Endoglin is an auxiliary receptor for ligands of TGF-β receptor superfamily, present in endothelial cells and the placental syncytiotrophoblast. The expression of placental membrane endoglin (mEng) is further increased during preeclampsia, a pregnancy-specific hypertensive syndrome. We hypothesize that the soluble form of endoglin (sEng) released from the placenta leads to endothelial dysfunction and hypertension by disrupting normal BMP-9 signaling. We show that cell surface mEng inhibits TGF-β1, BMP-2, and BMP-7 induced Smad1,5,8 phosphorylation while potentiating BMP-9 induced signaling. sEng has been shown to be elevated in the sera of preeclamptic women and is postulated to interfere with endothelial cell function. We show that sEng binds to BMP-9 with a 2 nM affinity and can compete for its binding to endothelial cells, inhibiting downstream Smad1,5,8 phosphorylation. Our results suggest that sEng is contributing to endothelial dysfunction by dysregulating BMP-9 signaling.

Endoglin is an auxiliary receptor for ligands of TGF-β receptor superfamily, present in endothelial cells and the placental syncytiotrophoblast. The expression of placental membrane endoglin (mEng) is further increased during preeclampsia, a pregnancy-specific hypertensive syndrome. We hypothesize that the soluble form of endoglin (sEng) released from the placenta leads to endothelial dysfunction and hypertension by disrupting normal BMP-9 signaling. We show that cell surface mEng inhibits TGF-β1, BMP-2, and BMP-7 induced Smad1,5,8 phosphorylation while potentiating BMP-9 induced signaling. sEng has been shown to be elevated in the sera of preeclamptic women and is postulated to interfere with endothelial cell function. We show that sEng binds to BMP-9 with a 2 nM affinity and can compete for its binding to endothelial cells, inhibiting downstream Smad1,5,8 phosphorylation. Our results suggest that sEng is contributing to endothelial dysfunction by dysregulating BMP-9 signaling.

Charles University in Prague Faculty of Pharmacy in HradecKrlov Department of Biological and Medical Sciences Evaluation of endoglin and P-selectin expression and co-expression in aortas of apoE-deficient mice Diploma thesis MonikaBrlicov Supervisor: Mgr.JanaRathousk Background: We observed the expression and the reciprocal co-expression of endoglin (receptor III for TGF- cytokine) and P-selectin (adhesion molecule and marker of endothelial dysfunction) in ascending aortas of apoE-deficient mice which were fed by standard diet for rodents and Western type diet (high-cholesterol diet) for achieving of different phases of the atherosclerotic process. The changes of total cholesterol levels in mice after administration of both types of diets were also evaluated. Methods: The modified strain C57BL/6J of mice with a deficiency of apolipoprotein E, which is prone to aterogenesis was used for this diploma thesis. Mice were divided into three groups. The first group was fed by standard diet (so-called "chow" diet) for a period of two months and the second two groups were fed by Western type diet for a period of two and four months. The levels of total cholesterol in the blood were biochemically determinated and then we statistically evaluated this levels in all groups. Immunohistochemical...

Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Evaluatiton of the expresion and co-expresion of endoglin and VCAM-1 in the aorta on apoE- deficient mice Diploma thesis Lucie Minaříková Supervisor: Mgr. Jana Rathouská Background: The aim of this work was to monitor the expression and a possible co-expression of endoglin (TGF-β receptor III) and VCAM-1 in mouse aortic endothelium. As an experimental model, we used the mouse strain C57BL/6J, that was genetically modified, and is characteristic by a deficit of apolipoprotein E. Methodes: In the study, we focused on testing the mouse strain C57BL/6J with gene knockout for apolipoprotein E in different stages of the atherosclerotic process. 10 weeks old female mice were divided into three groups and fed diets with different content of cholesterol. One experimental group was fed a standard diet (called "chow type diet") for a period of two months. The other two groups were fed a diet containing 21% fat (called "Western type diet") for a period of two and four months. For the determination of the levels of total cholesterol, a biochemical analysis of blood was performed. Obtained parts of ascending aorta were analyzed by ImmPRESSTM immunohistochemical method with the detection reagent DAB....

