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
An active pool of __Tissue Factor__ (TF) is required to initiate blood coagulation, but the mechanism that regulates the activation of TF is highly debated. The reduced coagulant activities of both... Show moreAn active pool of __Tissue Factor__ (TF) is required to initiate blood coagulation, but the mechanism that regulates the activation of TF is highly debated. The reduced coagulant activities of both human and mouse disulfide-mutated TF indicates that the formation of a Cys186-Cys209 or Cys190-Cys213 disulfide bond in the extracellular domain of TF controls its procoagulant function. Also the strong evolutionary conservation of this allosteric disulfide bond supports previous assumption. Procoagulant active TF is also found intravascularly on small vesicles (microparticles), and high levels of microparticle-associated TF is thought to increase the risk for thrombosis in cancer patients. Despite the fact that chemotherapy is an independent risk factor for thrombosis, chemotherapy-induced tumor destruction was not associated with an increase in microparticle-associated TF in testis cancer patients. The soluble isoform of TF, alternatively spliced TF (asTF), induces angiogenesis through interaction with integrins and independent of intracellular protease-activated receptor (PAR)-mediated signaling. PAR-2 but not PAR-1 plays a role in arteriogenesis in vivo. Furthermore, PAR-2 is involved in the anti-inflammatory response that may promote arteriogenesis. Show less
This thesis details our studies assessing the role of the endothelial-enriched miRNA-126 in the regulation of vascular homeostasis. In Chapter 2 the current insight in the role of miRNA-126 in... Show moreThis thesis details our studies assessing the role of the endothelial-enriched miRNA-126 in the regulation of vascular homeostasis. In Chapter 2 the current insight in the role of miRNA-126 in vascular homeostasis is reviewed. Chapter 3 focuses on the role of miRNA-126 in ischemia induced angiogenesis, followed by Chapter 4 which describes the potential role of miRNA-126 the mobilization of vasculogenic progenitor cells upon ischemia. Both chapters utilize antagomir-technology to specifically silence miRNA-126 in vivo. This approach to silence miRNA-126 was also used in Chapter 5 to elucidate the regulatory role of miRNA-126 in vascular cell adhesion molecule-1 expression in the kidney vasculature. Chapter 6 details our findings that circulating miRNA-126 in the periphery is not exclusively derived from endothelial cells but can also originate from platelets. Consequently, the use of aspirin has to be taken into account when relating circulating miRNA-126 levels to the progression of cardiovascular disease. Chapter 7 demonstrates that the angiogenic potential of miRNA-126 as described in Chapter 3 might reach beyond the presence of this pro-angiogenic miRNA in endothelium, but that neovascularization can also be supported by miRNA-126 expressed in circulating cells. Finally, Chapter 8 provides a summary of research presented in this thesis, presents the major conclusions that could be drawn and further discusses the role of miRNA-126 in vascular homeostasis. Show less
The aim of this work was to develop methods to measure structural changes in the skeleton using MicroCT. In addition, these new methods should be able to quantify biologically relevant changes. In... Show moreThe aim of this work was to develop methods to measure structural changes in the skeleton using MicroCT. In addition, these new methods should be able to quantify biologically relevant changes. In order to do this, normalized methods to analyse MicroCT scans and perform quantitative measurements within these datasets are described in this thesis. These techniques were combined with a biological angiogenesis assay and used as research tools in a study comparing various different combination treatments of bone metastases. Show less
