To improve the predictive capability of pre-clinical models and reduce the use of animal models in drug discovery and disease modelling, advanced in vitro models are being developed. These... Show moreTo improve the predictive capability of pre-clinical models and reduce the use of animal models in drug discovery and disease modelling, advanced in vitro models are being developed. These microphysiological systems (MPS) or “Organs-on-Chip” (OoC) are being developed to include all aspects of the human physiology to improve the in vitro cellular response. OoCs combined with differentiated human induced pluripotent stem cells (hiPSC) allow the use of cells with patient specific genotypes and aid the development of personalized and precision medicine.In this thesis, the development of tractable models of the vasculature is described. These models allow for the combination of hiPSC-derived vascular and tissue specific cells with haemodynamics to recapitulate essential stimuli of blood vessels. Show less
Ebola virus can trigger a release of pro-inflammatory cytokines with subsequent vascular leakage and impairment of clotting finally leading to multiorgan failure and shock after entering and... Show moreEbola virus can trigger a release of pro-inflammatory cytokines with subsequent vascular leakage and impairment of clotting finally leading to multiorgan failure and shock after entering and infecting patients. Ebola virus is known to directly target endothelial cells and macrophages, even without infecting them, through direct interactions with viral proteins. These interactions affect cellular mechanics and immune processes, which are tightly linked to other key cellular functions such as metabolism. However, research regarding metabolic activity of these cells upon viral exposure remains limited, hampering our understanding of its pathophysiology and progression. Therefore, in the present study, an untargeted cellular metabolomic approach was performed to investigate the metabolic alterations of primary human endothelial cells and M1 and M2 macrophages upon exposure to Ebola virus-like particles (VLP). The results show that Ebola VLP led to metabolic changes among endothelial, M1, and M2 cells. Differential metabolite abundance and perturbed signaling pathway analysis further identified specific metabolic features, mainly in fatty acid-, steroid-, and amino acid-related metabolism pathways for all the three cell types, in a host cell specific manner. Taken together, this work characterized for the first time the metabolic alternations of endothelial cells and two primary human macrophage subtypes after Ebola VLP exposure, and identified the potential metabolites and pathways differentially affected, highlighting the important role of those host cells in disease development and progression. Show less
BackgroundThree-dimensional (3D) human brain spheroids are instrumental to study central nervous system (CNS) development and (dys)function. Yet, in current brain spheroid models the limited... Show moreBackgroundThree-dimensional (3D) human brain spheroids are instrumental to study central nervous system (CNS) development and (dys)function. Yet, in current brain spheroid models the limited variety of cell types hampers an integrated exploration of CNS (disease) mechanisms.MethodsHere we report a 5-month culture protocol that reproducibly generates H9 embryonic stem cell-derived human cortical spheroids (hCSs) with a large cell-type variety.ResultsWe established the presence of not only neuroectoderm-derived neural progenitor populations, mature excitatory and inhibitory neurons, astrocytes and oligodendrocyte (precursor) cells, but also mesoderm-derived microglia and endothelial cell populations in the hCSs via RNA-sequencing, qPCR, immunocytochemistry and transmission electron microscopy. Transcriptomic analysis revealed resemblance between the 5-months-old hCSs and dorsal frontal rather than inferior regions of human fetal brains of 19–26 weeks of gestational age. Pro-inflammatory stimulation of the generated hCSs induced a neuroinflammatory response, offering a proof-of-principle of the applicability of the spheroids.ConclusionsOur protocol provides a 3D human brain cell model containing a wide variety of innately developing neuroectoderm- as well as mesoderm-derived cell types, furnishing a versatile platform for comprehensive examination of intercellular CNS communication and neurological disease mechanisms. Show less
