This doctoral thesis is an effort to understand how lipid phase-separation induced by diacylglycerol analogues in lipid-based nanoparticles affects their in vivo behavior, leading to specific... Show moreThis doctoral thesis is an effort to understand how lipid phase-separation induced by diacylglycerol analogues in lipid-based nanoparticles affects their in vivo behavior, leading to specific nanoparticle-protein communications and selective cell targeting. By studying how lipid composition affects morphology and this in turn affects the nano-bio interface, a comprehensive picture and prediction of nanoparticle behavior and cell selectivity is provided. More specifically, liposomes containing diacylglycerol analogues are found to phase separate and to be able to specifically target subsets of endothelial cells in zebrafish embryos. The mechanism behind this selective targeting is the result of a triglyceride lipase mediated mechanism due to phase separation and lipid composition, and is conserved in higher organisms (mice). Moreover, mRNA-based lipid nanoparticles that contain diacylglycerol analogues exhibit the same selectivity which leads to cell-specific mRNA delivery and transfection. Show less
β-Lactamases are enzymes that can break down β-lactam substrates, such as antibiotics, preventing the use of these antibiotics for the treatment of various infectious diseases. However, some... Show moreβ-Lactamases are enzymes that can break down β-lactam substrates, such as antibiotics, preventing the use of these antibiotics for the treatment of various infectious diseases. However, some compounds, β-lactamase inhibitors, can block these enzymes allowing for possible treatments using a combination of antibiotic and inhibitor. BlaC is the β-lactamase of Mycobacterium tuberculosis, the bacteria that cause tuberculosis, and is used as a model for protein evolution. To understand if and how BlaC can develop resistance against certain inhibitors we studied the evolutionary adaptability of this enzyme. We used laboratory evolution and various biochemical techniques to characterize several mutations in BlaC and subsequently tested the effect of combining mutations. One of the findings is that BlaC can easily become less sensitive to the inhibitor sulbactam by partially blocking the entrance to the active site. Interestingly, this was accompanied by increased sensitivity to another inhibitor, avibactam, that could not be compensated for by other mutations.Generally, Escherichia coli bacteria are used to test the effects of BlaC variants in cells, as they are easy and safe to use in the lab. We show that results obtained for E. coli can be extrapolated to conditions that resemble tuberculosis disease in humans: the M. marinum infection model of zebrafish. All these findings are of interest for the future development of combination therapies to treat tuberculosis. Show less
Cardiovascular diseases are still a major concern for the global health. The main underlying pathology of this disease is atherosclerosis which is characterized by the accumulation of lipids and... Show moreCardiovascular diseases are still a major concern for the global health. The main underlying pathology of this disease is atherosclerosis which is characterized by the accumulation of lipids and immune cells in the arterial wall leading to a chronic local inflammation and lesion formation. In this thesis, we aimed to (1) validate the use of zebrafish in cholesterol metabolism and atherosclerosis research, (2) study the role of certain classes of scavenger receptors in lipoprotein uptake and cholesterol-based functions, and (3) validated two immune-based potential targets for atherosclerosis. Show less
In this thesis, I study 1) metabolic alterations in tuberculosis related to wasting syndrome in human patients as well as in rodent and fish animal models. 2) effects of the mutation of the leptin... Show moreIn this thesis, I study 1) metabolic alterations in tuberculosis related to wasting syndrome in human patients as well as in rodent and fish animal models. 2) effects of the mutation of the leptin gene on cachexia and diabetes in rodent and zebrafish animal models. 3) how tuberculosis infection and resulting metabolic reprogramming are dependent on leptin signaling in mice and zebrafish larvae. Show less