Uveal melanoma (UM) is a disease with many faces: ophthalmologists treat the primary tumor, but the patient faces the problem of developing metastases, which are often deadly after a short period.... Show moreUveal melanoma (UM) is a disease with many faces: ophthalmologists treat the primary tumor, but the patient faces the problem of developing metastases, which are often deadly after a short period. Recent insight, indicates the need for knowledge-based treatment of UM. The __pseudohypoxic__ and tumor promoting effects of bevacizumab as described in this thesis is especially relevant. Bevacizumab is frequently used off-label to treat macular edema in UM patients suffering of radiation retinopathy, without the knowledge of possible effect on still viable UM cells. We further observed that specific peptides, such as UMAP1, which can successfully be internalized by targeted UM cells, have demonstrated potential for UM-targeted treatment. These peptides have to be investigated in vivo, to ascertain whether they are a viable clinical tool. Labeling the peptides with radionuclides, and demonstrating specificity for UM cells are some of the challenges which have to be overcome. Another aspect in the patient-specific treatment of UM is the in vitro analysis of primary UM samples to predict treatment responses. In case of Dasatinib, we describe treatment responses associated with monosomy 3 and __kinase__ activity in different primary UM samples. Show less
In peripheral arterial occlusive disease (PAOD), the formation of an atherosclerotic lesion eventually results in a significant stenosis of a major artery thereby disrupting blood flow in... Show moreIn peripheral arterial occlusive disease (PAOD), the formation of an atherosclerotic lesion eventually results in a significant stenosis of a major artery thereby disrupting blood flow in peripheral arteries towards lower limb tissue. Unfortunately, there is a substantial number of patients that suffer from severe critical limb ischemia, which is associated with a poor prognosis and high rates of amputation and mortality. Despite state-of-the-art revascularization treatment options and optimal control of co-morbidities these 'no option' patients remain at high risk for limb amputation. It is these patients that require new therapeutic applications to augment neovascularization and prevent them from limb amputation. Expansion of our current knowledge is required to gain insight in cellular and molecular mechanisms that are involved in neovascularization, to optimize revascularization therapies for patients with critical limb ischemia. This thesis provides a role for multiple leukocytes in blood flow recovery, in particular Natural Killer cells and CD4+ T lymphocytes, and shows that genetic differences in the Natural Killer gene complex induce differences in vascular remodeling. It was also shown how growth factor expression, signaling and regulation of gene expression modulate revascularization. Show less
Peripheral artery disease (PAD) remains a major cause of morbidity and mortality in the Western world. Therapeutic strategies to obtain revascularization of affected limbs are frequently needed in... Show morePeripheral artery disease (PAD) remains a major cause of morbidity and mortality in the Western world. Therapeutic strategies to obtain revascularization of affected limbs are frequently needed in these patients. Unfortunately, the initial success of surgical and/or endovascular revascularization can be threatened by progressive disease ultimately leading to an amputation. A better understanding of the complex cellular and molecular processes involved in post-ischemic neovascularization may reveal novel options for therapeutic interventions for PAD patients. This thesis describes reports that contribute to an increase in the knowledge and insights into different cell types involved in collateral artery growth in limb ischemia with the aim of developing new therapeutic strategies for PAD. Show less
The thesis is based on clinical research. Studies presented in the first part concentrate on the role of radiation therapy to reduce or to prevent (re)stenosis after percutaneous transluminal... Show moreThe thesis is based on clinical research. Studies presented in the first part concentrate on the role of radiation therapy to reduce or to prevent (re)stenosis after percutaneous transluminal angioplasty (PTA) and arteriovenous fistulas. After reviewing the concept, a multicenter study is reported in which the efficacy of endovascular brachytherapy (EBT) for prophylaxis of restenosis after femoropopliteal PTA is evaluated. Further, a randomised trial is discussed that assessed the presumed preventive effect of external beam radiation therapy on anastomotic intimal hyperplasia in prosthetic arteriovenous fistulas in patients with renal failure. The second part of this thesis focuses on stimulation of vascular growth in patients with severe limb ischemia