A bicuspid aortic valve (BAV) is a congenital heart defect in which the heart valve between the left ventricle and the aorta consists of two valve flaps instead of the normal three (tricuspid... Show moreA bicuspid aortic valve (BAV) is a congenital heart defect in which the heart valve between the left ventricle and the aorta consists of two valve flaps instead of the normal three (tricuspid aortic valve – TAV). In a large proportion of people with a BAV, calcification of the heart valves or a widening of the aorta occurs early in life. It is not yet clear why patients with a BAV are prone to develop valvular calcification and aortic dilatation. The aim of the research described in this thesis is to study the pathogenesis of aortic valve calcification and aortic dilatation in BAV patients with a focus on the role of endothelial cells in these processes. With the research described in this thesis, we have shown that endothelial cells from BAV patients respond differently than cells from people with a TAV. In addition, the endothelial cell activation in the vascular wall of BAV patients is different and dependent on the blood flow. We found two good tissue culture methods to study heart valve calcification and used them to study the role of the protein FHL2 in this process. 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
Netrin-4, recognized in neural and vascular development, is highly expressed by mature endothelial cells. The function of this netrin-4 in vascular biology after development has remained unclear.... Show moreNetrin-4, recognized in neural and vascular development, is highly expressed by mature endothelial cells. The function of this netrin-4 in vascular biology after development has remained unclear. We found that the expression of netrin-4 is highly regulated in endothelial cells and is important for quiescent healthy endothelium. Netrin-4 expression is upregulated in endothelial cells cultured under laminar flow conditions, while endothelial cells stimulated with tumor necrosis factor alpha resulted in decreased netrin-4 expression. Targeted reduction of netrin-4 in endothelial cells resulted in increased expression of vascular cell adhesion molecule 1 and intercellular adhesion molecule 1. Besides, these endothelial cells were more prone to monocyte adhesion and showed impaired barrier function, measured with electric cell-substrate impedance sensing, as well as in an ?organ-on-achip? microfluidic system. Importantly, endothelial cells with reduced levels of netrin-4 showed increased expression of the senescence-associated markers cyclin-dependent kinase inhibitor-1 and -2A, an increased cell size and decreased ability to proliferate. Consistent with the gene expression profile, netrin-4 reduction was accompanied with more senescent associated ?-galactosidase activity, which could be rescued by adding netrin-4 protein. Finally, using human decellularized kidney extracellular matrix scaffolds, we found that pre-treatment of the scaffolds with netrin-4 increased numbers of endothelial cells adhering to the matrix, showing a pro-survival effect of netrin-4. Taken together, netrin-4 acts as an anti-senescence and anti-inflammation factor in endothelial cell function and our results provide insights as to maintain endothelial homeostasis and supporting vascular health. Show less
In recent years, the study of endothelial cell (EC) metabolism has led to the discovery of novel regulatory mechanisms and potential new targets for vascular-related diseases. Despite the fact that... Show moreIn recent years, the study of endothelial cell (EC) metabolism has led to the discovery of novel regulatory mechanisms and potential new targets for vascular-related diseases. Despite the fact that ECs have readily available oxygen in the blood, they mainly generate ATP via anaerobic glycolysis rather than Krebs cycle. In the context of atherosclerosis, there has been growing interest in understanding how EC metabolism affects plaque formation and intraplaque (IP) angiogenesis, which has been identified as a contributing factor for plaque vulnerability in human atherosclerosis.Among the enzymes involved in glycolytic flux modulation, PFKFB3 plays a critical role for the proliferation and migration of ECs. PFKFB3 is upregulated in atheroprone regions of arterial vessels and in carotid plaques of patients with elevated levels of lipoprotein(a). The experimental work of this thesis focuses on PFKFB3 as a modulator of EC glycolysis and its effects on atherosclerosis progression and IP angiogenesis.In the first part of this thesis, a pharmacological study with partial glycolysis inhibitor 3PO in the context of advanced atherosclerotic plaques is described. 