"Medicine, Faculty of"@en . "Medicine, Department of"@en . "Experimental Medicine, Division of"@en . "DSpace"@en . "UBCV"@en . "Larrivee, Bruno"@en . "2009-12-23T18:07:24Z"@en . "2005"@en . "Doctor of Philosophy - PhD"@en . "University of British Columbia"@en . "Vascular endothelial growth factor (VEGF) and its receptors play an\r\nessential role in the formation and maintenance of the hematopoietic and vascular\r\ncompartments. Activation of the kinase activity of the VEGF receptor-2 (VEGFR-2) is\r\ntriggered by binding to VEGF, which affects endothelial cell proliferation, permeability,\r\nand migration. Accumulating evidence suggests that VEGFR-2 signalling may play an\r\nimportant role in post-natal hematopoiesis and vasculogenesis. One of the goals of this\r\nwork was to study some of the biological effects triggered by VEGFR-2 in isolation,\r\nwithout the interference of other VEGF receptors in the contexts of post-natal\r\nhematopoiesis and vasculogenesis. By inducing expression of the full length VEGFR-2 or\r\nof a VEGFR-2 construct that can be selectively activated in fibroblasts or hematopoietic\r\nprogenitors, we show that VEGFR-2 can induce activation of the Erkl/2 mitogen\r\nactivated protein (MAP) kinase, p38 MAP kinase and Akt signalling pathways.\r\nMoreover, VEGFR-2 activation can elicit biological responses such as cell proliferation,\r\nmigration and survival in vitro. Using a bone marrow transplantation model, we also\r\nshow that VEGFR-2 activation promotes the expansion of myeloid cells in vivo, in part\r\nthrough the up-regulation of the hematopoietic cytokine Granulocyte/Macrophage-\r\nColony Stimulating Factor (GM-CSF). In the second part of the thesis, we confirm the\r\nexistence of early endothelial progenitors in mice. These cells originate from the bone\r\nmarrow and can integrate in the vasculature of tumours, although at a low frequency.\r\nVEGF does not modulate the occurrence or mobilization of these progenitors. We also\r\ndemonstrate that these cells originate from hematopoietic stem cells and that they arise by cell differentiation, and not through cell fusion. The work presented in this thesis, by\r\nelucidating some of the effects triggered by VEGF signalling though VEGFR-2 in\r\nhematopoietic cells, could potentially lead to the development of therapies targeting the\r\ngrowth of malignant hematopoietic cells."@en . "https://circle.library.ubc.ca/rest/handle/2429/17216?expand=metadata"@en . "EFFECTS OF VEGFR-2 SIGNALLING IN POST-NATAL HEMATOPOIESIS AND VASCULOGENESIS by BRUNO LARRIVEE B.Sc, Universite Laval, 1996 M.Sc, Universite du Quebec a Montreal, 1999 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF T H E REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in T H E F A C U L T Y OF G R A D U A T E STUDIES Department of Experimental Medicine THE UNIVERSITY OF BRITISH COLUMBIA January 2005 \u00C2\u00A9 Bruno Larrivee, 2005 Abstract Vascular endothelial growth factor (VEGF) and its receptors play an essential role in the formation and maintenance of the hematopoietic and vascular compartments. Activation of the kinase activity of the VEGF receptor-2 (VEGFR-2) is triggered by binding to VEGF, which affects endothelial cell proliferation, permeability, and migration. Accumulating evidence suggests that VEGFR-2 signalling may play an important role in post-natal hematopoiesis and vasculogenesis. One of the goals of this work was to study some of the biological effects triggered by VEGFR-2 in isolation, without the interference of other VEGF receptors in the contexts of post-natal hematopoiesis and vasculogenesis. By inducing expression of the full length VEGFR-2 or of a VEGFR-2 construct that can be selectively activated in fibroblasts or hematopoietic progenitors, we show that VEGFR-2 can induce activation of the Erkl/2 mitogen activated protein (MAP) kinase, p38 MAP kinase and Akt signalling pathways. Moreover, VEGFR-2 activation can elicit biological responses such as cell proliferation, migration and survival in vitro. Using a bone marrow transplantation model, we also show that VEGFR-2 activation promotes the expansion of myeloid cells in vivo, in part through the up-regulation of the hematopoietic cytokine Granulocyte/Macrophage-Colony Stimulating Factor (GM-CSF). In the second part of the thesis, we confirm the existence of early endothelial progenitors in mice. These cells originate from the bone marrow and can integrate in the vasculature of tumours, although at a low frequency. VEGF does not modulate the occurrence or mobilization of these progenitors. We also demonstrate that these cells originate from hematopoietic stem cells and that they arise by ii cell differentiation, and not through cell fusion. The work presented in this thesis, by elucidating some of the effects triggered by V E G F signalling though VEGFR-2 in hematopoietic cells, could potentially lead to the development of therapies targeting the growth of malignant hematopoietic cells. iii Table of contents ABSTRACT ii TABLE OF CONTENTS iv LIST OF TABLES ix LIST OF FIGURES x LIST OF ABBREVIATIONS xii ACKNOWLEDGEMENTS xv CHAPTER 1 INTRODUCTION 1 1.1 Embryonic origins of hematopoiesis and vasculogenesis 2 1.1.1 Yolk sac hematopoiesis and vasculogenesis 2 1.1.2 The formation and maturation of definitive hematopoietic cells 4 1.1.3 The hemangioblast: a common progenitor of hematopoiesis and vasculogenesis 6 1.2 Hematopoiesis and Vasculogenesis in the adult 8 1.2.1. The hematopoietic stem cell 8 1.2.2 Hematopoietic hierarchy 11 1.2.3. Regulation of hematopoiesis 13 1.2.4. The angioblast and evidence of vasculogenesis in the adult 15 1.3 Vascular Endothelial Growth Factor (VEGF) 19 iv 1.3.1. Biological activities of V E G F 20 1.3.2. Regulation of V E G F expression 22 1.3.3. V E G F receptors 24 1.3.4. V E G F signal transduction 29 1.4 V E G F and hematopoiesis 37 1.4.1 Expression and induction of V E G F and its receptors in normal hematopoiesis.37 1.4.2 Role of V E G F and its receptors in hematopoietic cells 40 1.4.3 V E G F and its receptors in hematological malignancies 42 1.5 Rationale and thesis hypotheses 44 C H A P T E R 2 M A T E R I A L S A N D M E T H O D S 47 2.1 Cell culture 48 2.2 Plasmid construct 48 2.3 Gene transfer 49 2.4 HFF proliferation assay 50 2.5 Migration assays 50 2.6 In vitro MAP kinase assay 51 2.7 Isolation of murine bone marrow cells 51 2.8 Animals 52 2.8.1 Bone marrow transplants 52 2.8.2 Single cell transplants 53 2.8.3 CFU-Spleen assay 54 2.9 AP20187 formulation 54 2.10 Antibody staining for FACS analysis 55 2.11 Preparation of cDNA and RT-PCR 56 2.12ELISA 56 2.13 Bone marrow cells viability assays 57 2.14 Hematopoietic colony assays 57 2:15 Immunofluorescence and microscopy 58 2.16 Immunoblotting 59 2.17 Endothelial progenitor assays 59 2.18 Tumour tissue immunohistochemical staining 60 2.19 lacZ and hPLAP staining 60 2.20 Statistical analysis 61 C H A P T E R 3 B I O L O G I C A L E F F E C T S AND SIGNALLING P A T H W A Y S INDUCED B Y VEGFR-2 IN ISOLATION 62 3.1 Introduction 63 3.2 Results 64 3.2.1 VEGFR-2 transmits a mitogenic signal in response to V E G F stimulation 64 3.2.2 V E G F is chemotactic for HFF-VEGFR-2 but not HFF-Neo cells 66 3.2.3 V E G F activates Erkl/2 and p38 MAP kinases in HFF-VEGFR-2 cells 67 3.2.4 V E G F does not induce expression of endothelial markers in primary fibroblasts transduced with VEGFR-2 69 3.3 Discussion 71 C H A P T E R 4 E F F E C T S O F VEGFR-2 A C T I V A T I O N IN M U R I N E B O N E M A R R O W IN V I T R O 75 4.1 Introduction 76 VI 4.2 Results 77 4.2.1 Activation of VEGFR -2 delays loss of murine hematopoietic progenitors 77 4.2.2 VEGFR -2 does not increase S-phase entry in hematopoietic progenitors 85 4.2.3 VEGFR -2 activation reduces the number of apoptotic cells in hematopoietic precursors 87 4.2.4 VEGFR -2 activates the PI3-kinase and Erk kinase pathways 88 4.3 Discussion 92 CHAPTER 5 ACTIVATION OF VEGFR-2 IN BONE MARROW CELLS LEADS TO ACCUMULATION OF MYELOID CELLS IN VIVO: ROLE OF GM-CSF 96 5.1 Introduction 97 5.2 Results 99 5.2.1 Activation of VEGFR -2 induces expansion of bone marrow myeloid cells 99 5.2.2 VEGFR -2 increases the proportion of myeloid progenitors in the bone marrow 105 5.2.3 VEGFR -2 activation in bone marrow cells induces GM -CSF expression and secretion 106 5.3 Discussion 113 CHAPTER 6 IMPLICATION OF BONE MARROW-DERIVED CELLS TO TUMOUR VASCULOGENESIS 119 6.1 Introduction 120 6.2 Results 122 6.2.1 Determination of the existence of bone marrow-derived endothelial cells 122 vii 6.2.2 Determination of the existence of and adult hemangioblast 126 6.2.3 Role of V E G F and VEGFR-2 in the mobilization and differentiation of bone marrow-derived endothelial cells 131 6.2.4 Determination of the proportion of endothelial progenitors in human umbilical cord blood 136 6.3 Discussion 138 CHAPTER 7 CONCLUSIONS AND FUTURE PROSPECTIVES 144 BIBLIOGRAPHY 151 viii List of Tables Chapter 1 Table I Commonly used markers for the purification o f hematopoietic stem cells in human and mouse 10 Table II Common markers expressed on human endothelial progenitors, mature endothelial cells and subsets of hematopoietic cells 18 Table III Expression of V E G F and its receptors in hematopoietic malignancies 42 Chapter 2 Table IV Murine primers and P C R conditions used in this thesis 56 Chapter 5 Table V Peripheral blood counts..... 