who are without regular surgical or endovascular treatment options. After reviewing the concept, a clinical study evaluated feasibility and safety as well as efficacy of autologous cell therapy (bone marrow). The angiographic results of this study and hypotheses on the clinical usefulness of digital subtraction angiography in the assessment of the effects on possible inducement of collateral circulation after cell-based therapy are separately considered. In conclusion: The modest effect of vascular radiotherapy is too cumbersome for widespread clinical implementation. Cell therapy in vascular patients is promising. The mechanisms are not completely elucidated, but the ultimate success however will rest on its ability to demonstrate clinical efficacy. Show less
In this thesis we report novel studies on the molecular regulation of the transcriptional repressor Tel (Translocation Ets Leukemia). The work in this thesis is presented as follows: Chapter 1 is... Show moreIn this thesis we report novel studies on the molecular regulation of the transcriptional repressor Tel (Translocation Ets Leukemia). The work in this thesis is presented as follows: Chapter 1 is an introduction which summarizes the literature about Tel and its Drosophila orthologue Yan as it was known prior to the work presented here. Chapter 2 shows that Tel is modified by SUMO (Small Ubiquitin-like Modifier) on the highly conserved lysine 11 (K11), which serves to inhibit DNA binding of Tel. Chapter 3 describes the regulation of Tel and Yan by the F-box protein Fbl6, which mediates ubiquitination and subsequent degradation of Tel/Yan. Chapter 4 reports that Tel regulates angiogenesis through recruitment of the generic corepressor C-terminal Binding Protein (CtBP). This complex integrates intracellular Vascular Endothelial Growth Factor (VEGF) signaling and intercellular Delta-like 4 (Dll4)/Notch signaling. The impact of these findings is discussed in the general discussion in Chapter 5. Taken together the work in this thesis provides significant advances in the molecular details of Tel regulation. Furthermore, the work in chapter 4 should be of considerable importance to the field of angiogenesis. Show less
Vascularised, receptive endometrium is essential for implantation and for the success of the embryo-maternal interaction. Disturbances in vascular development may play an important role in... Show moreVascularised, receptive endometrium is essential for implantation and for the success of the embryo-maternal interaction. Disturbances in vascular development may play an important role in frequently occurring pathologies during pregnancy, such as early pregnancy wastage, pre-eclampsia and intrauterine growth restriction. Adaptation to implantation starts during the menstrual cycle by stromal decidualisation and the induction of angiogenesis, the formation of new vessels from existing vasculature. These processes continue throughout the 1st trimester. Angiogenesis is a multi-step process involving degradation of the basal membrane, activation, migration of endothelial cells and finally the formation of capillaries and the recruitment of pericellular smooth muscle cells. Numerous factors are involved in these processes. Three distinct groups of angiogenic factors, namely membrane bound proteases, vascular growth factors and its receptors and angiopoietins and its receptor, were analysed in this thesis. Pericellular proteolysis plays an important role in angiogenesis being required for endothelial cell migration, invasion and tube formation. In chapter 2 we studied the expression of proteases, matrix-metalloproteinase (MMPs) and uPA, by human endometrial microvascular endothelial cells (hEMVEC) and their involvement in the formation of capillary tubes in vitro. Inhibition of uPA and MMPs both reduced tube formation. hEMVEC expressed various MMPs mRNAs and proteins; in particular MMP-1, MMP-2, MT1-, MT3- and MT4-MMP. Immunohistochemistry confirmed the presence of MT3-MMP in endothelial cells of endometrial tissue. Overexpression of TIMP-1 or TIMP-3 by adenoviral transduction of hEMVEC reduced tube formation to the same extent. In addition, tube formation by hEMVEC was partly inhibited by the presence of anti-MT-3-MMP IgG. Thus, tube formation by hEMVEC is, at least in part, regulated by MT3-MMP. The study described in chapter 3 analyses the presence of MT-MMPs in human endometrium and their correlation with endometrial neovascularisation. The