3PO treatment restrains IP angiogenesis and plaque frequency but it does not affect plaque size and composition in ApoE-/-Fbn1C1039G+/- mice. In addition, a 3PO-mediated reduction in plaque formation is also observed in ApoE-/- mice that develop plaques without IP neovascularization.Furthermore, EC-specific PFKFB3 deletion leads to a significant reduction in plaque size, IP angiogenesis and hemorrhagic complications in a vein graft model. These findings suggest that endothelial glycolysis inhibition may represent a new therapeutic strategy to slow down plaque progression in vein grafts.A study performed in collaboration with the University of Aberdeen is also presented in this thesis. Here the development of a new PFKFB3-targeted PET radiotracer, [18F]ZCDD083, for in vivo plaque imaging is described. The specificity of the tracer for atherosclerotic plaques is demonstrated by a combination of ex vivo autoradiography and en face Oil Red O staining. This tracer is a promising non-invasive diagnostic tool to detect rupture-prone atherosclerotic plaques,Finally, a novel imaging method for a three-dimensional reconstruction of the IP vessel network is presented. This method is based on iDISCO immunolabeling and confocal microscopy. It may represent a novel tool to investigate the causal relationship between IP angiogenesis and atherogenesis.Overall, the experimental data generated in this thesis strongly argue for a critical role of EC metabolism in the formation and progression of atherosclerosis in addition to IP angiogenesis. Show less
Heart failure is a major health care problem with high mortality. Although advances have been made in treatment of patients suffering from heart failure with reduced ejection fraction, this is not... Show moreHeart failure is a major health care problem with high mortality. Although advances have been made in treatment of patients suffering from heart failure with reduced ejection fraction, this is not true for patients suffering from heart failure with preserved ejection fraction. The mechanism underlying heart failure with preserved ejection fraction is still unclear. Recent evidence suggests that factors circulating in blood might have an effect on the microvessels, including those in the heart. To diagnose and treat microvascular diseases, we aim to explore the association of circulating plasma factors with microvascular integrity. As current human 2D models with cultured endothelial cells lack sufficient complexity to assess the function of microvascular endothelial-pericyte interactions, research on microvascular loss largely depends on animal models. To mimic the microarchitecture and functions of the human blood vessel in a more efficient way for drug discovery, we developed the microvessel-on-a-chip. This system allowed us to screen microvascular destabilization factors in blood and study the efficacy of potential drugs for microvascular diseases. In conclusion, our platform may serve as a unique tool for microvascular destabilization studies as well as for the development of novel therapeutic strategies to combat microvascular complications. Show less
Abnormal vascular physiology and precipitating inflammatory pathways underlie many different diseases, including hemorrhage, stroke, vascular dementia and even cancer. Pluripotent stem cells (PSCs)... Show moreAbnormal vascular physiology and precipitating inflammatory pathways underlie many different diseases, including hemorrhage, stroke, vascular dementia and even cancer. Pluripotent stem cells (PSCs) can now be derived by reprogramming from any individual so that it is possible in principle to derive all somatic cells of the human body that would normally be difficult to access. In this thesis, I studied the derivation of myeloid cells from human induced pluripotent stem cells (hiPSCs) to model the inflammatory component of vascular disease and characterized the development path of hiPSC-derived endothelial cells (hiPSC-ECs) which form the vascular walls. Functional defects in either of these cell types can cause or exacerbate vascular disease. I then used these cell types to gain insight into the mechanisms underlying two genetic diseases: Hereditary Hemorrhagic Telangiectasia (HHT) which is caused by mutations in a gene called Endoglin expressed on cells of the vascular wall and inflammatory macrophages, and a vascular tumor called Pseudomyogenic hemangioendothelioma (PHE) in which endothelial cells are thought to be the tumor cell of origin. I developed new differentiation protocols to generate inflammatory cells from hiPSC, characterized these cells functionally and used Next-Generation Sequencing and bioinformatic analysis to gain insight into the molecular pathways controlling development of one particular type of endothelial cells from hiPSC and the underlying tumorigenic mechanisms of PHE. Show less