101 ix List of Figures Chapter 1 Figure 1 Hematopoietic sites in the developing embryo 3 Figure 2 Schematic representation of the hematopoietic hierarchy 12 Figure 3 Current concepts for neovascularization in adult ischemic tissues 16 Figure 4 Receptors of the V E G F family 24 Figure 5 Signalling pathways induced by V E G F 29 Chapter 3 Figure 6 Generation of a primary human foreskin fibroblast cell line expressing VEGFR-2 65 Figure 7 V E G F induces a proliferative response in HFF-VEGFR-2 cells but not in HFF-Neo cells 66 Figure 8 V E G F simulates migration of H M E C and HFF-VEGFR-2 cells, but not HFF-Neo cells 67 Figure 9 V E G F induces phosphorylation and activation of Erkl/2 M A P kinase in HFF-VEGFR-2 but not HFF-Neo cells 68 Figure 10 V E G F induces phosphorylation of p38 MAP kinase in HFF-VEGFR-2 but not HFF-Neo cells 69 Figure 11 V E G F does not induce expression of endothelial markers in HFF-VEGFR-2 cells 70 Chapter 4 Figure 12 MIG-FKBP/VEGFR-2 fusion construct 79 Figure 13 VEGFR-2 maintains hematopoietic cell numbers 81 Figure 14 VEGFR-2 delays the loss of CFCs 83 Figure 15 VEGFR-2 dimerization maintains the number of multipotential bone marrow progenitors 85 Figure 16 VEGFR-2 dimerization does not increase BrdU uptake in hematopoietic progenitors 86 Figure 17 VEGFR-2 dimerization inhibits apoptosis of cytokine-starved hematopoietic progenitors 88 Figure 18 VEGFR-2 dimerization activates Akt and Erkl/2 M A P kinases in hematopoietic progenitors 90 Figure 19 Effect of PI3-kinase or M E K inhibition on hematopoietic progenitor survival 92 Chapter 5 Figure 20 VEGFR-2 induces expansion of retrovirally transduced hematopoietic cells in vivo 100 Figure 21 VEGFR-2 induces expansion of myeloid cells, but not of erythroid and lymphoid cells in the bone marrow 103 Figure 22 FKBP-VEGFR-2-transduced cells are not mobilized in the peripheral blood in response to AP20187 104 Figure 23 VEGFR-2 induces expansion of bone marrow myeloid progenitors in vivo.. 105 Figure 24 Effects of VEGFR-2 activation on expression of hematopoietic factors in murine bone marrow cells 107 Figure 25 VEGFR-2 dimerization increases expression of GM-CSF at the protein level in murine bone marrow 109 Figure 26 Blocking of GM-CSF inhibits VEGFR-2-induced expansion of myeloid progenitors 112 Chapter 6 Figure 27 Bone marrow derived endothelial cells incorporate into tumour vasculature 124 Figure 28 Bone marrow-derived endothelial cells have a relatively small contribution to the formation of blood vessels in B6RV2 lymphomas 125 XI Figure 29 Bone marrow-derived endothelial cells arise by differentiation and not by cell fusion 128 Figure 30 A single hematopoietic stem cell can give rise to endothelial progenitor cells that incorporate into tumour blood vessels 130 Figure 31 V E G F secretion by tumour cells does not increase the contribution of bone marrow-derived cells to the formation of tumour blood vessels 132 Figure 32 VEGFR-2 activation in bone marrow cells does not result in endothelial progenitor mobilization or recruitment into tumour vasculature 135 Figure 33 Rarity of endothelial progenitors in post-natal human mononuclear cells 137 Chapter 7 Figure 34 Hypothetical model for the possible effects induced by VEGFR-2 activation in hematopoietic cells 148 xii List of Abbreviations 5-FU 5-fluorouracil A c L D L acetylated low density lipoprotein A G M aorta-gonad-mesonephros A M L acute myeloid leukemia BFU-E burst forming unit erythroid BL-CFC blast colony forming cell B M bone marrow BSA bovine serum albumin CD cluster of differentiation CFU-EC colony forming unit endothelial cell CFU-G colony forming unit granulocyte C F U - G E M M colony forming unit granulocyte, erythrocyte, macrophage, megakaryocyte C F U - G M colony forming unit granulocyte macrophage C F U - M colony forming unit macrophage CFU-S colony forming unit spleen C M L chronic myeloid leukemia D A G diacylglycerol D M E M Dulbecco's modified Eagle's medium DNA deoxyribonucleic acid eNOS endothelial nitric oxide synthase EPC endothelial progenitor cell Erk extracellular-regulated kinase ES embryonic stem Ets-1 endothelial specific transcription factor FACS fluorescence activated cell sorting FBS fetal bovine serum bFGF basic fibroblast growth factor FGFR fibroblast growth factor receptor FKBP FK506 binding protein GAPDH glyceraldehydes-3-phosphate-dehydrogenase G-CSF granulocyte colony stimulating factor GFP green fluorescent protein GM-CSF granulocyte-macrophage colony stimulating factor GM-CSFR granulocyte-macrophage colony stimulating factor receptor Gy Grey H A hemagglutinin HFF human foreskin fibroblast HIF hypoxia inducible factor H M E C human microvascular endothelial cell HSC hematopoietic stem cell HSP heat shock protein H U V E C human umbilical vein endothelial cell LAP inhibitor of apoptosis Ig immunoglobulin xiii IL interleukin IMDM Iscove's modified Dulbecco's medium IRES internal ribosomal entry site INK c-Jun NH-2-terminal kinase kDa kiloDalton KDR kinase domain region Lin\" lineage negative M A P K mitogen activated protein kinase MBP myelin basic protein M-CSF macrophage colony stimulating factor MIG mscv ires gfp MMP matrix metalloproteinase MSCV murine stem cell virus M T T 3-(4',5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide NO nitric oxide PAS para-aortic-splanchnopleura PBS phosphate buffered saline PCR polymerase chain reaction PDGF platelet-derived growth factor PDZ PSD- 95/Dlg/ZO-l PI3-kinase phosphatidylinositol-3 kinase PK protein kinase PLC phospholipase C P1GF placenta growth factor R A F T K related adhesion-focal tyrosine kinase RNA ribonucleic acid RT-PCR reverse transcriptase polymerase chain reaction SAPK stress-activated protein kinase SCF stem cell factor SCL stem cell leukemia SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis SP side population TGF-/3 transforming growth factor j8 TNF-a tumour necrosis factor-a Tpo thrombopoietin UTR untranslated region VE-cadherin vascular-endothelial cadherin V E G F vascular endothelial growth factor VEGFR vascular endothelial growth factor receptor vHL von Hippel-Lindau VPF vascular permeability factor vWF von Willebrand factor xiv Acknowledgements First, I would like to thank my supervisor, Dr. Aly Karsan, for giving me the opportunity to work on these projects, and for his many years of patient guidance and support with which I have come this far from knowing very little about the subjects discussed in this thesis. I would also like to thank all members of the Karsan lab, past and present, for providing a work environment that was fun and stimulating to work in. I would especially like to thank Ingrid Pollet for all the help she gave me over the years: your dedication, sense of organization and positive attitude were always greatly appreciated. I also thank Siun Murphy for drawing one of the figures presented in this thesis. Many thanks to my supervisory committee members, Drs. Peggy Olive, Keith Humphries and Vince Duronio for many invaluable discussions. I also want to thank members of the department of Medical Biophysics, from which I received many insights and useful technical tips. Special thanks to Denise McDougal and Fred Wong for taking care of the FACS analysis and cell sorting. I finally want to acknowledge the contribution of Dr. Fabio Rossi, Mike Long and Stephane Corbel who performed some of the transplants described in Chapter 6. I would like to thank my family: Gilles, Anne, Jean-Francois, even though you are far away, your support has meant a lot to me. I also want to express my gratitude to my aunt Madeleine and my cousin Matthew for their constant encouragement and for always being there for me, in good times and bad times. Finally, I am thankful to the Heart and Stroke foundation of Canada for the funding it provided me during my doctoral studies. XV Chapter 1 Introduction 1.1 Embryonic origins of hematopoiesis and vasculogenesis 1.1.1 Yolk sac hematopoiesis and vasculogenesis The development of a functional circulatory system is an early prerequisite for the survival and growth of the mammalian embryo. The first differentiated cells to form in the mammalian embryo are those of the hematopoietic and endothelial lineages, which, along with cardiac components, are the backbone of the developing circulatory system (Ema and Rossant, 2003). Cells from the hematopoietic and endothelial lineages are mesodermal in origin and are first generated in yolk sac blood islands beginning at embryonic day 7 (E7.0) in the mouse (Haar and Ackerman, 1971) and between the second and third week of human gestation. Thereafter, hematopoiesis shifts to the fetal liver and again, around the time of birth, to the bone marrow (Moore and Metcalf, 1970). The generation of blood cells in blood islands of the yolk sac is referred as primitive hematopoiesis and results primarily in the production of large, nucleated erythroblasts (Wong et al., 1986), as well as some megakaryocytes (Xu et al., 2001) and primitive macrophages (Shepard and Zon, 2000), as opposed to definitive hematopoiesis, which can generate mature cells of all the blood lineages. Blood islands, which consist of a central focus of hematopoietic cells surrounded by a layer of endothelial cells, arise in the mouse from proximal mesodermal cells in the visceral yolk sac between E7.0 and E7.5 (Shepard and Zon, 2000). 