Aims/hypothesis: Renal GLUT2 is increased in diabetes, thereby enhancing glucose reabsorption and worsening hyperglycaemia. Here, we determined whether loss of Glut2 (also known as Slc2a2)... Show moreAims/hypothesis: Renal GLUT2 is increased in diabetes, thereby enhancing glucose reabsorption and worsening hyperglycaemia. Here, we determined whether loss of Glut2 (also known as Slc2a2) specifically in the kidneys would reverse hyperglycaemia and normalise body weight in mouse models of diabetes and obesity. Methods: We used the tamoxifen-inducible CreERT2-Lox system in mice to knockout Glut2 specifically in the kidneys (Ks-Glut2 KO) to establish the contribution of renal GLUT2 to systemic glucose homeostasis in health and in insulin-dependent as well as non-insulin-dependent diabetes. We measured circulating glucose and insulin levels in response to OGTT or IVGTT under different experimental conditions in the Ks-Glut2 KO and their control mice. Moreover, we quantified urine glucose levels to explain the phenotype of the mice independently of insulin actions. We also used a transcription factor array to identify mechanisms underlying the crosstalk between renal GLUT2 and sodium-glucose cotransporter 2 (SGLT2). Results: The Ks-Glut2 KO mice exhibited improved glucose tolerance and massive glucosuria. Interestingly, this improvement in blood glucose control was eliminated when we knocked out Glut2 in the liver in addition to the kidneys, suggesting that the improvement is attributable to the lack of renal GLUT2. Remarkably, induction of renal Glut2 deficiency reversed hyperglycaemia and normalised body weight in mouse models of diabetes and obesity. Longitudinal monitoring of renal glucose transporters revealed that Sglt2 (also known as Slc5a2) expression was almost abolished 3 weeks after inducing renal Glut2 deficiency. To identify a molecular basis for this crosstalk, we screened for renal transcription factors that were downregulated in the Ks-Glut2 KO mice. Hnf1 alpha (also known as Hnf1a) was among the genes most downregulated and its recovery restored Sglt2 expression in primary renal proximal tubular cells isolated from the Ks-Glut2 KO mice. Conclusions/interpretation: Altogether, these results demonstrate a novel crosstalk between renal GLUT2 and SGLT2 in regulating systemic glucose homeostasis via glucose reabsorption. Our findings also indicate that inhibiting renal GLUT2 is a potential therapy for diabetes and obesity. Show less
Veneuze trombose is een ziekte gekenmerkt door het ontstaan van een ongewenst bloedstolsel. Om inzichten te verkrijgen in de pathofysiologie van dit ziektebeeld zijn voorheen genoomstudies... Show moreVeneuze trombose is een ziekte gekenmerkt door het ontstaan van een ongewenst bloedstolsel. Om inzichten te verkrijgen in de pathofysiologie van dit ziektebeeld zijn voorheen genoomstudies uitgevoerd. Deze studies hebben een nieuwe erfelijke factor voor veneuze trombose geïdentificeerd, namelijk het SLC44A2 gen. Dit was een opmerkelijke bevinding, aangezien SLC44A2 nooit eerder gekoppeld was aan de bloedstolling. Het onderzoek beschreven in dit proefschrift heeft als doel het mechanisme, onderliggend aan deze associatie, te ontrafelen. Er is hiervoor gebruik gemaakt van muizen die dit gen niet meer hebben, zogenaamde SLC44A2 knock-out muizen. Dierstudies maakten het mogelijk om de complexiteit van stromend bloed, de bloedvaatwand en het ontstaan van veneuze trombose nader te onderzoeken. We hebben aangetoond dat de afwezigheid van SLC44A2 de normale bloedstolling ongemoeid laat. De vorming van veneuze trombose in SLC44A2 knock-out muizen is echter afwijkend, met mogelijk betrokkenheid van neutrofielen en von Willebrand factor (VWF), een eiwit met een rol in veneuze trombose. Studies verricht aan SLC44A2 op de neutrofielen van mensen, met aandacht voor de twee verschillende vormen van SLC44A2 die bij mensen voorkomen, geven goede aanwijzingen dat SLC44A2 een rol speelt in de binding van neutrofielen aan VWF. Er werd ook geobserveerd dat de erfelijke variant van SLC44A2 op neutrofielen welke zwak bindt aan VWF, ook degene is die samengaat met een mindere kans op het krijgen van VT. Door deze studies begrijpen we beter hoe veneuze trombose ontstaat, met hopelijk in de nabije toekomst concrete aanknopingspunten voor alternatieve, betere en veiligere behandelingsstrategieën voor veneuze trombose. Show less