The work described in this dissertation contributes to a better mechanistic understanding of nanoparticles in vivo. To achieve that goal, we used the zebrafish as a highly predictive pre-screening... Show moreThe work described in this dissertation contributes to a better mechanistic understanding of nanoparticles in vivo. To achieve that goal, we used the zebrafish as a highly predictive pre-screening model of nanoparticles. This approach enables the investigation of the fundamental behavior of nanoparticles, correlation of the physicochemical properties of the formulated nanoparticles with their biodistribution and identification of important nano-bio interactions. Zebrafish established transgenic lines were used to study specific interactions. In addition, genetically modified zebrafish applying CRISPR/Cas9 were generated. These strategies not only show key mechanistic features of nanoparticles in circulation, but also promote the rational design of more efficient nanoparticles systems.After understanding the fundamental behavior of nanoparticles, this thesis describes the identification of a key interaction between stabilins receptors (expressed in liver sinusoidal endothelial cells) and nanoparticles. Next, the scope is changed to design nano-systems that target specific cell types showing liposomes capable of switching the surface charge in situ and in vivo using light as an external trigger and a rationally designed lipid nanoparticle formulation containing mRNA able to preferentially target the hepatic reticuloendothelial system. In addition, a phase-separated liposomes hijacking a lipase mediated transport to selectively target endothelial lipase in vivo was studied. Show less
In this thesis, two potential therapeutic targets for diabetic nephropathy were dentified and investigated. First, we show that glomerular clusterin is upregulated in diabetic nephropathy and... Show moreIn this thesis, two potential therapeutic targets for diabetic nephropathy were dentified and investigated. First, we show that glomerular clusterin is upregulated in diabetic nephropathy and demonstrated that recombinant clusterin protein can protect the podocytes against oxidative stress in vitro. Second, we reveal that hCN1 overexpression accelerated and aggravated diabetic nephropathy in BTBR ob/ob mice. We also studied two novel zebrafish models to investigate chronic kidney disease. We showed that lepb-/- adult zebrafish have the early signs of human diabetic nephropathy, and we demonstrated that ctns mutant adult zebrafish have the kidney pathologic features of human nephropathic cystinosis. Show less
This thesis focuses on two processes involved in fighting infections: metabolism and immune cell motility and navigation.Regarding metabolism, we present ZebraGEM 2.0, an improved whole-genome... Show moreThis thesis focuses on two processes involved in fighting infections: metabolism and immune cell motility and navigation.Regarding metabolism, we present ZebraGEM 2.0, an improved whole-genome scale metabolic reconstruction for zebrafish, that we used to study zebrafish metabolism upon infection with Mycobacterium marinum integrating gene expression data from control and infected zebrafish larvae. The chapters focusing on cell motility in response to the environment, revolve around the question of how the environmental inputs of cell-matrix interactions, cell-sized obstacles and cell-signalling upon wounding shape and guide cell motility. Show less
This thesis describes the possible applicability of mesenchymal stromal cells (MSCs) in the treatment of liver fibrosis. In CCL4 induced animal models for liver fibrosis, we showed that local... Show moreThis thesis describes the possible applicability of mesenchymal stromal cells (MSCs) in the treatment of liver fibrosis. In CCL4 induced animal models for liver fibrosis, we showed that local administration of MSCs after partial hepatectomy, results in a dose‐dependent on‐site amelioration of fibrosis. Furthermore, we compared the pro-regenerative and anti-fibrotic effects of four different subpopulations of MSCs, categorized on Endoglin (CD105) and VCAM (CD106) membrane expression. Our results showed that VCAM-positive subpopulations of MSCs are superior compared to VCAM-negative subpopulations in relation to their anti-fibrotic and pro-regenerative properties. In another study we showed that TAA induce liver fibrogenesis in zebrafish embryos through mechanisms similar to man and mice. In addition, we found that MSCs ameliorate fibrogenesis in this model.CRIPTO-1 is an (onco)foetal protein and is correlated to poor prognosis in HCC. The observations of our HCC study are suggestive for the existence of a more aggressive subgroup of HCCs recognized by their high CRIPTO-1 expression which also seems to be resistant to Sorafenib treatment. Cell survival and cell proliferation are some of the processes stimulated by CRIPTO-1, which are also known to be important during liver regeneration and fibrogenesis. We identified that multiple species show enhanced CRIPTO-1 during fibrogenesis and that elevated CRIPTO-1 plasma levels in humans with cirrhosis normalize after liver transplantation. Show less