2 Figure 1 Hematopoietic sites in the developing mouse embryo. Schematic representation of whole mouse embryos at (a) E8.5/9 and (b) E10.5/11. Abbreviations: A G M , aorta-gonad-mesonephros. Adapted from Dzierzak et al. (Dzierzak et al., 1998). Between E8.0 and E9.0, the cells comprising the outer layer ofthe blood island cell aggregates assume a spindle shape as they differentiate into endothelial cells (Shepard and Zon, 2000). The vast majority of the inner cells of the blood islands progressively lose their intercellular attachments as they differentiate into primitive erythroblasts. The simultaneous spatial and temporal appearance of hematopoietic and endothelial cells in the yolk sac blood islands has led to the concept of the hemangioblast, a common precursor for the hematopoietic and endothelial lineages (Ema and Rossant, 2003). Moreover, many known markers of endothelial cells are also expressed on hematopoietic cells (Fina et al., 1990; Kallianpur et al., 1994; Matthews et al., 1991). Recent studies with cells from chick embryos and with embryonic stem cells have led credence to this concept (Eichmann et al., 1997). While histological studies indicate a restricted hematopoietic potential within the yolk sac, precursor analysis in vitro and transplantation studies in vivo have provided clear evidence that this tissue is able to generate multiple definitive lineages as well (Yoder and Hiatt, 1997). Macrophage precursors were detected in low numbers as early 3 as those of the primitive erythroid lineage (Takahashi and Naito, 1993). Definitive erythroid precursors were found at E8.25 and also showed a dramatic increase followed by a general decline of their numbers (Palis et al., 1999). Precursors of the mast cell lineage developed slightly later at E8.5 (Palis et al., 1999). Unlike primitive erythroblasts, definitive erythroid precursors and mast cell precursors do not mature in the yolk sac, suggesting that they are produced for export to other sites, presumably the fetal liver (Palis et al , 1999). These findings suggest a dual role for the yolk sac: the generation of a functional primitive erythroid lineage as well as the production of a cohort of definitive precursors which migrate to the fetal liver and establish the initial stage of definitive hematopoiesis in this tissue (Galloway and Zon, 2003). Yolk sac hematopoiesis is transient and shows a dramatic decline in activity between E l l and E12. This decline coincides with the onset of activity in the developing liver, which becomes the predominant hematopoietic tissue throughout the remainder of fetal life. In contrast to the restricted program observed in the yolk sac, the fetal liver is a site of multilineage definitive hematopoiesis which includes erythropoiesis, myelopoiesis and lymphopoiesis (Galloway and Zon, 2003). 1.1.2 The formation and maturation of definitive hematopoietic cells Shortly before the onset of organogenesis the embryo starts to generate a transitory population of embryonic hematopoietic cells that serve its immediate needs. These first hematopoietic cells, consisting mainly of primitive erythroid cells, appear in the embryonic circulation in growing numbers and then colonize the initially inactive fetal liver (Medvinsky and Dzierzak, 1996). Definitive hematopoiesis, which results in the production of all hematopoietic lineages, develops slightly later and gradually forms a 4 massive pool in the fetal liver, which becomes the main source of hematopoietic stem cells which subsequently colonize the bone marrow (Dzierzak et al., 1998). Hematopoietic stem cells are defined within the context of stringent transplantation assays used for adult bone marrow cells and are characterized by the following properties: 1) they clonally give rise to all differentiated lineages of the blood cells; 2) they are self-renewing; 3) they possess high proliferative/expansion potential contributing to high level hematopoietic repopulation of the recipient; and 4) they are active long term/over the lifespan of the individual (Wognum et al., 2003). Definitive hematopoietic stem cells are produced both in the mature yolk sac and within the para-aortic splanchnopleura (PAS, E8.5-E9.5) and the aorta-gonad-mesonephros region (AGM, E10.5-E11.5) (Galloway and Zon, 2003). When A G M , yolk sac and fetal liver cells are directly compared throughout development for repopulation capabilities by the criteria previously mentioned, the A G M region consistently demonstrates more hematopoietic stem cells than the yolk sac or fetal liver at E10 and E l l (Muller et al., 1994). The definitive hematopoietic stem cells formed in the yolk sac and the A G M region do not mature in situ but instead are believed to migrate and seed the fetal liver, where they undergo terminal differentiation (Palis and Yoder, 2001). Thus, within the yolk sac, there is a temporal, if not spatial, overlap between primitive and definitive hematopoiesis. Although it was previously believed that the definitive hematopoietic stem cells formed in the yolk sac contribute only to primitive hematopoiesis (Muller et al., 1994), it was later shown that yolk sac cells isolated around day 9 and later can engraft and repopulate recipient animals following transplantation into the livers of newborn mice (Yoder and Hiatt, 1997; Yoder et al., 1997a; Yoder et al., 1997b). Furthermore, yolk sac 5 hematopoietic cells have the ability to repopulate adult bone marrow following coculture on certain types of stromal cells (Matsuoka et al., 2001) or when HoxB4 is ectopically expressed (Kyba et al., 2002). Yolk sac and A G M definitive hematopoietic stem cells appear to circulate (Kumaravelu et al., 2002) and can presumably seed intraembryonic tissues such as the liver and large arteries. 1.1.3 The hemangioblast: a common progenitor of hematopoiesis and vasculogenesis The close spatial and temporal appearance of hematopoietic and endothelial cells in the embryo has led to the hypothesis that a common precursor exists for both of these lineages. The term hemangioblast was first introduced to describe discrete cell masses that develop in chick embryo cultures and displayed both hematopoietic and endothelial potential (Haar and Ackerman, 1971). Since originally introduced, the concept of the hemangioblast has gained support from studies demonstrating that the hematopoietic and endothelial lineages share expression of a number of different genes such as MB1/QH1 in the quail (Pardanaud et al., 1987; Peault et al., 1983), CD31, CD34, SCL/Tal-1 and VEGFR-2 (Kabrun et al., 1997; Kallianpur et al., 1994; Watt et al., 1995; Young et al., 1995). Gene targeting studies in the mouse have provided further evidence for this bi-potential precursor in showing that some of these genes are essential for the development of both lineages (Robb et al., 1995; Shalaby et al., 1995; Shivdasani et al., 1995). Furthermore, overexpression of SCL/Tal-1 in zebrafish embryos results in overproduction of common hematopoietic and endothelial precursors, perturbation of vasculogenesis and concomitant loss of pronephric duct and somitic tissue (Gering et al., 1998). Analysis of knock-in embryos in which SCL/Tal-1 is expressed under the control of the VEGFR-2 locus suggests that VEGFR-2 and SCL/Tal-1 act in combination to 6 regulate cell fate decisions for formation of endothelial and hematopoietic cells in early development (Ema et al., 2003). Additional support for the existence of the hemangioblast comes from analysis of a specific mutation in zebrafish known as cloche, which affects the development of hematopoietic cells and endocardium (Stainier et al., 1995). The most compelling and direct evidence for the presence of the hemangioblast comes from recent studies utilizing the in vitro ES cell system (Choi et al., 1998). Embryoid bodies differentiated for 2.5-3.5 days contain a unique cell population, the blast colony-forming cell (BL-CFC). BL-CFCs form blast colonies in the presence of vascular endothelial growth factor (VEGF) in methylcellulose cultures (Choi et al., 1998). Gene expression analysis indicates that cells within blast colonies express a number of genes common to both hematopoietic and endothelial lineages, including SCL, CD34, and the V E G F receptor-2 (VEGFR-2) (Kennedy et al., 1997a). Most importantly, BL-CFCs give rise to primitive, definitive hematopoietic and endothelial cells when replated in medium with both hematopoietic and endothelial cell growth factors (Choi et al., 1998; Kennedy et al., 1997a). Moreover, in the quail embryo, VEGFR-2 + cells isolated from the mesoderm can give rise to cells of the hematopoietic or endothelial lineages depending on the culture conditions (Eichmann et al., 1997). Blast colonies have recently been shown to give rise to a third lineage, the smooth muscle cell (Ema et al., 2003). These characteristics of the BL-CFC suggest that it represents the in vitro equivalent of the hemangioblast and, as such, one of the earliest stages of hematovascular development described to date. BL-CFCs have been detected in small numbers in dissected mouse embryos (Baron, 2003), suggesting that hemangioblasts may form during normal mouse development. The technical difficulties encountered in identifying cells with the properties of the hemangioblast in mouse embryos suggests that these cells are produced in very small numbers and for a very short time, indicating that these cells might be short-lived, differentiating soon after their formation. 1.2 Hematopoiesis and Vasculogenesis in the adult 1.2.1. The hematopoietic stem cell The scale of the hematopoietic system is quite remarkable, considering that daily some 2 x 1011 erythrocytes and 5 x 1010 granulocytes, in addition to platelets, lymphocytes, and monocytes enter the circulation (Finch et al., 1977). Despite the magnitude of this production system, dysregulation is uncommon and external influences can rapidly induce changes in the blood cell count of a specific lineage. For example, hypoxia can induce an increase in erythrocyte production, but does not affect the neutrophil count, whereas the opposite is true in an acute bacterial infection. The regulation of hematopoiesis must be exquisitely fine to be able to maintain blood cells counts within a relatively narrow range. This prodigious output of mature cells is ultimately dependent on the precise regulation of primitive hematopoietic stem cells, which, in turn, give rise to an ordered series of transit populations of progenitor and precursor cells of progressively more restricted proliferative and differentiative potentiality. By definition, a hematopoietic stem cell is a cell isolated from the blood or bone marrow that can renew itself, can differentiate to a variety of specialized cells, can 8 mobilize out of the bone marrow into circulating blood and can undergo apoptosis (Krause, 2002). Definitive proof of the existence of a hematopoietic stem cell requires the demonstration of its ability to produce a long-lasting multilineage clone in vivo. Identifying and characterizing the properties of hematopoietic stem cells has been a formidable task, since it is estimated