Preeclampsia is a pregnancy related disorder, characterized by proteinuria and hypertension. The pathogenesis of preeclampsia is poorly understood; however, two proteins, called sFlt-1 and sEng, were found to be highly elevated in the maternal plasma weeks prior to the onset of clinical symptoms. sFlt-1 and sEng are thought to inhibit VEGF and TGF-β receptor signalling respectively. In order to elucidate how these proteins may contribute to preeclampsia, we looked at their ability to affect the secretion of endothelin-1 (ET-1), a powerful vasoconstrictor, shown to be dysregulated in preeclampsia. We found that both TGF-β1 and BMP9, a recently described ligand for sEng, induce ET-1 secretion through Smad1/5/8 and p38 pathways. Moreover, we report that sEng and VEGF can efficiently block ET-1 secretion, induced by BMP9. We propose that the balance between VEGF and BMP9 signalling is disturbed during preeclampsia, leading to excessive release of ET-1, which in turn may cause hypertension.

Preeclampsia is a pregnancy related disorder, characterized by proteinuria and hypertension. The pathogenesis of preeclampsia is poorly understood; however, two proteins, called sFlt-1 and sEng, were found to be highly elevated in the maternal plasma weeks prior to the onset of clinical symptoms. sFlt-1 and sEng are thought to inhibit VEGF and TGF-β receptor signalling respectively. In order to elucidate how these proteins may contribute to preeclampsia, we looked at their ability to affect the secretion of endothelin-1 (ET-1), a powerful vasoconstrictor, shown to be dysregulated in preeclampsia. We found that both TGF-β1 and BMP9, a recently described ligand for sEng, induce ET-1 secretion through Smad1/5/8 and p38 pathways. Moreover, we report that sEng and VEGF can efficiently block ET-1 secretion, induced by BMP9. We propose that the balance between VEGF and BMP9 signalling is disturbed during preeclampsia, leading to excessive release of ET-1, which in turn may cause hypertension.

Charles University, Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Title of Diploma Thesis: Effect of long-term exposure to soluble endoglin on the expression of adhesion molecules on endothelial cells Author of Diploma Thesis: Dominika Tuschlová Supervisor of Diploma Thesis: PharmDr. Iveta Najmanová, Ph.D. Background: The aim of this thesis was to find out the effect of long-term exposure to soluble endoglin (sEng) on the expression of adhesion molecules on endothelial cells (ICAM-1, VCAM-1, P-selectin). We have worked with a control and experimental group of mice on a standard diet, with different levels of sEng. Methods: Genetically modified female mice from the CBAxC57BL/6J strain with high production of human sEng were used for analysis. These mice formed an experimental group, while mice with a low level of sEng formed a control group. They were 12 months old females. We used biochemical analysis to determine the level of total cholesterol and triacylglycerols (TAG). Levels of sVCAM-1 markers in all mice studied were determined by ELISA. The expression of the adhesion molecules ICAM-1, VCAM-1 and P-selectin was monitored and evaluated by Western blotting, where the structural protein GAPDH was used as a control. Results: Biochemical analysis did not show a...

