan biosynthesis and function. Show less
Despite the available treatment options and sophisticated imaging technologies for monitoring lesion development, the morbidity and mortality from acute cardiovascular events remain unacceptably... Show moreDespite the available treatment options and sophisticated imaging technologies for monitoring lesion development, the morbidity and mortality from acute cardiovascular events remain unacceptably high.While cholesterol-lowering, anti-inflammatory and anti-platelet therapies benefits can increase survival as a primary or secondary prevention, they are not sufficient for plaque rupture prevention. Moreover, the most advance imaging technologies to detect high-risk atherosclerotic patients fail to visualize and explore cellular events in small preclinical models. Therefore, there is a clear need for the development of new therapies and the application of high-resolution imaging modalities.In the current thesis, we evaluated new possibilities to inhibit and image intraplaque angiogenesis. Show less
Myocardial infarction results in a permanent loss of function in the heart. Currently, there is no therapy available that addresses the heart of the problem: the loss of cardiomyocytes. Cell... Show moreMyocardial infarction results in a permanent loss of function in the heart. Currently, there is no therapy available that addresses the heart of the problem: the loss of cardiomyocytes. Cell transplantation has been a focus of cardiac regenerative studies. Interestingly, cell transplantation has effect on cardiac function and vessel formation in the absence of cardiac differentiation, suggesting a role for the paracrine factors. Besides replacing cardiomyocytes, restoring blood flow to the infarcted area is vital. We showed that vasculogenesis is not hampered by the loss of endoglin, but angiogenesis, and network formation was impaired with reduced endoglin expression. We also studied the effect of extracellular vesicles (EVs) secreted by mesenchymal stromal cells (MSC) and cardiac progenitor cells (CPC) on angiogenesis and infarct size. Both in vitro and in vivo angiogenesis was significantly improved in the presence of these EVs. Knockdown of the pro-angiogenic factor EMMPRIN resulted in a reduction in angiogenesis. Injection of the CPC derived EVs into the heart after MI resulted in a decrease in infarct size. Furthermore, total proliferation was increase in the border zone and infarcted area as seen by an increase in Ki67 and Yap. These results show therapeutic potential of EVs for cardiac regeneration. Show less
Vascular remodeling is an active process of structural changes in the vasculature due to changes in the blood flow. It comprises diseases and processes such peripheral artery disease (PAD),... Show moreVascular remodeling is an active process of structural changes in the vasculature due to changes in the blood flow. It comprises diseases and processes such peripheral artery disease (PAD), coronary artery disease (CAD), neovascularization and vein graft disease (VGD), that are covered by cardiovascular diseases (CVD). The main underlying pathology of CVD is atherosclerosis, which can cause ischemia distal to atherosclerotic occlusions. Neovascularization (angiogenesis and arteriogenesis) naturally occurs in the body to form new blood vessels and restore blood flow to ischemic tissue. In case of severe atherosclerotic lesions, when the neovascularization capability of the body is not sufficient and revascularization interventions are no longer possible, bypass surgery is indicated with preferaby a vein graft. However, due to VGD, patency rates of vein grafts after 10 years are low. The aim of this thesis was to elucidate the role of the innate and adaptive immune system on vascular remodeling. We investigated the role of Toll-like receptors, Interferon regulatory factors and T cells on neovascularisation and VGD. In conclusion, new knowledge was obtained on several contributing factors in vascular remodeling. Described molecules of the innate and adaptive immune system might be used as targets to prevent VGD and stimulate neovascularization. Show less
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
This thesis describes the respective contribution of the expression of Tissue factor isoforms full length Tissue Factor (flTF) and alternatively spliced Tissue Factor (asTF) as well as Factor... Show moreThis thesis describes the respective contribution of the expression of Tissue factor isoforms full length Tissue Factor (flTF) and alternatively spliced Tissue Factor (asTF) as well as Factor VII by tumor cells to promote cancer progression. Cohorts of breast, colon, and bone cancer specimens and a multitude of in vitro and in vivo models were used to explore the mechanism behind enhanced cell proliferation and metastasis in in vitro and in vivo models, as well as decreased patient survival associated with TF and FVII expression in cancer patients. Show less