Muscular dystrophies (MDs) encompass a wide variety of inherited disorders that are characterized by loss of muscle tissue associated with a progressive reduction in muscle function. With a cure... Show moreMuscular dystrophies (MDs) encompass a wide variety of inherited disorders that are characterized by loss of muscle tissue associated with a progressive reduction in muscle function. With a cure lacking for MDs, preclinical developments of therapeutic approaches depend on well-characterized animal models that recapitulate the specific pathology in patients. The mouse is the most widely and extensively used model for MDs, and it has played a key role in our understanding of the molecular mechanisms underlying MD pathogenesis. This has enabled the development of therapeutic strategies. Owing to advancements in genetic engineering, a wide variety of mouse models are available for the majority of MDs. Here, we summarize the characteristics of the most commonly used mouse models for a subset of highly studied MDs, collated into a table. Together with references to key publications describing these models, this brief but detailed overview would be useful for those interested in, or working with, mouse models of MD. Show less
During my research project we mainly focussed on studying the pathophysiology of venous and arterial thrombosis in mice. When we transiently lowered plasma protein levels of natural anticoagulants... Show moreDuring my research project we mainly focussed on studying the pathophysiology of venous and arterial thrombosis in mice. When we transiently lowered plasma protein levels of natural anticoagulants antithrombin and protein C using RNA interference, mice developed venous thrombosis in the head. In contrast to other mouse models for venous thrombosis where surgery is required for provoking the disease, mice injected with RNA interference against the mRNA of Serpinc1 and Proc (antithrombin and protein C, respectively) developed venous thrombosis without additional handlings. In this unique form of venous thrombosis, we studied the roles of platelets, neutrophils, and coagulation factor XII. These factors have been shown to be indispensable in experimental venous thrombosis in other mouse models, and they have been introduced as novel therapeutic targets. For the second part of my thesis we again used the RNA interference approach, to lower natural anticoagulation in atherosclerotic mice. When we lowered protein C in these mice, they developed atherothrombosis in the aortic root without any additional intervention. This unique form of atherothrombosis has been showed in multiple independent experiments, and we aimed to further characterize the process to learn more about prevention atherothrombosis in atherosclerotic mice and the role of protein C. Show less
The muscular dystrophies are a heterogeneous collection of inherited and progressive myogenic disorders sharing similar clinical “dystrophic” characteristics. Many therapies are currently... Show moreThe muscular dystrophies are a heterogeneous collection of inherited and progressive myogenic disorders sharing similar clinical “dystrophic” characteristics. Many therapies are currently being tested in clinical trials and several of them achieved marketing authorisation. Additionally, many promising treatments are currently being tested in preclinical animal models. At a pathological level, patients show chronic inflammation and degeneration of muscle fibers, which eventually are replaced by adipose and fibrotic tissue leading to loss of muscle tissue and function. Targeting pathways involved in regulation of muscle regeneration and fibrosis can improve muscle quality and function. This would be beneficial for many patients with neuromuscular disorders, which share similar pathology. There is a great demand to accelerate the translation of potential new treatments from the bench to the clinic. Therefore, natural history studies and reliable outcome measures are required for preclinical mouse models improving study design and comparison between studies. To address the lack of a therapy targeting muscle pathology, the thesis focuses on evaluating therapeutic potential of antisense oligonucleotides inhibition of TGF-β and myostatin type I receptors and elucidating their function in muscles. Additionally, this thesis contains a natural history dataset that can be useful to design preclinical studies in dystrophic mouse models. Show less