data show that all MT-MMP antigens are expressed in endometrium in a cycle-dependent pattern. The vascular expression of MT2- MT3- and MT4-MMP correlated with angiogenic episodes of the cycle. Since MT2- and MT3-MMP are known to regulate capillary-like tube formation, these findings support earlier in vitro data on the regulatory role of MT3-MMP in endometrial angiogenesis. Additionally, MT2-MMP appears to be associated with endometrial neovascularisation as well. Chapter 4 focuses on the role of proteases in decidual angiogenesis and remodelling in general. The expression of membrane-type matrix metalloproteinases (MT-MMPs) and urokinase/urokinase receptor (uPA/uPAR) were studied in decidua basalis (DB), decidua parietalis (DP) and decidual secretory endometrium (DSE) of human 1st trimester pregnancies. This enabled evaluation of decidual expression of the proteases, their regulation by pregnancy-induced hormones and/or the extra-villous trophoblast (EVT) and their relationship with decidual remodelling, trophoblast invasion and vascularisation. Pericellular-acting proteases appeared to be regulated by both pregnancy-induced hormones and EVT; uPAR and MT1-MMP by pregnancy-induced hormones and uPA, MT2-, MT3-, and MT5-MMP by EVT. All proteases were expressed by the EVT themselves and might be involved in trophoblast invasion. MT2- and MT3-MMP protein expression in endothelium was reduced in DB. Since these MT-MMPs are known regulators of angiogenesis, they may not only support decidual remodelling and trophoblast invasion but also partially regulate vascularisation at the embryonic implantation site. Chapter 5 describes the effect of human embryo-conditioned medium on in vitro endometrial angiogenesis. The conditioned media were shown to contain significant amounts of VEGF-A and stimulated in vitro tube formation by hEMVEC. This inducing effect was counteracted by adding soluble VEGF-R1. The other mediators, which have been described as produced by the early embryo/trophoblast, for instance Il-10, TGF_, PlGF, hCG and TGF_ had no effect on tube formation by hEMVEC. In conclusion, we show that the human embryo is able, via VEGF-A, to stimulate in vitro endometrial angiogenesis at the time of implantation. Chapter 6 evaluates vascular adaptation to implantation. We studied vascularisation and the expression of angiogenic factors in decidua basalis (DB), decidua parietalis (DP) and decidual secretory endometrium (DSE) of 1st trimester pregnancies. This enabled not only evaluation of decidual vascularisation and the expression of VEGF-A, PlGF, Flt-1, KDR, Angiopoietin-1 (Ang-1), Angiopoietin-2 (Ang-2) and TIE-2, but also their regulation by pregnancy-induced hormones and/or the extra-villous trophoblast (EVT). Both pregnancy-induced hormones and EVT influence vascularisation by enhancing vascular and luminal surface and reducing vessel density at the implantation site. These changes correlate with differences in gene and protein expression. PlGF mRNA and PlGF and Flt-1 protein expressions were elevated in DB under influence of EVT. Also the angiopoietins were differentially expressed, in favour of Ang-2, in DB. These data suggest that PlGF, Flt-1, Ang-1 and Ang-2 may be regulators of angiogenesis at the implantation site. Chapter 7 describes vascular adaptation to implantation in Missed Abortions by studying vascularisation and the expression of angiogenic factors and proteases in decidua basalis (DB), decidua parietalis (DP) and decidual secretory endometrium (DSE) of 1st trimester pregnancies which are complicated by a missed abortion. This enabled evaluation of decidual vascularisation, expression of VEGF-A, PlGF, Flt-1, KDR, Angiopoietin-1 (Ang-1), Angiopoietin-2 (Ang-2), TIE-2, uPA, uPAR, MT1-, MT2-, MT3- and MT5-MMP and their regulation by pregnancy-induced hormones and/or the extra-villous trophoblast (EVT). In Chapter 8, the expression of angiogenic factors in the 1st trimester, obtained during chorion villus sampling, were correlated with the pregnancy outcome. This was performed to study the hypothesis that early imperfections in the formation of the placental bed may result in pre-eclampsia, eclampsia or intra uterine growth retardation (IUGR). The mRNA levels of VEGF-A, PlGF, Flt-1, KDR, Angiopoietin-1 (Ang-1), Angiopoietin-2 (Ang-2) and TIE-2 did not show a correlation with pregnancy outcome. This chapter is incorporated as a preliminary report since the provided samples were not sufficient but we do think this study is of value to the thesis. In Chapter 9 the results of the studies of this thesis are discussed and Chapter 10 gives a final conclusion and recommendations for future research. Show less