The studies included in this thesis demonstrated a preclinical murine model to study neovascularization in vivo and subsequently a number of potential targets to stimulate therapeutic... Show moreThe studies included in this thesis demonstrated a preclinical murine model to study neovascularization in vivo and subsequently a number of potential targets to stimulate therapeutic neovascularization. This thesis contributes to a better insight into mechanisms underlying post-ischemic neovascularization and offers new therapeutic perspective to current treatment strategies for patients with critical limb ischemia. Whether stagnated blood flow recovery after an occlusive event is due to restricted pre-existing collateral bed or due to decreased collateral remodeling, we are now closer to a tailor made treatment available for each patient with peripheral arterial disease. Show less
In dit proefschrift worden studies besproken naar de rol van de TGF-β signaleringsroute in de tumor micro-omgeving in colorectaal kanker. Deze studies hebben zich voornamelijk gefocust op de rol... Show moreIn dit proefschrift worden studies besproken naar de rol van de TGF-β signaleringsroute in de tumor micro-omgeving in colorectaal kanker. Deze studies hebben zich voornamelijk gefocust op de rol van kanker-geassocieerde fibroblasten (CAFs) in kanker. Wij hebben aangetoond dat een co-receptor voor TGF-β, endoglin, een grote rol speelt in het CAF-gemedieerd uitzaaien van darmkanker en dat de expressie van endoglin op CAFs een prognostische marker is voor metastase-vrije overleving in vroeg stadium darmkankerpatienten. Daarnaast werd een nieuwe muizenstam ontwikkeld om specifiek op fibroblasten endoglin uit te schakelen, zodat de rol hiervan in het ontstaan van darmkanker bestudeerd kon worden in een chemisch geinduceerd model voor darmkanker. Samengevat laten de studies in dit proefschrift zien dat endoglin expressie op CAFs een belangrijke rol speelt in het metastaseren van colorectaalkanker en opent het deuren naar therapeutische toepassingen. Show less
In this thesis computational modeling is used to help unravel the mechanisms of key steps in angiogenesis, the formation of new capillaries from existing blood vessels. The first step in... Show moreIn this thesis computational modeling is used to help unravel the mechanisms of key steps in angiogenesis, the formation of new capillaries from existing blood vessels. The first step in angiogenesis is the invasion of new branches into the surrounding tissue by degradation of extracellular matrix proteins, e.g. fibrin. A first model describes how invading sprouts use the so called plasminogen system, which dissolves fibrin matrices. A next model asks how endothelial cells can dynamically switch position during angiogenesis. Based on experimental observations, several authors suggest that dynamic cell shuffling is under strict, genetic control. Our simulations show, however, that shuffling can emerge as a side effect of sprouting. Once a sprout is formed, it needs to hollow to allow blood flow. The mechanisms responsible for this hollowing, or lumen formation, are debated: vacuoles may punch a hole through the cell, or cells might repulse one another. In our simulations, both these hypotheses can work synergistically in lumen formation, suggesting that both hypotheses might work together. In a final chapter, we introduce a workflow to simultaneously test the impact of changes in the value of multiple parameters on the outcome of the type of models used in this thesis. Show less
Von Willebrand disease is the most common inherited bleeding disorder and is characterized by reduced plasma von Willebrand factor (VWF) levels or functionally abnormal VWF. VWF is best known for... Show moreVon Willebrand disease is the most common inherited bleeding disorder and is characterized by reduced plasma von Willebrand factor (VWF) levels or functionally abnormal VWF. VWF is best known for its three classical hemostatic functions: (i) as a carrier protein for coagulation factor VIII, (ii) binding to exposed collagen upon vascular damage and (iii) as a mediator of the recruitment of platelets to sites of vascular injury. However, in recent years it has become clear that VWF has a versatile function beyond hemostasis, and has been implicated in several pathophysiological processes, such as tumor metastasis, angiogenesis, cell proliferation and inflammatory processes. So, apart from its evident role in hemostasis, it has become clear that VWF is a much more complex multifaceted protein then initially thought. Several aspects __ from synthesis, secretion and clearance, to functions in the circulation __ of this protein have been studied and the results are described in this thesis. These results will advance our knowledge about the mechanism(s) of VWF biosynthesis, clearance and its role in angiogenesis, which might lead to a more personalized treatment for von Willebrand disease and its complications in the future. Show less