Tuberculosis (TB) is the most prevalent bacterial infectious disease in the world, caused by the pathogen Mycobacterium tuberculosis (Mtb). In this study, we have used Mycobacterium marinum (Mm)... Show moreTuberculosis (TB) is the most prevalent bacterial infectious disease in the world, caused by the pathogen Mycobacterium tuberculosis (Mtb). In this study, we have used Mycobacterium marinum (Mm) infection in zebrafish larvae as an animal model for this disease to study the role of the myeloid differentiation factor 88 (Myd88), the key adapter protein of Toll-like receptors. Previously, Myd88 has been shown to enhance innate immune responses against bacterial infections, and in the present study, we have investigated the effect of Myd88 deficiency on the granuloma morphology and the intracellular distribution of bacteria during Mm infection. Our results show that granulomas formed in the tail fin from myd88 mutant larvae have a more compact structure and contain a reduced number of leukocytes compared to the granulomas observed in wild-type larvae. These morphological differences were associated with an increased bacterial burden in the myd88 mutant. Electron microscopy analysis showed that the majority of Mm in the myd88 mutant are located extracellularly, whereas in the wild type, most bacteria were intracellular. In the myd88 mutant, intracellular bacteria were mainly present in compartments that were not electron-dense, suggesting that these compartments had not undergone fusion with a lysosome. In contrast, approximately half of the intracellular bacteria in wild-type larvae were found in electron-dense compartments. These observations in a zebrafish model for tuberculosis suggest a role for Myd88-dependent signalling in two important phenomena that limit mycobacterial growth in the infected tissue. It reduces the number of leukocytes at the site of infection and the acidification of bacteria-containing compartments inside these cells. Show less
Glucocorticoids (GCs) are widely prescribed as anti-inflammatory drugs due to their well-established immunosuppressive effects. However, their utilization is severely limited by the occurrence of... Show moreGlucocorticoids (GCs) are widely prescribed as anti-inflammatory drugs due to their well-established immunosuppressive effects. However, their utilization is severely limited by the occurrence of side effects and drug resistance. Therefore, there is still a major need to investigate the molecular and cellular mechanisms underlying the effects of GCs. Zebrafish are increasingly used as an in vivo model system for studying the immune system, in particular the inflammatory response. In Chapter 2, an overview is provided of the available inflammation models in zebrafish, and how they are used to unravel molecular mechanisms underlying the inflammatory response and for testing of potential novel anti-inflammatory drugs, in particular GCs. In this thesis, we have used zebrafish model system to study molecular and cellular mechanisms of GC action on the immune system and to develop a model for in vivo screening of the anti-inflammatory effects as well as possible adverse effects of novel GC therapies. For this purpose, we have studied the effect of GCs on leukocyte migration and differentiation during an inflammatory response (Chapter 3), how GCs modulate the immune response to a mycobacterial infection (Chapter 4), and we have investigated targeting of GCs to inflamed tissue by liposomal delivery (Chapter 5). Show less
Lysosomal storage disorders (LSDs) are a group of orphan diseases characterized by lysosomal dysfunction or impaired lysosomal catabolism and affect collectively about 1 in 5000 live births. A... Show moreLysosomal storage disorders (LSDs) are a group of orphan diseases characterized by lysosomal dysfunction or impaired lysosomal catabolism and affect collectively about 1 in 5000 live births. A common LSD is Gaucher disease, which is characterized by a defect in glucocerebrosidase (GCase) degrading glucosylceramide (GlcCer) in lysosomes. In this thesis, the zebrafish is evaluated as vertebrate animal model for the investigation of lysosomal storage disorders, in particular Gaucher disease. Zebrafish are an appealing model organism to study genetic disorders with a high evolutionary conservation of genes and proteins compared to humans, easy maintenance and simple genetic and pharmacological manipulation. Zebrafish larvae are of particular use as zebrafish can generate hundreds of off-spring which have a rapid embryonal development, are transparent and fit in a 96-wells plate. In this thesis several biochemical and genetic techniques have been developed in order to 1) compare the catalytic features of zebrafish GCase with human GCase, 2) investigate the consequences of its defect in zebrafish larvae and adults as well as a concomitant defect in non-lysosomal GBA2 and 3) study the potential toxicity of excessive glucosylsphingosine during GCase deficiency as consequence of a defect in lysosomal acid ceramidase. GCase-deficient zebrafish showed similar symptoms and affected molecular mechanisms as patients and mouse models. Therefore the zebrafish offers exciting new possibilities to study molecular mechanisms underlying pathological processes during lysosomal hydrolase deficiencies. Show less