that about 1 in every 10,000 to 15,000 bone marrow cells is thought to be a stem cell (Coulombel, 2004). In the bloodstream, the proportion falls to 1 in 100,000 blood cells (Coulombel, 2004). Despite the immense burden of producing over 10 n cells per day in the human adult, the great majority of stem cells are not dividing at any one time (Lajtha et al., 1971). The current picture of the stem cell pool is that of a small, but potent group of cells, able to maintain tremendous hematopoietic cell supplies through the division of a small fraction of its members, keeping the remainder of the stem cells in reserve. There appear to be two kinds of hematopoietic stem cells. If bone marrow cells from a transplanted mouse can, in turn, be transplanted to another lethally irradiated mouse and restore its hematopoietic system over some months, they are considered to be long-term stem cells that are capable of self-renewal (Coulombel, 2004). Other cells from bone marrow can immediately regenerate all the different types of blood cells, but under normal circumstances cannot renew themselves over the long term, and these are referred to as short-term progenitor or precursor cells (Coulombel, 2004). Progenitor or precursor cells are relatively immature cells that are precursors to a fully differentiated cell of the same tissue type. They are capable of proliferating, but they have a limited capacity to differentiate into more than one cell type, as hematopoietic stem cells do (Messner, 1998). 9 The primitive hematopoietic stem cells and their immediate progeny are morphologically indistinguishable (Messner, 1998). Despite continued refinement of multiparameter cell separation strategies, unique phenotypic markers that are able to unmistakably characterize, resolve, and purify hematopoietic stem cells have not been identified. However, several makers have been used in combination in order to separate hematopoietic stem cells. Some of the markers most frequently used to separate human and murine hematopoietic stem cells are listed in Table 1 (Wognum et al., 2003). Table I Commonly used markers for the purification of hematopoietic stem cells in human and mouse. Lin: Lineage markers (CD1 lb, B220, Gr-1, TER119) Mouse Human CD34 l o w / ' CD34 + Sca-1 + CD59 + Thyl + / l o w Thyl + CD38 + CD38 l o w / \" c-kit + c-kit - / l o w Lin\" Lin\" FGFR + CD 133 CD201 VEGFR-1 VEGFR-1 VEGFR-2 Fluorescent dye staining approaches have also been used to define hematopoietic stem cells based on efflux capacity and staining profile. The combination of both Rhodamine 123 and Hoechst 33342 staining has demonstrated that Hoechst low -10 Rhodamine low cells are highly enriched for hematopoietic stem cell activity (Goodell et al., 1996). A dual-wavelength flow cytometric analysis of bone marrow based on differential Hoechst 33342 staining that identifies a \"side population\" (SP) which gives rise to all mature blood lineages in transplanted mice has attracted much interest in the field of hematopoietic stem cells (Goodell et al., 1996). To detect hematopoietic stem cell functional activity, in vivo transplantation is generally considered to be the most appropriate assay because the ability of cells to reconstitute blood cell production in a myeloablated recipient measures the capacity of stem cells for both extensive proliferation and multilineage differentiation over extended time periods (Coulombel, 2004). 1.2.2 Hematopoietic hierarchy The bone marrow contains a bewildering array of dividing and intermediate or immature cell types. These immature types can be recognized by morphology, antibodies to specific surface markers or stains. In this way, it is possible to arrange the immature cells into lineages and construct a path by which an immature cell becomes fully mature. li Common Lymphoid Progenitor T Lymphocyte Figure 2 Schematic representation of the hematopoietic hierarchy: all the final mature blood cells (extreme right) are derived from a multipotential hematopoietic stem cell (extreme left). Self-renewal potential decreases from left to right co-ordinately with increasing differentiation; therefore, cells on the left are more primitive than those on the right. Successive divisions of the hematopoietic stem cell generate a series of lineage-limited divisions, and differentiation generates the end cells. The mature bone marrow cells within each cell lineage are believed to derive from a small pool of undifferentiated cells (progenitors), whose destiny is to divide and differentiate along a single hematopoietic pathway, resulting in the unique production of mature, differentiated cells (Messner, 1998). When hematopoietic progenitors are placed into in vitro cultures in methylcellulose in the presence of optimal combinations of hematopoietic growth factors, each progenitor cell repeatedly divides and differentiates, giving rise to colonies of one or 12 more hematopoietic lineages. Progenitor cells constitute approximatively 0.1% of total bone marrow mononuclear cells (Messner, 1998). Progenitors for B and T cells appear to diverge early and follow separate modes and sites of differentiation: progeny of B-committed progenitors differentiate partly in the bone marrow, then migrate to the lymph nodes. T cell progenitors migrate to the thymus where independent proliferation, differentiation, and selection for immune function take place (Petrie, 2003). The common myeloid progenitors (CFU-GEMM) are unipotential progenitors committed to granulocytopoiesis, erythropoiesis and megakaryocytopoiesis. The granulocytic colonies which emerge in vitro are usually made up of both neutrophils and monocytes and originate from a single bipotential cell, named C F U - G M (Barreda et al., 2004). Progenitor cells committed to the erythroid cell line will also grow in vitro but will differentiate to haemoglobin-containing erythroblasts only if erythropoietin is present (Koury et al., 2002). 1.2.3 Regulation of hematopoiesis The regulation of lineage commitment and expansion is clearly physiologically critical, since this determines what sorts of blood cells are finally produced by the bone marrow. Hematopoietic stem cells either self-renew, thereby maintaining stem cell properties, or alternatively give rise to cells increasingly committed to differentiation into various hematopoietic lineages. It is unclear if this cell fate decision is controlled by a purely stochastic mechanism or is the result of environmental cues mediated at least in part through specific receptor-ligand interaction. It is likely that cell fate is influenced both by the stochastic nature of gene expression and by soluble factors and cell-cell interactions. 13 The processes of self-renewal and differentiation occur in vivo within bone marrow microenvironments. These niches are populated by densely packed differentiating hematopoietic cells and by stromal endothelial cells, fibroblasts and adipocytes, as well as macrophages and lymphocytes (Sensebe et al., 1997). These stromal cells not only provide physical support and points of adhesion, but actually direct the processes of hematopoietic differentiation, by secreting positive hematopoietic growth factors such as colony stimulating factors, tyrosine kinase receptor ligands, or negative regulators such as transforming growth factor B (TGF-/3) (Ogawa, 1993; Sensebe et al., 1997). These cytokines can act independently or in combination to maintain or stimulate hematopoietic stem cell lineage commitment by binding to cell surface receptors. Some cytokines are known to have various biological functions on different types of progenitors, but how they elicit these functions and what signal transduction pathways they activate remains unclear. In addition to cytokines, lineage commitment and maturation of hematopoietic cells is driven by the action of a few lineage-specific transcription factors (Friedman, 2002). As cells differentiate, there is an orchestrated silencing of some genes and activation of others. Experimental data suggest that pluripotent stem/progenitor cells are primed to differentiate down several different lineages by low level transcription of many genes that are characteristic of multiple independent discrete lineages. Over the past years, gene targeting experiments have demonstrated the essential functions of individual transcription factors in specifying commitment to the mature blood lineages. Transcription factors such as SCL/Tal-1, LM02 and GATA-2 are required for the formation and maintenance of stem cells (Robb et al., 1996; Tsai et al., 1994; Yamada et 14 al., 1998). GATA-1 and PU.l serve as the dominant factors for erythroid/megakaryocytic and myeloid development, respectively (Nerlov and Graf, 1998; Rekhtman et al., 1999). Other transcription factors such as GATA-3, Dcaros (T-lymphoid cells) and Pax-5 (B-lymphoid cells) are required for normal lymphopoiesis (Busslinger, 2004; Pai et al., 2003; Smith and Sigvardsson, 2004). Other proteins, such as the Notch receptors, play a central role in the fate decisions of multipotent precursor cells. The Notch pathway is an evolutionarily conserved mechanism that plays a fundamental role in regulating cell-fate decisions of various types of progenitors in both invertebrates and vertebrates (Harper et al., 2003). Notch signalling is involved in multiple developmental processes, including neurogenesis, myogenesis, eye development, and oogenesis (Artavanis-Tsakonas et al., 1999). Hematopoietic cells and bone marrow stromal cells have been shown to express Notch receptors and their ligands, and Notch signalling affects the survival, proliferation and fate choices of progenitors at various stages of hematopoietic development (Ohishi et al., 2003), including whether hematopoietic stem cells self-renew or differentiate (Varnum-Finney et al., 2000), whether common lymphoid precursors undergo T or B cell differentiation (Izon et al., 2002), and whether monocytes differentiate into macrophages or dendritic cells (Ohishi et al., 2001). These observations suggest that Notch signalling can play a fundamental role in regulating hematopoietic development. 