HHT wird in verschiedene Typen unterteilt, wobei Typ 1 am häufigsten vorkommt und durch vaskuläre Unregelmäßigkeiten und Blutungsepisoden aufgrund von CD105-Haploinsuffizienz gekennzeichnet ist. Da CD105 vor allem in ECs exprimiert wird, nutzte unsere Gruppe das VE-cadh-ERT2:cre-CD105f/f-Mausmodell, um die Auswirkungen des EC-CD105-Mangels auf das vaskuläre und hämatopoetische System des Knochenmarks von erwachsenen Mäusen zu untersuchen. Dieses Modell schuf eine Umgebung, in der ECs in erwachsenen Mäusen mit zuvor regulärer CD105-Expression in einer In-vivo-Umgebung reduzierte Werte aufwiesen, was zum ersten Mal eine Zeitraffer-Analyse der EC-Umstrukturierung als Reaktion auf CD105-Mangel ermöglichte. In der vorliegenden Studie untersuchten wir das Zusammenspiel zwischen Blutgefäßumbau und hämatopoetischen und Immunzellaktivitäten nach EC-CD105-Mangel. Insbesondere konnten wir einen zeitabhängigen vaskulären Umbauprozess im Knochenmark feststellen, der etwa vier Wochen andauerte. Dieser Umbau war durch eine allmähliche Abnahme der Größe einzelner Blutgefäße gekennzeichnet, die in Woche 4 zum Stillstand kam. Bemerkenswert ist, dass diese Schrumpfung mit Veränderungen des EC-Zyklus und der Lebensfähigkeit einherging. Insbesondere waren die ECs von cKO-Mäusen in den Wochen 2 und 3 weniger ruhig und in Woche 4 nach der Induktion des EC-CD105-Mangels weniger lebensfähig. Bemerkenswert ist, dass der Anstieg der Proliferation in Woche 2 mit einer vorübergehenden Zunahme der Bindung der VEGFR2-Gensignatur korrelierte. Neben diesen Veränderungen in der Proliferation und Lebensfähigkeit zeigten die ECs zeitabhängige Veränderungen in ihrer Adhäsions, MMP- und Integrin-Aktivität. Darüber hinaus wurden diese Veränderungen der Endothelaktivität von einer erhöhten Menge an Wachstumsfaktoren im extrazellulären Proteingehalt des Knochenmarks in Woche 2 begleitet, die in Woche 4 nicht mehr vorhanden waren, einschließlich VEGF-A. Nach intrakardialer Injektion von Brustkrebszellen wiesen Mäuse mit EC-CD105-Mangel 72 Stunden nach der Injektion weniger metastatische Knoten in ihrem Knochenmark auf als ihre WT-Wurfgeschwister, was darauf hindeutet, dass EC-CD105 ein wichtiger Akteur bei der Regulierung der Extravasation von Brustkrebs in das Knochenmark ist. Darüber hinaus stellt Woche 3 möglicherweise einen entscheidenden Zeitraum dar, in dem das Endothel des Knochenmarks anfälliger für die Infiltration verschiedener Zelltypen wird, wie der vorübergehende Anstieg von B-Zellen und Monozyten und der dauerhafte Anstieg der Neutrophilen- und Eosinophilenzahlen im Blutkreislauf zeigen. Darüber hinaus stieg die Extravasation von Dextran in das Knochenmark nach Woche 3 ebenfalls dauerhaft an. Bemerkenswert ist, dass die Zahl der Eosinophilen und Neutrophilen auch nach heterozygoter Deletion von EC-CD105 anstieg, während die Zahl der Monozyten und B-Zellen davon unberührt blieb. Als Teil der Auswirkungen des homozygoten EC-CD105-Mangels wurde die Sequestrierung von Erythrozyten und PLTs durch die Milz erhöht, was zu Splenomegalie und einem Rückgang der Erythrozyten und PLTs im Kreislauf führte. Gleichzeitig stieg die Zahl der HSPCs, die zu Erythrozyten und PLTs im Knochenmark führen, was darauf hindeutet, dass das Knochenmark die Produktion von Erythrozyten und PLTs als Ausgleichsmechanismus erhöht. Heterozygoter EC-CD105-Mangel führte auch zu einem dauerhaften Anstieg der Neutrophilen- und Eosinophilenzahlen im Blutkreislauf in Woche 3. Die Zahl der B-Zellen und Monozyten blieb jedoch unverändert. Dies korrelierte mit einem fehlenden Unterschied in der Größe der Blutgefäße in Woche 4. Dies deutet darauf hin, dass das Gefäßsystem des Knochenmarks nach einem homozygoten EC-CD105-Mangel aufgrund des Gefäßumbaus eine erhöhte Anfälligkeit für die Infiltration von B-Zellen und Monozyten aufweist. Darüber hinaus führte die heterozygote EC-CD105-Deletion nicht zu signifikanten Unterschieden in der Anzahl der Erythrozyten und PLT im Blutkreislauf. Dieser fehlende Unterschied ging einher mit einer fehlenden Splenomegalie bei diesen Mäusen und keinen signifikanten