Tissue Factor (TF) is a membrane protein that is responsible for the initiation of the coagulation. In addition to its coagulant activity, it can also signal through a member of G-protein coupled... Show moreTissue Factor (TF) is a membrane protein that is responsible for the initiation of the coagulation. In addition to its coagulant activity, it can also signal through a member of G-protein coupled receptor family, PARs, thus play a role in breast cancer growth and angiogenesis. The switch between signaling and coagulant TF regulated by the oxidation/reduction of an allosteric disulfide bond which resides in TF antigen. A decade ago, it was discovered that TF RNA can be alternatively spliced to form a soluble protein called Alternatively Spliced Tissue Factor (asTF). This protein is non-coagulant. However, it plays a major role in breast cancer growth via inducing cancer cell proliferation. The mechanism lying behind behind this phenomenon is asTF's capability to ligate integrins thus it can initiate integrin signaling. Moreover, both TF and asTF can synergize with estrogen pathway thus providing a complex regulation of breast cancer progression. Show less
The transforming growth factor (TGF)-_ signalling pathway plays a major role in angiogenesis. Aberration of the TGF-_ signalling cascade leads to abnormal remodelling and maturation of the... Show moreThe transforming growth factor (TGF)-_ signalling pathway plays a major role in angiogenesis. Aberration of the TGF-_ signalling cascade leads to abnormal remodelling and maturation of the primitive vascular plexus and decreased vessel wall integrity in adults. Targeted deletion of TGF-_ signalling receptors in mice, such as ALK1, ALK5, T_RII or endoglin, results in embryonic lethality due to impaired vascular development. In humans, mutations in ALK1, ALK5, T_RII or endoglin are associated with human vascular diseases such as HHT and pulmonary hypertension (PAH). Vascular endothelial growth factor (VEGF) is a multifunctional molecule that is involved in vascular growth and remodeling. Perturbation in VEGF signalling also contributes to the pathology of tumor angiogenesis and cardiovascular diseases in humans. This thesis is focused on the characterization of the crosstalk between the TGF-_ and VEGF signalling pathways, on EC function, the effect of bone morphogenetic protein (BMP)9 on EC function and the role of endoglin in VEGF-induced angiogenesis. The results of these studies may give us insights into the impacts/effects of these two angiogenic signalling cascades on EC function. This can be beneficial for the understanding of the etiology of certain vascular diseases and the development of new treatment modalities in the future Show less
Growth and progression of cervical carcinoma is dependent on a complex interaction between cervical carcinoma cells and composition of the extracellular matrix. For local progression as well as... Show moreGrowth and progression of cervical carcinoma is dependent on a complex interaction between cervical carcinoma cells and composition of the extracellular matrix. For local progression as well as metastasizing, the extracellular matrix needs to be rearranged creating space for tumor cells to expand and angiogenesis to secure supply of nutrients and oxygen and removal of waste products. The net result of all contributing factors will lead to either progression or degradation of cervical cancer. In this thesis the role of contributing factors is investigated, e.g. cytokines, chemokines, inflammatory cells, the role of extracellular matrix and angiogenesis Show less
Preeclampsia is a pregnancy-specific condition that originates in the placenta. Despite decades of research, its pathogenesis remains largely unknown. However, several risk factors for preeclampsia... Show morePreeclampsia is a pregnancy-specific condition that originates in the placenta. Despite decades of research, its pathogenesis remains largely unknown. However, several risk factors for preeclampsia have been identified, including a (family) history of preeclampsia, autoimmune disease and conditions associated with endothelial damage, including hypertension, diabetes mellitus and preexistent renal disease. This thesis aims to further investigate through which mechanisms these risk factors increase the risk for preeclampsia. It deals with both the genetic background of preeclampsia, as well as the role of complement activation in its pathogenesis. Finally, it touches upon the role of angiogenic factors in the development of preeclampsia. Show less