Vascular maladaptation prior and during implantation may lead to serious complications during pregnancy, perinatally, but also later in life (Barker hypothesis). The consequences later in life... Show moreVascular maladaptation prior and during implantation may lead to serious complications during pregnancy, perinatally, but also later in life (Barker hypothesis). The consequences later in life often appear to be related to endothelial dysfunction. Angiogenesis, the formation of new blood vessels from pre-existing ones, is an important endothelial function and plays a key role in the process of implantation and placentation. Two epidemiological studies described here, show that myocardial infarction is related to low birth weight and that assisted procreation adversely affects birth weight. An optimal intra-uterine environment forms the basis for a good perinatal outcome and is created by a receptive endometrium in which angiogenesis is crucial. To study endometrial angiogenesis, human endometrial endothelial cells were isolated. The high expression of u-PA by these cells was found to contribute to their high angiogenic properties. Furthermore, these cells depend on MT3-MMP to form tubes. The ovarian steroids overall regulate endometrial angiogenesis indirectly via the endometrial stromal cells. During implantation, the embryo takes over as the main (local) regulator by inducing angiogenesis at its implantation site through the expression of VEGF. These results provide more insight in the (patho-)physiology of endometrial angiogenesis and in the role of the embryo in this. Show less
Aderverkalking (atherosclerose) is een ziekte waarbij door verdikking van de vaatwand vernauwing van slagaderen optreedt. Door deze vernauwing is het mogelijk dat er te weinig zuurstofrijk bloed de... Show moreAderverkalking (atherosclerose) is een ziekte waarbij door verdikking van de vaatwand vernauwing van slagaderen optreedt. Door deze vernauwing is het mogelijk dat er te weinig zuurstofrijk bloed de organen bereikt. In het geval van atherosclerose in slagaderen die hart of hersen van bloed moeten verzien leidt de vernauwing niet zelden tot de dood. Het feit dat atherosclerose de belangrijkste onderliggende oorzaak is van wereldwijde sterfte onderstreept het maatschappelijk belang van ontwikkeling van een adequate therapie tegen atherosclerose. In dit proefschrift werden nieuwe vaccinatietechnieken aangewend om het proces van atherosclerose tegen te gaan. Door de tolerantie voor lichaamseigen stoffen of cellen te doorbreken met deze vaccinatietechnieken bleek het mogelijk te zijn atherosclerose in muizen sterk af te remmen. Het wegnemen van de functie van ontstekingsbevorderende eiwitten (interleukinen 12 en 17) met deze vaccinatietechieken leidde tot een 70-90% afname in atherosclerose. Ook het met vaccinatie specifiek opruimen van cellen die betrokkken zijn bij atherosclerose-geassocieerde processen, zoals nieuwvaatvorming, bleek een geschikte methode te zijn om atherosclerose tegen te gaan. Het voordeel van de nieuwe vaccinatietechnieken die bij dit onderzoek zijn gebruikt is gelegen in het feit dat met enkele injecties voor lange tijd (ten minste 24 weken) bescherming tegen atherosclerose bewerkstelligd kan worden. Andere voordelen van deze vaccinaties zijn het gebrek aan afweerreacties tegen lichaamsvreemde medicijnen en de lage productiekosten van dergelijke vaccins. De combinatie van de sterke afname in atherosclerose en de bijkomende voordelen van de vaccinatietechnieken leidde in dit proefschrift tot de conclusie dat vaccinatie een belangrijke bijdrage kan leveren aan de ontwikkeling van therapieën tegen hart- en vaatziekten in mensen. Show less
This thesis describes several topics on angiogenesis and screening in uveal melanoma, the most frequent eye tumour in adults. The expression of vascular endothelial growth factor and other... Show moreThis thesis describes several topics on angiogenesis and screening in uveal melanoma, the most frequent eye tumour in adults. The expression of vascular endothelial growth factor and other angiostimulating factors in this tumour are described. In a second part, the use of different screening tests in screening for metastases of uveal melanoma is investigated. Show less