Prostate cancer (PCa) is one of the most prevalent cancer in males. Although the majority of the patients can benefit from the present clinical treatments, 20%-30% of the patients who originally... Show moreProstate cancer (PCa) is one of the most prevalent cancer in males. Although the majority of the patients can benefit from the present clinical treatments, 20%-30% of the patients who originally respond to the therapy still develop incurable, castration-resistance bone metastases, which is a main cause of death in PCa . In this thesis, I combined an advanced zebrafish xenograft model with in vitro cellular approaches and mice xenografts to study the early stage of PCa metastasis. Using this comprehensive esearch platform, I identified multiple key signaling pathways that play essential roles in promoting the onset of PCa metastatis. The pathways I discovered include Cripto-associated EMT plasticity, CDC-42-N-Wasp-Cortactin associated mechanosensing and mechanotransduction, microenvironment dependent NF-ĸB-Activin A signaling pathway, and AMPK-Autophagy dependent metabolic stress coping pathway. Show less
Hoeksma, J.; Zon, G.C.M. van der; Dijke, P. ten; Hertog, J. den 2020
Zebrafish models are well-established tools for investigating the underlying mechanisms of diseases. Here, we identified cercosporamide, a metabolite from the fungus Ascochyta aquiliqiae, as a... Show moreZebrafish models are well-established tools for investigating the underlying mechanisms of diseases. Here, we identified cercosporamide, a metabolite from the fungus Ascochyta aquiliqiae, as a potent bone morphogenetic protein receptor (BMPR) type I kinase inhibitor through a zebrafish embryo phenotypic screen. The developmental defects in zebrafish, including lack of the ventral fin, induced by cercosporamide were strikingly similar to the phenotypes caused by renowned small-molecule BMPR type I kinase inhibitors and inactivating mutations in zebrafish BMPRs. In mammalian cell-based assays, cercosporamide blocked BMP/SMAD-dependent transcriptional reporter activity and BMP-induced SMAD1/5-phosphorylation. Biochemical assays with a panel of purified recombinant kinases demonstrated that cercosporamide directly inhibited kinase activity of type I BMPRs [also called activin receptor-like kinases (ALKs)]. In mammalian cells, cercosporamide selectively inhibited constitutively active BMPR type I-induced SMAD1/5 phosphorylation. Importantly, cercosporamide rescued the developmental defects caused by constitutively active Alk2 in zebrafish embryos. We believe that cercosporamide could be the first of a new class of molecules with potential to be developed further for clinical use against diseases that are causally linked to overactivation of BMPR signaling, including fibrodysplasia ossificans progressiva and diffuse intrinsic pontine glioma.This article has an associated First Person interview with the first author of the paper. Show less
In this thesis, we will utilize embryonic zebrafish tumour models to understand the interaction between engrafted human cancer cells and macrophages from the host, test drug administration... Show moreIn this thesis, we will utilize embryonic zebrafish tumour models to understand the interaction between engrafted human cancer cells and macrophages from the host, test drug administration modalities and anti-cancer efficacies of newly-developed PDT and PACT compounds, and test a light-triggered liposomal system for targeted drug delivery specifically to cancer cells in vivo. In chapter 2, we investigate the role of macrophages in tumour-induced angiogenesis. We show that macrophage-dependent angiogenesis is driven by macrophage recruitment to lactic acid secreted by glycolytic B16 melanoma cells. Chemical inhibition of macrophages and glycolysis blocks the initiation of angiogenesis in these models, suggesting that macrophages attracted to glycolytic melanoma cells contribute to the tumour-induced angiogenesis process.In chapters 3 and 4, we explore novel PDT and PACT compounds, respectively, for treatment of conjunctival melanoma in zebrafish. We inject conjunctival melanoma cells into the retro-orbital site to establish an orthotopic model and into the Duct of Cuvier to generate an ectopic model. Our results prove that zebrafish provides a fast vertebrate cancer model to test the optimal administration regimen of drugs, conditions of light irradiation, host toxicity and anti-cancer efficacy of PDT and PACT drugs against conjunctival melanoma.In chapter 5, we focus on modifying liposomes to be light triggered in order to deliver drugs specifically to cancer cells. We inject MDA231 breast cancer cells into the Duct of Cuvier at 2 days post fertilization (dpf) to initiate metastasis to the CHT. We successfully demonstrate that light-triggered, cell-specific delivery of liposome-encapsulated doxorubicin reduces the xenograft cancer cell burden without enhanced cytotoxicity of the zebrafish embryos. In chapter 6, we summarize the novel anti-cancer strategies, which we have developed using zebrafish xenograft models. In the same chapter, we frame our findings in the current scientific landscape and discuss future perspectives. Show less