1.2.4 The angioblast and evidence of vasculogenesis in the adult Until recently, neovascularization in the adult was thought to occur by angiogenesis only, which represents the sprouting of new blood vessels from pre-existing blood vessels (Tonini et al., 2003). The other means of neovascularization, 15 vasculogenesis, which refers to the de novo formation of blood vessels from endothelial progenitors (angioblasts), was thought to occur only in the embryo (Figure-3). A Angiogenic factors (VEGF, bFGF) B Figure 3 Current concepts for neovascularization in adult ischemic tissues. Angiogenesis (A) and vasculogenesis (B). In recent years, several groups have shown that such endothelial progenitors can be isolated from adult sources such as peripheral blood, bone marrow and umbilical cord blood mononuclear cells (Asahara et al , 1997; Nieda et al , 1997; Peichev et al , 2000; Shi et al., 1998). Likewise hematopoietic cells positive for the hematopoietic stem cell marker CD133 (previously named AC133) are capable of differentiating into endothelial cells in vitro (Gehling et al., 2000). 16 Under the current status, it is impossible to differentiate endothelial progenitors from hematopoietic cells or endothelial cells, since the markers used to isolate endothelial progenitors are also expressed on subsets of hematopoietic cells (CD 133, CD34) and endothelial cells (VEGFR-2, VE-cadherin) (Bhatia, 2001). In circulation, the endothelial progenitors are considered to be included in the cell population expressing CD 133 and VEGFR-2 markers within the subset of CD34 + cells (Gill et al., 2001). When isolated and cultured in vitro, CD133 +VEGFR-2 + endothelial progenitors loose expression of CD133, but acquire features of mature endothelial cells, such as cobblestone morphology, uptake of acetylated low density lipoproteins (AcLDL) and expression of von Willebrand factor (Peichev et al., 2000). Mature endothelial cells derived in vitro from endothelial progenitors also display higher proliferative potential than endothelial cells such as HUVECs (human umbilical vein endothelial cells) (Quirici et al., 2001). It has also been reported that VEGFR-3 and CD133 identify a population of lymphatic endothelial progenitors in fetal liver, bone marrow and peripheral blood (Salven et al., 2003). Table II displays some of the markers found to be expressed on endothelial progenitors, and how these markers overlap with markers found on endothelial cells and subsets of hematopoietic cells. 17 Table II Common markers expressed on human endothelial progenitors, mature endothelial cells and subsets of hematopoietic cells. Surface antigen Hematopoietic cells Endothelial progenitors Mature endothelial cells CD133 Subset (primitive hematopoietic stem cells) + -CD117(cKit) Subset (Hematopoietic stem/progenitor cells) + + CD34 Subset (Hematopoietic stem/progenitor cells) + + VEGFR-1 Subset ? + VEGFR-2 Subset + + VEGFR-3 ? Lymphatic endothelial progenitors + (lymphatic endothelial cells) Tie-1 ? ? + Tie-2 Subset - + VE-cadherin - + + CD31 (PECAM) + + + AcLDL uptake Subset (mainly monocytes/macrophages) + + E-selectin - - + (activated endothelium) Some groups have demonstrated in a mouse model that the recruitment of bone marrow-derived endothelial precursors plays an essential role in tumour vascularization and tumour growth (Lyden et al., 2001). These bone marrow-derived endothelial cells are thought to have the capacity to form new vessels in cancer and ischemic tissues. The relative contribution of bone marrow-derived endothelial cells to tumour vasculature remains unclear however. Studies report a relative contribution of bone marrow-derived endothelial progenitors to neovessel formation ranging from 5 to 25% in response to granulation tissue formation (Crosby et al., 2000) or growth factor-induced neovascularization (Murayama et al., 2002). In tumour neovascularization, the reported ranges are from 35-65% higher than the former events (Hilbe et al., 2004; Lyden et al., 18 2001; Rafii et al., 2002). This issue remains controversial however, as recent studies report no significant contribution of bone marrow-derived cells to tumour vascularization (Gothert et al., 2004a; Machein et al., 2003; Rajantie et al., 2004). 1.3 Vascular Endothelial Growth factor (VEGF) The formation and maintenance of the vascular system is a fundamental requirement for organ development and differentiation during embryonic life and for processes such as wound healing and reproduction in the adult. Many types of cancer cells secrete angiogenic factors that attract endothelial cells to neovascularize solid tumours (Dvorak, 2002; Folkman, 1995). Angiogenesis is the formation of a new vascular network from preexisting vessels. Other than cancer, abnormal angiogenesis is implicated in diseases such as rheumatoid arthritis, diabetic retinopathy and psoriasis (Folkman, 1995). In contrast, in ischemic diseases such as angina and peripheral vascular disease, the relative lack of neovasculature may worsen symptoms. Inhibition or promotion of angiogenesis may therefore offer a new approach for the treatment of many diseases. Many secreted molecules have been shown to promote angiogenesis, including transforming growth factors (TGF) a and p\ tumour necrosis factor a (TNF-a), angiogenin, interleukin 8 (Folkman and Shing, 1992; Risau, 1997), angiopoietins (Maisonpierre et al., 1997; Suri et al., 1996), members of the fibroblast growth factor family (FGF) (Mergia et al., 1989) and vascular endothelial growth factor (VEGF) (Neufeld et al., 1999). These factors can induce a diverse array of signals and initiate downstream intracellular pathways that affect the survival, proliferation, transcription, 19 adherence, permeability and migration of endothelial cells. Some of these signals can be elicited through transmembrane tyrosine kinase receptors that are expressed at high levels in endothelial cells, including VEGFR-1 (flt-1), VEGFR-2 (KDR/flk-1), VEGFR-3 (flt-4), Tie-1 and Tie-2/Tek (Mustonen and Alitalo, 1995; Terman et al., 1992). V E G F was first purified in 1989 by Ferrara and Henzel (Ferrara and Henzel, 1989). Cloning and expression of V E G F revealed that it was the same molecule as vascular permeability factor (VPF) (Keck et al., 1989), a protein first described in 1983 (Senger et al., 1983) which was able to promote extravasation of proteins from tumour-associated blood vessels. It was later reported that the V E G F gene could produce several isoforms by alternative splicing to form active disulfide-linked homodimers (Poltorak et al., 1997; Robinson and Stringer, 2001; Tischer et al., 1989; Tischer et al., 1991). Studies have shown that the loss of a single V E G F allele results in embryonic lethality, pointing to the indispensable role of this protein in development and maintenance of the whole organism (Carmeliet and Collen, 1999; Carmeliet et al., 1996; Ferrara et al., 1996; Patterson et al , 1996). Over the past few years, several members of the V E G F gene family have been identified, including placenta growth factor (P1GF) (Maglione et al., 1991), VEGF-B (Olofsson et al., 1996a; Olofsson et al., 1996b), VEGF-C (Joukov et al., 1996), VEGF-D (Achen et al., 1998) and V E G F - E , a viral protein that binds specifically to VEGFR-2 (Meyer et al , 1999; Ogawa et al., 1998; Wise et al., 1999). 1.3.1 Biological activities of V E G F V E G F is a secreted mitogen whose target cell specificity is mainly restricted to endothelial cells (Partanen et al., 1999). V E G F was first purified from conditioned 20 medium of several cell lines as a glycosylated homodimer of 46 to 48 kDa (Ferrara and Henzel, 1989; Leung et al., 1997). Differentially spliced mRNA transcripts encode five V E G F transcripts coding for polypeptides of 121, 145, 165, 189 and 206 amino acids (Houck et al., 1991; Poltorak et al., 1997; Tischer et al., 1989). V E G F , 6 5 is the predominant V E G F isoform produced by a variety of normal and transformed cells, whereas V E G F 1 2 1 , V E G F 1 4 5 , VEGFigg and V E G F 2 0 6 are rarer forms encountered in some of the tissues expressing the V E G F gene (Houck et al., 1991; Poltorak et al., 1997). V E G F 1 6 5 is a basic, homodimeric glycoprotein of 45 kDa which has affinity for heparan sulfates, and is partially sequestered in the pericellular matrix. In contrast, V E G F 1 2 1 is a weakly acidic polypeptide which lacks the heparin binding domain, and is secreted as a freely diffusible protein (Houck et al., 1992). VEGFigg and V E G F 2 0 6 are more basic, and bind heparan sulfates with greater affinity than V E G F 1 6 5 (Houck et al., 1992). These two molecules are almost completely sequestered in the extracellular matrix, but can be released as soluble forms by heparinase or by plasmin, which generate a bioactive proteolytic fragment (Park et al., 1993). This suggests that V E G F may become available to endothelial cells in two ways: as freely diffusible proteins ( V E G F 1 2 1 and VEGF165) or following protease activation and cleavage of the two longer isoforms ( V E G F ] 8 9 and VEGF 2 o6) - Thus, plasminogen activation and generation of plasmin could play an important role in angiogenesis. Although V E G F is a mitogen for vascular endothelial cells, it lacks significant mitogenic activity for most other cell types, because the expression of V E G F receptors is mainly restricted to endothelial cells (Barleon et al., 1994; Plouet and Bayard, 1994). Moreover, it can act as a survival factor for endothelial cells cultured in serum-deprived 21 conditions (Karsan et al., 1997). The mechanisms of the V E G F cytoprotective effect have not been well-studied but may involve the PI3-kinase pathway and upregulation of members of the Bcl-2 and LAP (Inhibitor of Apoptosis) family of anti-apoptotic proteins (Tran et al., 1999). V E G F also promotes expression of intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 in endothelial cells, which can result in the adhesion of activated leukocytes to endothelial cells (Melder etal., 1996a; Melder et al., 1996b). In hematopoiesis, V E G F can inhibit the maturation of antigen-presenting cells, such as dendritic cells, thereby inhibiting the ability of the immune system to recognize and target tumour cells (Gabrilovich et al., 1998; Kusmartsev and Gabrilovich, 2002; Takahashi et al., 2004). In vivo, V E G F is known for its ability to induce vascular leakage, thus causing the exudation of plasma proteins (Bates et al., 1999). This leakage results in the laying down of a provisional matrix of fibrin and fibronectin allowing endothelial migration (Dvorak et al., 1995). V E G F possesses a potent ability to increase microvessel permeability to a 50,000-fold higher level than histamine (Dvorak et al., 1995). 