Abdominal aortic aneurysms (AAAs) are potentially lethal due to rupture. Rupture occurs mainly in AAA greater than 55mm and acute repair still results in mortality over 30%. Although the... Show moreAbdominal aortic aneurysms (AAAs) are potentially lethal due to rupture. Rupture occurs mainly in AAA greater than 55mm and acute repair still results in mortality over 30%. Although the results of elective treatment have significantly improved over the years and mortality is low (<3%), there is a considerable risk of morbidity. AAAs are prevalent mostly in elderly patients and generally only progress slowly in size. Therefore, treatment that slows aneurysm growth would allow patients to avoid aneurysm repair, in particularly elderly patients. Insight into the pathophysiology of the disease has improved over the past few years and continuing research has led the focus towards finding pharmaceutical means to inhibit or even abrogate aneurysm growth. The aim of this thesis was to identify new possible targets for pharmacological treatment of AAAs and to apply this insight to the development of new therapies in a preclinical setting. Besides, understanding the cause of AAA progression can help identify secondary prevention strategies aimed at slowing down expansion. Show less
Duchenne muscular dystrophy (DMD) is the most prevalent neuromuscular disorder, caused by mutations in the DMD gene that prevent synthesis of dystrophin. Fibers that lack dystrophin are sensitive... Show moreDuchenne muscular dystrophy (DMD) is the most prevalent neuromuscular disorder, caused by mutations in the DMD gene that prevent synthesis of dystrophin. Fibers that lack dystrophin are sensitive to exercise-induced damage, resulting in progressive muscle wasting, loss of ambulation and premature death. There is no cure, but several therapeutic approaches are clinically tested. At best, these clinical interventions result in the expression of low dystrophin levels. Fortunately, expression of wild type levels is not needed, as both humans and mice expressing ~50% of dystrophin do not show pathology. Detailed studies on which dystrophin levels are needed to prevent pathology and improve muscle function have been performed in this thesis. After the set-up of good outcome measures and serum biomarkers to monitor disease progression, two new innovative mouse models expressing low levels of dystrophin based on skewed X-inactivation were generated. In the mdx-Xist__hs model we observed that <15% dystrophin already improved muscle performance, while histopathology was largely with >15% dystrophin. To protect muscles from exercise-induced damage >22% dystrophin was needed. Dystrophin levels between 3-21% prevent the development of dilated cardiomyopathy in 10 months old mice. Mice lacking both dystrophin and its homologue utrophin, mimic the human phenotype and die before the age of 12 weeks. In these mice, <10% dystrophin improved life expectancy and muscle function while >10% dystrophin was needed to improve histopathology. These findings are encouraging for ongoing and future clinical trails. Show less
Allogeneic hematopoietic stem cell transplantation (alloSCT) is a potentially curative treatment for various hematological malignancies. The beneficial Graft-versus-Leukemia (GvL) effect of alloSCT... Show moreAllogeneic hematopoietic stem cell transplantation (alloSCT) is a potentially curative treatment for various hematological malignancies. The beneficial Graft-versus-Leukemia (GvL) effect of alloSCT is mediated by donor-derived allo-reactive T cells targeting the malignant cells of the patient. Unfortunately, detrimental Graft-versus-Host-Disease (GvHD) often co-develops due to recognition of allo-antigens by donor-derived T cells on non-hematopoietic tissues. To prevent the development of GvHD, donor T cells can be depleted from the alloSCT graft. Although the risk and severity of GvHD are effectively reduced by T cell depleted (TCD) alloSCT, the absence of donor T cells in the stem cell graft also leads to an increased risk of relapses of malignancies. Early intervention with unmanipulated donor lymphocyte infusion (DLI) may effectively prevent or treat post-transplant relapses, but is frequently associated with re-introduction of GvHD. Although post-transplant relapses of chronic myeloid leukemia (CML) in chronic phase can be effectively treated with DLI, patients with relapsed acute leukemia often fail to respond to DLI and their prognosis remains poor. This disease-specific difference in efficacy of DLI may be explained by the poor capacity of acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML) and CML in blast crisis (CML-BC) to function as professional antigen presenting cells (APC) and induce primary T cell responses. In addition, acute leukemias are generally rapidly proliferating