Verwilligen, R.A.F.; Bussmann, J.; Eck, M. van 2020
This thesis elucidated the possibility of manipulating BMP/TGFβ signaling to achieve inhibition of breast cancer metastasis, including boosting BMP signaling via blockade of the BMP antagonist... Show moreThis thesis elucidated the possibility of manipulating BMP/TGFβ signaling to achieve inhibition of breast cancer metastasis, including boosting BMP signaling via blockade of the BMP antagonist Grem1 extracellularly or via stimulation of small-molecule compounds intracellularly, preventing TGFβ signaling to allow accumulation of pro-oncogenic stimuli. We also highlight the importance of selecting appropriate cancer types when adopting dual inhibition of PD-L1 and TGFβ signaling. I hope my research will aid in more efficient clinical cancer therapies. Show less
The zebrafish is a promising vertebrate model organism in early drug discovery and development. Translation of pharmacological findings to higher vertebrates requires quantification of the... Show moreThe zebrafish is a promising vertebrate model organism in early drug discovery and development. Translation of pharmacological findings to higher vertebrates requires quantification of the underlying pharmacological and (patho)physiological processes. In this thesis, we therefore developed and integrated innovative experimental and computational methods for the successful quantification of 1) the internal exposure over time after waterborne drug treatment, 2) disease dynamics and drug-induced changes therein, and 3) between-species differences in disease mechanisms. The state-of-the-art methods that we developed included nanoscale blood sampling, sensitive LC-MS/MS methods for drugs and their isomers and metabolites, and three-dimensional microscopy, integrated with non-linear mixed effects modelling to quantify the pharmacological processes in this small vertebrate. This multidisciplinarity enabled quantification of internal drug exposure-response relationships, contributed to positioning the zebrafish in the preclinical drug development pipeline, and inspired continuous collaborations between experimental and computational scientists. Show less
Ren, J.; Smid, M.; Iaria, J.; Salvatori, D.C.F.; Dam, H. van; Zhu, H.J.; ... ; Dijke, P. ten 2019
The aim of this study was to investigate the hepatotoxic effect and its underlying mechanism of aloe emodin (AE). AE was docked with the targets of NF-kappa B inflammatory pathway and P53 apoptosis... Show moreThe aim of this study was to investigate the hepatotoxic effect and its underlying mechanism of aloe emodin (AE). AE was docked with the targets of NF-kappa B inflammatory pathway and P53 apoptosis pathway respectively by using molecular docking technique. To verify the results of molecular docking and further investigate the hepatotoxicity mechanism of AE, the zebrafish Tg (fabp10: EGFP) was used as an animal model in vivo. The pathological sections of zebrafish liver were analyzed to observe the histopathological changes and Sudan black B was used to study whether there were inflammatory reactions in zebrafish liver or not. Then TdT-mediated dUTP Nick-End Labeling (TUNEL) was used to detect the apoptotic signal of zebrafish liver cells, finally the mRNA expression levels as well as the protein expression levels of the targets in NF-kappa B and P53 pathways in zebrafish were measured by quantitative Real-Time PCR (qRT-PCR) and western blot. Molecular docking results showed that AE could successfully dock with all the targets of NF-kappa B and P53 pathways, and the docking scores of most of the targets were equal to or higher than that of the corresponding ligands. Pathological sections showed AE could cause zebrafish liver lesions and the result of Sudan black B staining revealed that AE blackened the liver of zebrafish with Sudan black B. Then TUNEL assay showed that a large number of dense apoptotic signals were observed in AE group, mainly distributed in the liver and yolk sac of zebrafish. The results of qRT-PCR and western blot showed that AE increased the mRNA and protein expression levels of pro-inflammatory and pro-apoptotic targets in NF-kappa B and P53 pathways. AE could activate the NF-kappa B inflammatory pathway and the P53 apoptosis pathway, and its hepatotoxic mechanism was related to activation of NF-.B-P53 inflammation-apoptosis pathways. Show less