1.3.2 Regulation of V E G F expression V E G F is a key regulator of angiogenesis, and its expression is regulated by many external factors. Many cytokines, which do not promote angiogenesis, can modulate angiogenesis indirectly by regulating V E G F expression in specific cell types. Molecules that can increase V E G F expression include epidermal growth factor (EGF), insulin-like growth factor 1, fibroblast factor 4 (FGF-4) (Deroanne et al., 1997), TGFp (Pertovaara et al., 1994), TNFa (Ryuto et al., 1996), PDGF (Finkenzeller et al., 1997) and IL-6 (Cohen et al., 1996). On the other hand, cytokines such as IL-10 and IL-13 can inhibit V E G F 22 expression (Matsumoto et al., 1997). In addition, hydrogen peroxide, which is produced by neutrophils that invade a wound in the healing process, or U V - B radiation also potentiate V E G F production by keratinocytes (Bielenberg et al., 1998; Brauchle et al., 1996; Mildner et al., 1999). Nitric oxide can also increase V E G F production, which, in turn upregulates the production of nitric oxide in a positive feedback loop (Frank et al., 1999; Kroll and Waltenberger, 1999; Shen et al., 1999). Hypoxia is also a major stimulator of V E G F expression. Since cells in a solid tumour are often hypoxic, this could account, at least in part, for the increased V E G F expression in many types of cancer cells (Richard et al., 1999). The transcription ofthe V E G F gene, under hypoxic conditions is mediated by the binding of hypoxia-inducible factor 1 (HIF-1) to a hypoxic responsive element localized in the V E G F promoter (Levy et al., 1995; Liu et al., 1995; Semenza et al., 1999). Hypoxia also stabilizes the V E G F transcript by inducing proteins that bind and stabilize the 3' untranslated region (UTR) of V E G F mRNA (Stein et al., 1998; Stein et al., 1995). Specific transforming events can also result in induction of V E G F gene expression. For example, oncogenic mutations or amplification of ras lead to V E G F mRNA stabilization (Okada et al., 1998). The von Hippel-Lindau (vHL) tumour suppressor gene has also been implicated in up-regulation of V E G F (Brieger et al., 1999; Siemeister et al., 1996). Wild type vHL inhibits the production of several hypoxia-regulated proteins such as V E G F at the post-transcriptional level. vHL is known to inhibit the activity of protein kinase C zeta and delta (Pal et al., 1997). In the absence of wild-type vHL, these kinases remain active and V E G F mRNA is stabilized as a result of 23 constititutive interactions of various proteins that are normally induced by hypoxia with the 3'UTR (Pal etal., 1997). Finally, loss of the wild-type tumour suppressor p53 also correlates with up-regulation of V E G F expression, and with increased angiogenesis in developing tumours (Kieser et al., 1994; Saito et al., 1999). 1.3.3 V E G F receptors The V E G F family members are ligands for a set of mammalian tyrosine kinase receptors (VEGF receptors, VEGFRs) (Figure 4). Three V E G F tyrosine kinase receptors have been identified and cloned: VEGFR-1 also known as fit-1 (fins-like tyrosine kinase) (de Vries et al , 1992), VEGFR-2 (KDR/flk-1) (Terman et al., 1992) and VEGFR-3 (flt-4) (Neufeld et al , 1994). VEGFR - 1 V E G F R - 2 V E G F R - 3 Neuropil in-1 Neurop i l in -2 (flt-1) (KDR/flk-1) (flt-4) Figure 4 Receptors of the V E G F family. These receptors include three tyrosine kinase receptors: VEGFR-1 (flt-1), VEGFR-2 (KDR/flk-1) and VEGFR-3 (flt-4). Other receptors for V E G F also include neuropilin-1, which binds VEGFRs and P1GF-2, and neuropilin-2, which binds VEGFRs- Ligand binding causes dimerization and transphosphorylation of V E G F receptors. 24 These receptors form a subfamily distinguished by the presence of immunoglobulin-like loops in their extracellular portion, a single transmembrane region and a split tyrosine-kinase domain in the intracellular region. The presence of the split kinase domain places these receptors in the subfamily of class III receptor tyrosine kinases, which includes c-Fms, c-Kit and the a and p chains of the PDGF receptor (Shibuya et al., 1999). V E G F binds and activates both VEGFR-1 (de Vries et al., 1992) and VEGFR-2 (Kendall et al., 1999), whereas P1GF (Athanassiades and Lala, 1998; Shore et al., 1997) and VEGF-B (Olofsson et al., 1998) bind only VEGFR-1. V E G F - C (Joukov et al., 1996; Kukk et al., 1996) and VEGF-D (Achen et al., 1998) bind VEGFR-2 in addition to VEGFR-3 (Figure 4). VEGFR-1 has a higher binding affinity for V E G F (Kd of 10-20 pM) than VEGFR-2 (Kd of 75-125 pM) (de Vries et al., 1992; Terman et al., 1992). VEGFR-3 binds VEGF-C and VEGF-D (Achen et al., 1998; Ristimaki et al., 1998). VEGFR-1 and VEGFR-2 are expressed mainly on endothelial cells, although other cell types have also been shown to express these receptors. VEGFR-1 has been shown to be expressed in monocytes (Barleon et al., 1996), megakaryocytes (Casella et al., 2003) and hematopoietic stem cells (Hattori et al., 2002) whereas VEGFR-2 is also expressed in hematopoietic stem cells (Ziegler et al., 1999) and megakaryocytes (Katoh et al., 1995). In addition, some tumour cells, such as melanoma cells, leukemic blasts, lymphoma cells and multiple myeloma cells, express VEGFR-1 or VEGFR-2 (Bellamy et al., 1999; Gitay-Goren et al., 1993; Graeven et al., 1999; Herold-Mende et al., 1999; Padro et al., 2002; Ria et al., 2003; Zhang et al , 2004). VEGFR-3, which is expressed mainly in lymphatic endothelium, has been shown to be involved in the regulation of lymphatic angiogenesis (Jussila et al., 1998). However, recent evidence indicates that 25 VEGFR-3 is required for vasculogenesis in mouse embryos, thus indicating that this receptor plays an essential role in the development of the cardiovascular system (Dumont etal., 1998). To provide evidence of the proposed role of VEGFR-1 and VEGFR-2 in vascular development, the genes encoding VEGFR-1 and VEGFR-2 have been disrupted in mice. Because VEGFR-2 is expressed in the primitive blood islands, it has been proposed that this receptor may play an essential role during the initial stages of vasculogenesis, when both endothelial and blood progenitor cells are presumed to differentiate from a common precursor, the hemangioblast (Hatva et al., 1996). Embryos lacking wild-type VEGFR-2 died in utero by day 9. Morphological analysis revealed an absence of blood islands and hemopoietic progenitor cells. These embryos were also characterized by the absence of mature endothelial cells (Shalaby et al., 1997; Shalaby et al., 1995). Hence, VEGFR-2 is required for the formation, migration and/or proliferation of endothelial and hemopoietic cells during vasculogenesis. In contrast to the critical role of VEGFR-2 in vasculogenesis, endothelial and hemopoietic cell differentiation was found to occur in embryos lacking the VEGFR-1 gene. However, the organization of vascular structures was grossly abnormal, and the mutant embryos also died in utero by day 9. Instead of fusing into typical blood vessels, the endothelia in VEGFR-1 mutants formed abnormal vascular channels, characterized by large lumens containing trapped endothelial cells mixed with blood cells. Moreover, an excessive number of endothelial cells accumulated in the VEGFR-1 knockout embryos (Fong et al., 1996; Fong et al., 1995; Fong et al , 1999). These studies suggest that VEGFR-1 is necessary to repress excessive endothelial cell proliferation. 26 As with VEGF, the expression of VEGFR-1 and VEGFR-2 is reported to be affected by hypoxia, although to a lesser extent than V E G F (Gerber et al., 1997; Tuder et al., 1995). VEGFR-1 transcription is upregulated by hypoxia (Suzuki et al., 1999) whereas VEGFR-2 production is also up-regulated by hypoxia, but at the post-transcriptional level (Waltenberger et al., 1996). This hypoxia-induced upregulation of VEGFR-1 and VEGFR-2 may be indirectly triggered, since V E G F is known to potentiate the expression of these two receptors (Kremer et al., 1997; Shen et al., 1998; Tsopanoglou and Maragoudakis, 1999). Of the seven IgG-like domains of the extracellular portion of VEGFR-1 and VEGFR-2, only domains 2 and 3 are required for the tight binding of V E G F to these two receptors (Fuh et al., 1998). Several studies have mapped the binding site for V E G F to the second Ig-like domain of the receptors. V E G F also interacts with the neuronal cell-guidance receptors, neuropilin-1 and neuropilin-2 (Miao et al., 1999; Soker et al., 1998). These receptors were initially described to bind several types of semaphorins, which are factors that act as axonal repellents (Chedotal et al., 1998; Chen et al., 1998; He and Tessier-Lavigne, 1997). The neuropilins, which are known to be expressed in neurons, some cancer cell lines and endothelial cells, bind to V E G F i 6 5 , but not VEGF121 (Neufeld et al., 1999; Soker et al., 1998). In addition to V E G F 1 6 5 , neuropilin-1 has also been found to bind the heparin binding form of