aggressive malignancies that may outpace the donor T cell responses as induced after DLI. Therefore, novel T cell based immunotherapeutic strategies with potent efficacy, but limited treatment-related toxicity are highly relevant to improve the clinical outcome for patients with aggressive acute malignancies. In contrast to constitutive expression of HLA-class I molecules on all nucleated cells, HLAclass II molecules are only constitutively expressed on normal hematopoietic cells as well as most B-lineage and myeloid malignancies, while most non-hematopoietic cells only express HLA-class II under inflammatory conditions. Therefore, CD4+ T cells recognizing allo-antigens in the context of HLA-class II molecules under non-inflammatory circumstances are likely to mediate selective GvL reactivity without GvHD. Clinical application of HLA-identical DLI depleted of CD8+ T cells has demonstrated to induce conversion to donor hematopoiesis and disease remissions in patients with relapsed malignancies, in the absence of induction of severe GvHD. In addition, it has been demonstrated that allo-HLA-DPB1 specific CD4+ T cells can mediate profound GvL reactivity in the absence of clinically significant GvHD after DLI following HLA-DPB1 mismatched TCD alloSCT, indicating that allo-HLA-class II molecules can also serve as malignancy-specific targets. Therefore, a possible strategy to induce profound and selective GvL immunity against acute leukemia without causing GvHD may be achieved by targeting (disparate) HLA-class II molecules by CD4+ T cell based immunotherapy following TCD alloSCT. This thesis explored the benefits and threats of this approach in a preclinical mouse model and in patients. Show less
In dit proefschrift wordt onderzoek beschreven waarin een aantal door lipiden gereguleerde genen op bepaalde witte bloedcellen (macrofagen) zijn onderzocht voor nieuwe behandelmethoden ter... Show moreIn dit proefschrift wordt onderzoek beschreven waarin een aantal door lipiden gereguleerde genen op bepaalde witte bloedcellen (macrofagen) zijn onderzocht voor nieuwe behandelmethoden ter voorkoming of behandeling van atherosclerose (slagaderverkalking). Dit wordt gedaan met behulp van de beenmergtransplantatie techniek, waarmee specifiek witte bloedcellen __ waaronder ook macrofagen __ worden vervangen. De in het proefschrift beschreven genen die met behulp van de beenmergtransplantatietechniek zijn onderzocht zijn: ATP-binding cassette transporter (ABC)A1, ABCG1, apolipoproteine (apo)E, en adipose triglyceride lipase (ATGL). Deze genen worden gereguleerd door lipiden en spelen een belangrijke rol in de lipiden homeostase van cellen. Allereerst bleek dat ABCA1 en apoE in macrofagen op verschillende wijze een beschermende werking hebben met betrekking tot atherosclerose, en dat ze gezamenlijk ontstekingsremmend werken. In overeenstemming met deze resultaten bleken ABCG1 en apoE in macrofagen geheel onafhankelijk van elkaar een beschermende rol te spelen in de ontwikkeling van atherosclerose. Afwezigheid van ATGL __ verantwoordelijk voor de afbraak van vetten - in macrofagen leidde verrassenderwijs tot een vermindering van atherosclerose. De afwezigheid van ABCA1 bleek te beschermen tegen een acute hartaanval. Daarnaast bleek dat macrofaag ABCA1 veranderingen in de milt teweegbrengt, maar dat dit geen gevolgen heeft voor de ontwikkeling van atherosclerose. Show less
A mesenchymal stem cell (MSC) based osteosarcoma model was established. The model provided evidence for a MSC origin of osteosarcoma. Normal MSCs transformed spontaneously to osteosarcoma-like... Show moreA mesenchymal stem cell (MSC) based osteosarcoma model was established. The model provided evidence for a MSC origin of osteosarcoma. Normal MSCs transformed spontaneously to osteosarcoma-like cells which was always accompanied by genomic instability and loss of the Cdkn2a locus. Accordingly loss of human CDKN2A/p16 protein expression in patients__ samples identified a subgroup of patients which did not respond to the chemotherapy and all showed poor survival (< 50 months). Moreover the CDKN2A/p16 gene expression loss in the human samples was caused by the same mechanism as in the mice cells, i.e. homozygous genomic deletion, validating the mouse model. Subsequently other useful models were identified by extensive characterization of osteosarcoma cell lines. From analyses of important signal transduction pathways (Wnt, BMP, TGF_ and Ihh) candidate targets for therapy, like a GSK3_-inhibitor, were identified. Furthermore a novel zebrafish model was established to study the progression of normal MSCs towards osteosarcoma-like cells in a high throughput manner. In this model both the behavior of the cells as well as the response of the host involved in processes like angiogenesis and migration were studied. The exploration of osteosarcoma at different levels was essential to broaden our knowledge about its origin, etiology and behavior. A better understanding of this complex malignancy offers more accurate targets to hit. Moreover it provides better opportunities to model the tumor which in turn allows for discovering and assaying novel treatment strategies Show less