Von Willebrand disease (VWD) is the most common inherited bleeding disorder. Patients mainly develop mucocutaneous bleeding, like bruises, epistaxis and menorrhagia. The more severely affected... Show moreVon Willebrand disease (VWD) is the most common inherited bleeding disorder. Patients mainly develop mucocutaneous bleeding, like bruises, epistaxis and menorrhagia. The more severely affected patients may also develop joint bleeding, or bleeding from the gastrointestinal tract. Also, trauma, surgery or dental procedures may lead to critical bleeding events. VWD-related bleeding are caused by defects in von Willebrand factor (VWF), a large multimeric protein that is produced by endothelial cells and megakaryocytes. Most VWD patients develop the disease because of dominant-negative mutations in VWF. In this thesis we investigated whether inhibition of production of mutant VWF with limited inhibition of wildtype VWF positively affects the function of VWF and improves VWD phenotypes. We used small interfering RNAs (siRNAs) to selectively inhibit production of mutant VWF. These siRNAs were tested in several models for VWD. We indeed prove that siRNAs can distinguish a mutant and wildtype VWF allele in vitro in heterologous cell systems, ex vivo in patient-derived endothelial cells, and in vivo in a VWD mouse model. We also show in these disease models that we can improve several VWD phenotypes. These results are promising for further development of allele-specific siRNAs as a new treatment strategy for VWD. Show less
Koning, M.; Berg, C.W. van den; Rabelink, T.J. 2019
Kidney organoids can be generated from human pluripotent stem cells (PSCs) using protocols that resemble the embryonic development of the kidney. The renal structures thus generated offer great... Show moreKidney organoids can be generated from human pluripotent stem cells (PSCs) using protocols that resemble the embryonic development of the kidney. The renal structures thus generated offer great potential for disease modeling, drug screening, and possibly future therapeutic application. At the same time, use of these PSC-derived organoids is hampered by lack of maturation and off-target differentiation. Here, we review the main protocols for the generation of kidney organoids from human-induced PSCs, discussing their advantages and limitations. In particular, we will focus on the vascularization of the kidney organoids, which appears to be one of the critical factors to achieve maturation and functionality of the organoids. Show less
One of the major limitations in culturing complex tissues or organs is the lack of vascularization in the cultured tissue. Development of a functional capillary bed could overcome this problem.... Show moreOne of the major limitations in culturing complex tissues or organs is the lack of vascularization in the cultured tissue. Development of a functional capillary bed could overcome this problem. The zebrafish is a promising model for in vitro vasculogenesis and angiogenesis studies, as a replacement for currently used mammalian models. However, the culture of endothelial cells from this species is not well characterized. Here, we test different culture strategies, medium supplementations and culture substrates for their effect on the generation of putative endothelial (fli:GFP+ and kdrl:GFP+) cells and vascular morphogenesis in zebrafish blastocyst cell derived embryoid body culture. we have also developed a perfused culture model, using microfluidic technology, to culture zebrafish vascular networks. This study is a step forward to the development of zebrafish vascular networks in vitro. Show less
Solingen, C. van; Bijkerk, R.; Boer, H.C. de; Rabelink, T.J.; Zonneveld, A.J. van 2015