P1GF, P1GF-2 (Migdal et al., 1998). The neuropilins have only a short intracellular domain. It has recently been shown that the cytoplasmic domain of neuropilin-1 contains a central PSD- 95/Dlg/ZO-l (PDZ) domain, which may act as a protein interaction module (Jelen et al., 2003), and a C-terminal acyl carrier protein 27 domain (Cai and Reed, 1999), thus indicating that neuropilin-1 might be able to transduce a signal. However, no responses to VEGF165 were observed when cells expressing neuropilin-1 but no other V E G F receptors were stimulated with V E G F 1 6 5 (Soker et al., 1998). It is not known whether V E G F binding is sufficient for signal transduction via a neuropilin-1-associated hypothetical signal-transducing polypeptide chain although the enhanced mitogenic signalling of VEGFR-2 in neuropilin-1 -overexpressing cells suggests such a possibility. Mouse embryos lacking a functional neuropilin-1 die in utero because their cardiovascular system fails to develop properly, indicating that this receptor is probably an important regulator of blood vessel development (Kawasaki et al., 1999; Kitsukawa et al., 1997). Furthermore, overexpression of neuropilin-1 under the p-actin promoter is lethal because of severe anomalies of the nervous and cardiovascular systems (Kitsukawa et al., 1995). The cardiovascular defects may result from modulation of V E G F bioactivity and VEGF-induced angiogenesis by abnormal neuropilin-1 levels. Therefore, it has been proposed that neuropilin-1 acts as a VEGF]65 co-receptor. This postulate is supported by studies showing that VEGFR-2 binds V E G F 165 more efficiently in cells expressing neuropilin-1, resulting in a better migratory response to V E G F 1 6 5 (Soker et al., 1998). However it does not seem that neuropilin-1 acts as a VEGFR-1 co-receptor, since P1GF-1 and P1GF-2, which both bind VEGFR-1, promote migration of endothelial cells equally well (Clauss et al., 1996). Neuropilin-2 also binds V E G F i 6 5 , but its expression pattern is different from that of neuropilin-1 or VEGFR-1, in that it is absent from endothelial cells of capillaries (Chen et al., 1997; Giger et al., 1998). 28 1.3.4 V E G F signal transduction Studies of receptor tyrosine kinases in a number of different systems have clearly shown that the pathways activated by these molecules can lead to drastic changes in cells, including their fate, survival, proliferation, migration and adhesion (van der Geer et al., 1994). Although VEGFR-1 and VEGFR-2 are both activated by the same ligand, their downstream signalling pathways cause different cellular responses. V E G F SMOOTH MUSCLE RELAXATION/ VASODILATION V E G F R PLCy Rac \u00E2\u0080\u00A2 SAPK-2 RAFTK IP3 V C a 2 I eNOS \ \ JNK N n DAG \ G C cGMP P K C I RAF t P K G / Grb2/Shc/S0S - > RAS - > MEK I PI3 Kinase \u00E2\u0080\u00A2 MAPK(ERK) PKB/Akt PROLIFERATION MKK3 I p38 I MAPKAPK-2 I H S P 2 7 I ACTIN REORGANIZATION CELL SURVIVAL/ PROLIFERATION Jt E t s - 1 ^ JNK90% of the wells were found to contain single cells, and the remaining wells were empty. Wells containing two cells were found with an approximate frequency of 1 in 200. Only wells containing a single cell were used for transplantation. To guarantee the survival of the recipients during the lag phase between the injection of the donor cell and the development of sufficient functional single cell-derived peripheral blood cells, stem cell-depleted bone marrow from GFP\" congenic mice was coinjected with the single hematopoietic stem cells. Stem cell-depleted bone marrow was obtained by removal of Sca-1+ cells using Sca-l-phycoerythrin (clone D7, PharMingen), followed by anti-phycoerythrin magnetic beads (Miltenyi Biotec) and an automated magnetic cell sorter (autoMACS, Miltenyi Biotec). One million of these helper cells were added to each well containing a single hematopoietic stem cell, and the total volume of the well was injected into lethally irradiated (950 cGy) CD45.2 C57BL/6 mice. 2-8-3 CFU-Spleen (CFU-S t 2 ) assay Transduced GFP + bone marrow cells were cultured in EVIDM supplemented with 10% FBS with or without 100 nM AP20187 for 7 days. 25,000 cells were injected into the tail vein of lethally irradiated B6C3 mice. 12 days later, mice were sacrificed and spleens were harvested, fixed in Telleyesniczky's fixative (ethanol 70%:glacial acetic acid:formalin / 20:1:1) and hematopoietic colonies counted. 2-9 AP20187 formulation AP20187 was a gift from Ariad Pharmaceuticals. Lyophilized AP20187 was dissolved in 100% ethanol at a concentration of 62.5 mg/ml and stored at - 2 0 \u00C2\u00B0 C . For in vitro use, the ethanol stock was diluted in complete culture medium to the desired concentration 54 immediately before use. The final concentration of ethanol in the culture medium was below 0.5%. For in vivo use, peritoneal injections were prepared from the 62.5 mg/ml ethanol stock diluted to 2.5 mg/ml in an injection solution consisting of 4% ethanol, 10% PEG-400, and 1.7% Tween 20 in water. All injections were administered to mice within 30 min of dilution into the injection solution. The volume of injection solution was adjusted according to mouse body weight to deliver 10 mg AP20187 per kg mouse. The average injection volume was 100 ul per mouse. For in vivo experiments, mice were injected with AP20187 daily for 10 consecutive days. 2-10 Antibody staining for Fluorescence Activating Cell Sorting (FACS) analysis After red blood cells lysis, cells from bone marrow and peripheral blood were washed and resuspended in PBS containing 4% goat serum (Sigma) followed by monoclonal primary antibody staining for 1 hour at room temperature. Antibodies used for analysis were anti-Mac-1/CDllb (monocytes), anti-Gr-1 (granulocytes), anti-B220 (B lymphocytes), anti-CD5 (T lymphocytes), anti-Terll9 (erythroid progenitors), anti-CD 144/VE-cadherin and anti-VEGFR-2 (BD Pharmingen). Isotype rat IgG was used for negative control. Cells were then washed in PBS containing 4% goat serum and incubated for 30 minutes with phycoerythrin (PE)-conjugated secondary antibody. After subsequent washes, cells were resuspended in PBS for FACS analysis. Samples were run on an EPICS ELITE-ESP flow cytometer (Beckman Coulter), and data were analyzed with FCS Express, version 2 (De Novo softwares, Thornhill, ON). 55 2-11 Preparation of cDNA and reverse transcription-polymerase chain reaction (RT-PCR) Total cellular RNA was extracted from murine bone marrow using Qiagen RNeasay Quick spin columns (QIAGEN) as described by the manufacturer. The purified total RNA prep was used as a template to generate first strand cDNA synthesis using random hexamer (Invitrogen) priming and reverse transcriptase (Superscript II; Invitrogen). PCR was performed using the following primer pairs: Table IV Murine primers and PCR conditions used in this thesis Target Sense Anti-sense Tm(\u00C2\u00B0C) Cycles Size (bp) M-CSF agctgcttcaccaaggactatgag ctctgtcaacggcctgtctgttat 55 35 737 Tpo tgtggactttagcctgggagaatg ttgactctgaatccctgaagcctg 55 35 491 SCF ctgcgggaatcctgtgactgataa cgggacctaatgttgaagagagca 55 35 430 Flt3-ligand gacacctgactgttacttcagcca acgaatcgcagacattctggtagg 55 35 290 IL-6 gttctctgggaaatcgtgga tgtactccaggtagctatgg 53 35 207 GM-CSF cttggaagcatgtagaggccatca cttgtgtttcacagtccgtttccg 55 35 254 V E G F gctttactgctgtacctccaccat atctctcctatgtgctggctttgg 55 35 319 BMP-2 atcaactagaagccgtggaggaac catggttagtggagttcaggtggt 55 35 686 BMP-4 cagaaatggttcctggacacctca cacaatccaatcattccagcccac 55 32 415 Jagged-1 aatggagactccttcacctgt cgtccattcaggcactgg 53 35 383 Delta-1 tggttctctcagagttagcagag agacccgaagtgcctttgta 55 35 409 Delta-4 gcattgtttacattgcatcctg gtagctcctgcttaatgccaaa 55 30 473 GAPDH gcatggccttccgtgt gggccgagttgggatagg 53 22 256 PCR products were detected by electrophoresis on 2.0% agarose gels. 2-12 ELISA Murine GM-CSF levels were determined by standard sandwich ELISA according to the instructions of the manufacturer (R&D Systems, Minneapolis, M N , limit of detection: 1 pg/ml). For detection of human V E G F 1 6 5 in mouse serum, the following protocol was used: a 96-well plate (Costar) was coated with an anti-VEGF capture antibody (R&D Systems, Minneapolis, MN) overnight. Wells were blocked for 1 hour with blocking 56 buffer (PBS containing 1% BSA, 5% sucrose and 0.05% sodium azide) and then were incubated with serum samples or V E G F standards for 2 hours at room temperature. Wells were washed 3 times with PBS containing 0.05% Tween, and a biotinylated anti-VEGF detection antibody (R&D Systems) was added and incubated at room temperature for 2 hours. After washing, the plate was incubated with a streptavidin-Horseradish peroxidase (HRP) conjugate (Vector Laboratories, Burlingame, CA) and incubated at room temperature for 20 min. Plates were washed and incubated with a substrate solution (0.1 mg/ml 3,5,3',5'-tetramethylbenzidine (TMB) and 0.05% H2O2 in sodium acetate buffer pH 5.5). The colorimetric reaction was stopped by addition of 50 ul stop solution (1M H2SO4). Absorbance at 450 nm was read on a microplate reader with wavelength correction set at 540 nm. Good linear correlation was observed with standards in the range between 7.8 and 1000 pg/ml. 2-13 Bone marrow cells viability assays Sorted bone marrow cells were plated in IMDM supplemented with 10% FBS with or without the addition of 100 nM AP20187. Cells were harvested at various times and counted on a hemacytometer. 2-14 Hematopoietic colonies assay For in vitro assays, transduced GFP + bone marrow cells were grown in vitro in JJVIDM supplemented with 10% FBS, with or without 100 nM AP20187, for 7 and 14 days. At these time points, hematopoietic clonogenic progenitor frequencies were determined by plating 20,000 bone marrow cells in methylcellulose medium containing 50 ng/ml SCF, 10 ng/ml IL-3, 10 ng/ml IL-6 and 3 U/ml erythropoietin (Methocult GF M3434, Stem Cell Technologies). For quantitation of in vivo murine progenitors, GFP + and GFP\" bone 57 marrow cells were sorted for GFP expression and plated in methylcellulose medium. Resultant colonies were scored after 10 days of incubation. 