Unterschieden in der Anzahl der HSPCs, die zu Erythrozyten und PLTs in ihrem Knochenmark führen. Diese Ergebnisse unterstützen die Annahme, dass ein homozygoter EC-CD105-Mangel zu einer verstärkten Sequestrierung von PLTs und RBCs in der Milz und damit zu Splenomegalie führt.:Table of Contents Acknowledgements 3 1 INTRODUCTION 9 1.1 Endoglin (CD105) 9 1.2 The endothelium and angiogenesis 12 1.3 The hematopoietic niche 17 1.4 Vascular control of hematopoiesis 21 1.4.1 EC control of oxygenation 22 1.4.2 Endothelial secretory factors and the perivascular niche 22 1.4. Remodeling of the ECM 23 1.4.4 Physical barrier separating marrow and lumen 24 1.5 β-integrin family 25 2 Aims of this thesis 28 3 MATERIALS AND METHODS 30 3.1 Tamoxifen treatment 30 3.2 Blood analysis 30 3.3 Flow cytometry and cell sorting 30 3.4 Next generation sequencing 30 3.5 Bone isolation for histological analysis 31 3.6 Bone sectioning and microscopy image acquisition 31 3.7 Dextran injection 31 3.8 Dextran area analysis 32 3.9 Breast cancer homing to bone marrow 32 3.10 Blood vessel histological analysis and calculation of distribution peak 32 3.11 Flow cytometry 33 3.12 Cell cycle analysis with Ki67 33 3.13 Cell viability analysis with Annexin V 33 3.14 Extracellular integrin staining 34 3.15 Intracellular integrin staining 34 3.16 Proteome profiler 35 3.17 Statistics 35 3.18 Breast cancer homing to bone marrow 36 3.19 Micro-computed tomography 36 4 RESULTS 39 4.1 Induced CD105-deficiency in endothelial cells of the bone marrow reduces blood vessel size 39 4.2 EC-CD105 deficiency differentially affects circulating cell populations throughout time 41 4. 3 EC-CD105 deficiency reorganizes hematopoietic progenitors in the bone marrow 45 4.4 CD105 deficiency causes vessels to remodel through endothelial cell proliferation and consequent apoptosis 48 4.5 CD105 deficiency differentially switches EC activity from angiogenic to apoptotic in a time dependent manner 51 4.6 EC-CD105 deficiency reduces EC adhesion and regulates integrin dynamics in a time dependent manner 57 4.7 Partial EC-CD105 deficiency affects immune cell dynamics in a time dependent manner 61 4.8 EC-CD105 deficiency affects cancer homing to bone marrow 65 5 DISCUSSION 67 5.1 EC-CD105 deficiency induces temporally orchestrated vasculature remodeling in the bone marrow 67 5.2 EC-CD105 deficiency affects circulating cells and the hematopoietic niche 70 5.3 EC-CD105 deficiency affects EC activity in a time dependent manner, cascading into affecting EC-circulating cell dynamics 73 5.4 EC-CD105 deficiency impairs breast cancer metastasis to bone marrow 76 6.1 Summary 79 6.2 Zussamenfassung 81 Bibliography 83 Deklaration 96 List of abbreviations 99

Author: Nikola Jozefčeková Title: Endoglin and liver Form: Diploma Thesis University: Charles University, Faculty of Pharmacy in Hradec Králové Degree: Pharmacy This diploma thesis concludes the available information about endoglin, its isoforms in the liver and its impact on the liver during various pathological conditions. In the first part of the thesis describes morphology and physiology of the liver, its structure, histology and metabolic functions. Second part contains an information about endoglin, its isoforms and role in TGF-β signaling complex, expression of endoglin and regulation of the expression. In the third part are described liver diseases during which is expression of endoglin changed. This part deals with the significance of endoglin as a diagnostic and prognostic marker. Endoglin (CD105, TGF-β receptor III) is a homodimeric transmembrane glycoprotein and a membrane co-receptor TGF-β with high expression in endothelial cells. Endoglin plays an important role in the vascular development. Endoglin is involved in the processes of angiogenesis, vascular homeostasis and TGF-β signalization. It affects activity of TGF-βRII, ALK1 and ALK5 receptors. Due to the interactions with TGF-β complex, modulation of activity of ALK receptors and Smads, endoglin controls fibrotic and anti-...

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