Blood coagulation is vital for life by reducing blood loss upon injury. One of the key players in the initial steps of coagulation is Von Willebrand Factor (VWF). VWF is mainly produced and stored... Show moreBlood coagulation is vital for life by reducing blood loss upon injury. One of the key players in the initial steps of coagulation is Von Willebrand Factor (VWF). VWF is mainly produced and stored by the cells that line the blood vessels and functions upon secretion by creating a platelet plug at the side of injury. Mutations in VWF can disturb this process, for instance through insufficient VWF secretion, which causes Von Willebrand disease, a hereditary bleeding disorder characterized by prolonged bleeding. As the morphology of VWF is highly related to its functionality, we aimed to obtain more knowledge on the mechanisms that can be affected in Von Willebrand disease by studying the normal lifecycle of VWF using correlative light and electron microscopy (CLEM). CLEM combines fluorescent light microscopy with electron microscopy on the same sample to obtain structural information of specific biological events that are difficult to study with electron microscopy alone. Using CLEM, novel information was obtained regarding the formation of the VWF storage organelle, in addition we also discovered novel pathophysiological mechanisms acting during VWF secretion that may be effectuated in Von Willebrand disease. Show less
Aims High-fat diet-induced obesity (DIO) is amajor contributor to type II diabetes and micro-andmacro-vascular complications leading to peripheral vascular disease (PVD). Metabolic abnormalities of... Show moreAims High-fat diet-induced obesity (DIO) is amajor contributor to type II diabetes and micro-andmacro-vascular complications leading to peripheral vascular disease (PVD). Metabolic abnormalities of induced pluripotent stemcell-derived endothelial cells (iPSC-ECs) fromobese individuals could potentially limit their therapeutic efficacy forPVD. The aimof this studywas to compare the function of iPSC-ECs from normal and DIO mice using comprehensive in vitro and in vivo assays.Methods and results Six-week-old C57Bl/6 micewere fed with a normal or high-fat diet. At 24weeks, iPSCs were generated fromtail tip fibroblasts and differentiated into iPSC-ECs using a directed monolayerapproach. In vitro functional analysis revealed that iPSCECs from DIO mice had significantly decreased capacity to form capillary-like networks, diminished migration, and lower proliferation. Microarray and ELISA confirmed elevated apoptotic, inflammatory, and oxidative stress pathways in DIO iPSC-ECs. Following hindlimb ischaemia, mice receiving intramuscular injections of DIO iPSC-ECs had significantly decreased reperfusion compared with mice injected with control healthy iPSC-ECs. Hindlimb sections revealed increased muscle atrophy and presence of inflammatory cells in mice receiving DIO iPSC-ECs. When pravastatin was co-administered to mice receiving DIO iPSC-ECs, a significant increase in reperfusionwas observed; however, this beneficial effect was blunted by co-administration of the nitric oxide synthase inhibitor, Nv-nitro-L-arginine methyl ester.Conclusion This is the first study to provide evidence that iPSC-ECs from DIO mice exhibit signs of endothelial dysfunction and have suboptimal efficacy following transplantation in a hindlimb ischaemia model. These findings may have important implications for future treatment of PVD using iPSC-ECs in the obese population. Show less
This thesis describes a role for microRNAs in kidney (patho)physiology. First, it is shown that miR-155 negatively regulates RhoA signaling in TGF-induced endothelial-to-mesenchymal-transition ... Show moreThis thesis describes a role for microRNAs in kidney (patho)physiology. First, it is shown that miR-155 negatively regulates RhoA signaling in TGF-induced endothelial-to-mesenchymal-transition (EndoMT). EndoMT associates with reorganization of the cytoskeleton and production of extracellular matrix, and is potentially involved in the loss of microvascular capillaries in renal fibrosis. Second, we focused on ischemia/reperfusion-injury (IRI), a central event in conditions such as acute kidney injury and organ transplantation, where the renal peritubular capillary network is considered the primary site of injury. Consequently, capillary integrity is a key determinant for preservation of renal function. Systemic silencing of miR-126 impairs ischemia-induced angiogenesis, and affects mobilization of vasculogenic progenitor cells, potentially by modulating SDF-1 expression. Furthermore, overexpression of miR-126 in the hematopoietic compartment protects against renal IRI by promoting vascular integrity. We identified miR-132 to be involved in renal fibrosis. Lineage analysis of kidney stroma showed that pericytes are the major source of myofibroblasts Silencing miR-132 results in decreased pericyte differentiation towards myofibroblasts. Subsequently we demonstrated that miR-132 regulates diuresis by altering Aquaporin-2 localization in collecting ducts, which is responsible for water-reabsorption, by modulating vasopressin- and prostaglandin-dependent pathways. This thesis illustrates the essential role that microRNAs play in kidney health and disease. Show less