Over the years, a number of acquired risk factors for venous thrombosis have been identified in large epidemiological studies. We aimed to identify the biological mechanisms by which acquired risk... Show moreOver the years, a number of acquired risk factors for venous thrombosis have been identified in large epidemiological studies. We aimed to identify the biological mechanisms by which acquired risk factors like female hormones, thyroid hormone and obesity result in a hypercoagulable state and increased risk for venous thrombosis, since these are currently poorly understood. As these risk factors are all, to a certain extent, able to interfere with liver metabolism we hypothesized that they modulate hepatic transcription of coagulation genes, either directly via nuclear hormone receptors and hormone response elements in target genes (female hormones and thyroid hormone), or indirectly as a result of altered liver homeostasis (obesity). To study these hypotheses, we used an in vivo approach, which not only gives the opportunity to study the risk factor-mediated transcriptional modulation of coagulation genes, but also allowed us to study the relation between transcriptional changes on the one hand and plasma protein levels and a thrombotic tendency on the other. The data presented in this thesis clearly demonstrate that modulation of hepatic coagulation gene transcription is a key mechanism by which acquired risk factors for venous thrombosis impact the hemostatic balance. Show less
This dissertation deals with the role of local immune stimulation in the lymph node and tumor microenvironment and its effect on systemic CD8+ T cell responses, in particular the anti-tumor CD8+ T... Show moreThis dissertation deals with the role of local immune stimulation in the lymph node and tumor microenvironment and its effect on systemic CD8+ T cell responses, in particular the anti-tumor CD8+ T cell responses. In chapter 2 the use of a slow-release system is described to deliver the immune-activating agonistic CD40 antibody to the tumor-draining area, and the advantages of this method over systemic administration of the antibody. The local, slow-release administration was very effective in activating a systemic anti-tumor effector CD8+ T cell response, to such an extent that a tenfold lower dose of antibody could be used without loss of efficacy. Adverse side-effects, analyzed by organ histology and liver enzymes in the blood, were much lower upon local anti-CD40 antibody delivery compared to systemic administration. The local delivery of anti-CD40 antibody resulted in a systemic anti-tumor CD8+ T cell response, capable of clearing distant tumors expressing identical tumor antigens. Chapter 3 shows that slow-release local administration of CTLA-4 blocking antibody can also activate a tumor-specific CD8+ T cell response and cause tumor regression, while lowering systemic adverse side-effect as compared to systemic administration. CTLA-4 blocking antibody is being widely used in clinical trials, and its use has been complicated by induction of auto-immune disease. Here we show that using a local low dose injection of CTLA-4 blocking antibody in a slow-release formulation is equally effective in activating a tumor-specific CD8+ T cell response, capable of eradicating tumor cells as systemic high dose treatment. The influence of local lymph node activation on systemic T cell responses is further analyzed in chapter 4. CD8+ T cell priming generally occurs in a locally inflamed lymph node, called a reactive LN, due to the presence of pathogens. The role of the inflammatory milieu on the priming and fate of CD8+ T cells was studied by separating the TCR-MHC interaction from the inflammatory cues, by priming briefly in vitro followed by transfer to mice with or without a CpG-induced reactive lymph node. The primary CD8+ T cell response was not influenced by the presence of a reactive lymph node, however, after a boost vaccination in the memory phase, CD8+ T cells primed in the presence of a reactive LN displayed a strong quantitative advantage over control CD8+ T cells. The reactive LN, which remained swollen with enhanced cellularity for a pronounced period of time, was envisaged to act as a shelter for CD8+ T cells while undergoing contraction after the primary response. In chapter 5, the advantages and disadvantages of the use of dextran-based microparticles as slow-release system for the delivery of immune-activating antibodies such as agonistic CD40 in the tumor-draining area are described. Dextran-based microparticles can be tailored to release antibodies in desired pharmacokinetics, leading to an even further decrease of adverse side-effects, as compared to previously described Montanide-ISA 51. However, dextran-based particles were unexpectedly found to have a stimulating effect on tumor-outgrowth. This effect coincided with the appearance of large, ulcerated swellings at the site of injection. In chapter 6, the issues presented in this thesis are discussed. The knowledge gained in the work shown here, compared with and strengthened by related published work, is used to state the opinion that targeting the tumor-draining lymph node and/or tumor microenvironment for immune-activating therapy against tumors must be seriously considered. Show less
In this thesis we analyzed the pathogenetic sequence of renal cyst and aneurysm formation in PKD mouse models. Cyst formation in the mouse model, Pkd1nl/nl with reduced Pkd1 transcripts was studied... Show moreIn this thesis we analyzed the pathogenetic sequence of renal cyst and aneurysm formation in PKD mouse models. Cyst formation in the mouse model, Pkd1nl/nl with reduced Pkd1 transcripts was studied by analyzing tubular cell proliferation, apoptosis, extracellular matrix remodeling, and the expression the Na+K+2Cl- co-transporter. These mice presented with a rapidly progressive phenotype and, in time, cyst formation was nephron segment-dependent. Interestingly, the expression of Na+K+2Cl- co-transporters was also segment-dependent. TGF__ signaling, the most important cytokine involved in fibrosis, is activated at the progressive stages of the disease and coincides with mild fibrosis and increased expression of TGF__ target genes. These results suggest that the TGF__ signaling pathway is probably not implicated in initial steps of cyst formation, but indicate an important role during cyst progression and in fibrogenesis of progressive ADPKD. Aneurysm formation was found in the Pkd1nl/nl, indicating a direct association between Pkd1 and vessel wall integrity. But, selective disruption of Pkd1 in vascular smooth muscle cells (SM22-Pkd1del/de micel) did not induce any gross structural blood vessel abnormalities. However, SM22-Pkd1del/del mice significantly showed reduced decrease in heart rate upon Angiotensin II-induced hypertension. These findings further demonstrate in vivo, that adaptation to hypertension is altered in SM22-Pkd1del/del mice. Show less
In this thesis the role of murine FcgammaR in autoimmune diseases is analyzed with special focus on arthritis and lupus. Series of studies performed with a variety of arthritis models established... Show moreIn this thesis the role of murine FcgammaR in autoimmune diseases is analyzed with special focus on arthritis and lupus. Series of studies performed with a variety of arthritis models established in mice deficient for one or more FcgammaR provided direct evidence for a prominent role of FcgammaR in arthritis (Chapter 2). Chapters 3 to 5 provide new insights into the role of individual FcgammaR in arthritis pathology. The precise contribution of FcgammaRIIB to the development of autoimmunity has been a matter of debate. In Chapter 6, we provide direct evidence that FcgammaRIIB on its own plays a limited role in the development of spontaneous autoimmunity in C57Bl/6 mice. Our results suggest that the role of FcgammaRIIB in the development of spontaneous autoimmunity (Systemic Lupus Erythematosus) is more restricted as originally postulated on the basis of the phenotype of FcgammaRIIB129-/- mice, whereas its role in induced autoimmunity is prominent, supporting the hypothesis that FcgammaRIIB represents a late checkpoint in B cell tolerance. In Chapter 7 we describe the generation and characterization of a novel B cell-specific inducible Cre transgenic mouse line. The results presented in this thesis provide new insights in the role of FcgammaRs in protective immunity and immuno-pathology. Show less