2-15 Immunofluorescence and microscopy For BrdU staining, sorted GFP + bone marrow cells were cultured in EVIDM supplemented with 10% FBS for 2 days, then treated for 2 hours with 10 uM BrdU with or without 100 nM AP20187. Cytospin preparations of bone marrow cells were fixed with 4% paraformaldehyde for 5 minutes, washed with PBS, and permeabilized with ice cold methanol for 1 min. Slides were then incubated for 20 min at 37\u00C2\u00B0C with 2N HC1 to denature DNA. Slides were blocked in PBS containing 5% Goat serum and 0.1% Triton X-100 for ten minutes, followed by 1 hour incubation with primary antibody (Anti-BrdU conjugated with AlexaFluor 594 (Molecular Probes, Eugene, OR), 1:50 dilution in PBS containing 5% goat serum and 0.1% Triton X-100). After washing, nuclear DNA was stained with DAPI (1 u.g/ml), and slides were mounted in anti-fading solution. For activated caspase 3 staining, cytospin preparations of hematopoietic progenitors grown in culture for 14 days in IMDM containing 10% FBS were stained using the same protocol as above (DNA denaturation step was omitted) and the following antibodies were used: anti-activated caspase 3 (BD Pharmingen, San Diego, CA) and goat anti-rabbit Ig conjugated with Texas Red (Molecular Probes). ). Cells were visualized through a 40x Neofluor objective (numerical objective 0.75) using a Zeiss Axioplan II Imaging inverted microscope (Carl Zeiss, Toronto, Canada), and images were captured with a 1350EX cooled charge-coupled device (CCD) digital camera (Qlmaging, Burnaby, BC, Canada) using Northern Eclipse software (Empix Imaging, Mississauga, ON, Canada). 58 2-16 Immunoblotting Proteins from total cellular extracts were separated by SDS-PAGE and assessed by immunoblotting. Antibodies against phosphorylated VEGFR-2 and total and phosphorylated Akt and Erk MAP kinase were obtained from Cell Signalling Technology (Mississauga, ON). Anti-HA antibody was obtained from B A B C O (Richmond, CA). The kinase inhibitors LY294002 and U0126 were obtained from Calbiochem (La Jolla, CA). 2-17 Endothelial progenitor assays Human mononuclear cells were obtained from umbilical cord blood. Cord blood samples, which were collected during standard or caesarean delivery of full-term infants, were harvested in 200 ml plastic bottles containing 40 ml of IMDM containing 800 U/ml heparin. Ammonium chloride lysis was performed to remove red blood cells. Mononuclear cells were either selected for the surface marker CD133 using anti CD133-coupled magnetic micro-beads (Miltenyi Biotech) as indicated by the company or lineage depleted (Lin-) using lineage panel antibodies (CD2, CD3, CD 14, CD 16, CD 19, CD24, CD56, CD66b, Glycophorin A; Stem cell technologies, Vancouver, Canada) and separated using a magnetic separation device, StemSep (Stem Cell technologies) or further used without selection. For endothelial progenitor assays, cells were plated onto tissue culture dishes in MCDB131 medium supplemented with 20% FBS, endothelial cell growth supplement (ECGS, Sigma), heparin, 5 ng/ml human bFGF and 30 ng/ml human VEGF. For 3 consecutive days, non-adherent cells were replated onto a new tissue culture dish in order to remove any contaminating endothelial cells. After 3 days, non-adherent cells were harvested, counted and resuspended in fresh medium and plated onto tissue culture dishes coated with 0.2% gelatin. Half of the medium was replaced twice 59 weekly. Endothelial colonies were scored after 4 weeks, as identified by staining with the P1H12 antibody (Chemicon). 2-18 Tumour tissue immunohistochemical staining Tumour tissues were fixed overnight at -20\u00C2\u00B0C in 2% paraformaldheyde / 30% glycerol. After washing with PBS, tumours were embedded in OCT compound and sectioned with a cryostat. Sections (8 Lim) were stained with antibodies against VE-cadherin, CD31 and C D l l b (BD pharmingen). Secondary antibodies were either Alexa 594- or Alexa 633-conjugated (Molecular Probes). 2-19 lacZ and hPLAP staining Tumour tissues were fixed in 4% paraformaldehyde for 4 h, and cryopreserved by incubating overnight in 15% sucrose in PBS at 4\u00C2\u00B0C. The samples were washed in PBS before being embedded in OCT compound. Sections were fixed in 0.2% glutaraldehyde for 10 min, washed in 100 mM sodium phosphate (pH7.3) and washing buffer solution (2 mM MgCb, 0.01% sodium deoxycholate and 0.02% NP-40 in lOOmM sodium phosphate pH 7.3) and stained in fresh X-gal solution (0.5 mg/ml X-gal, 5mM potassium ferrocyanide, 5mM potassium ferricyanide in washing buffer solution) at 37\u00C2\u00B0C overnight. For alkaline phosphatase staining, slides were washed three times in PBS for 5 min, and endogenous alkaline phosphatase was inactivated by incubating slides at 70\u00C2\u00B0C in PBS for 30 min. Slides were rinsed in PBS, washed in alkaline phosphatase buffer (100 mM Tris-HCl pH9.5, 100 mM NaCl, 10 mM MgCl 2) and incubated in NBT/BCIP stain (Sigma) for 15 to 40 min at room temperature, in the dark. Slides were washed in PBS and either stained for VE-cadherin or dehydrated through an ethanol series and mounted with coverslips. 60 2-20 Statistical Analysis Results were analyzed by analysis of variance (ANOVA) to ascertain differences between groups, followed by a Tukey test for multiple comparisons. P<0.05 was considered significant. 61 Chapter 3 Biological effects and signalling pathways induced by VEGFR-2 isolation 3.1 Introduction The effects of V E G F on a variety of cell types have been studied in recent years. V E G F stimulates angiogenesis by induction of endothelial cell proliferation and by prevention o f endothelial cell death, i.e., anti-apoptotic effects (Ruhrberg, 2003). It was shown that V E G F stimulation leads to the activation of signalling pathways such as the PI3-kinase/Akt-signalling pathway and the Erk 1/2 M A P kinase pathway, which mediate V E G F effects (Ferrara, 1999b). Due to the fact that numerous receptors can bind V E G F ( V E G F R - 1 , V E G F R - 2 , V E G F R - 3 , neuropilin-1 and neuropilin-2) and that most cell types responsive to V E G F express more than one receptor, the role of each receptor in transmitting V E G F signals and biological effects remains unclear. Due to the critical importance o f V E G F R - 2 in the generation of the endothelial and hematopoietic lineages, and the increasing evidence o f its role in adult vasculogenesis, angiogenesis and hematopoiesis, we chose to study the biological effects triggered by V E G F R - 2 in isolation. In the first part of this thesis, we studied the specific signalling pathways and some of the biological effects triggered by V E G F R - 2 activation. We used primary fibroblasts transduced with V E G F R - 2 to analyze the role of this receptor in endothelial differentiation, proliferation and migration. Primary fibroblasts were used as a model since they do not express endogenous V E G F receptors, which makes studying V E G F R - 2 signalling without the interference o f other V E G F receptors possible. Furthermore, fibroblasts have been reported to be able to transdifferentiate into endothelial cells in vivo (Kon and Fujiwara, 1994). Others have shown that adenoviral-mediated M y o D gene transfer into cultured human fibroblasts induces myogenic conversion (Lattanzi et al., 63 1998). Since VEGFR-2 is critical in the establishment of the endothelial lineage, we also studied whether signals transmitted through VEGFR-2 are sufficient to promote an endothelial phenotype in fibroblasts. 3.2 Results 3.2.1 VEGFR-2 transmits a mitogenic signal in response to V E G F stimulation in primary fibroblasts. To determine the biological responses induced by VEGFR-2 in the absence of other V E G F receptors, we generated a cell line of primary human foreskin fibroblasts expressing VEGFR-2 (HFF-VEGFR-2) by retroviral transduction. Expression of VEGFR-2 in transduced HFF was verified by Western blot (Figure 6). Phase contrast micrographs of the cell lines are seen in Figure 6B. 64 Figure 6 Generation of a primary human foreskin fibroblast cell line expressing VEGFR-2. (A) Human foreskin fibroblasts (HFF) were transduced with either the empty L N C X retroviral vector or L N C X containing the VEGFR-2 cDNA. Level of protein expression was determined by Western blot of cellular extracts from H M E C , HFF-Neo and HFF-VEGFR-2 cells. (B) Phase contrast micrographs showing the morphology of monolayers of HFF-Neo and HFF-VEGFR-2 cells incubated with 20 ng/ml V E G F for four days. In all experiments, HFF transduced with the empty vector pLNCX (HFF-Neo) were used as a negative control. Several studies indicate that VEGFR-2, rather than VEGFR-1 stimulation is responsible for the mitogenic effect of V E G F in endothelial cells (Meyer et al., 1999; Takahashi and Shibuya, 1997). To assess this, we examined the proliferative effect of V E G F in HFF-VEGFR-2 cells. HFF-VEGFR-2 cells were found to increase in number, when stimulated with increasing concentrations of V E G F for 48 hours (Figure 7A). By contrast, HFF-Neo cells did not show a proliferative response when stimulated 6 5 with V E G F for the same amount of time. These results were confirmed by BrdU incorporation assays (Figure 7B). 0.5- : 0 10 20 30 40 50 VEGF (ng/ml) HFF-Neo HFF-Neo HFF- HFF-+VEGF VEGFR-2 VEGFR-2 +VEGF Figure 7 V E G F induces a proliferative response in HFF-VEGFR-2 cells but not in HFF-Neo cells. The proliferative effect of V E G F on HFF-Neo and HFF-VEGFR-2 was measured by either M T T assay (A) or BrdU incorporation assay (B). (A) HFF-Neo (\u00E2\u0080\u00A2) and HFF-VEGFR-2 cells (\u00E2\u0080\u00A2) (5000 cells/well) were incubated in serum-free medium in the presence of V E G F for 48 hours. Relative cell number in each well was estimated by measuring absorbance at 570 nm following incubation with MTT. (B) Quiescent cells were incubated with BrdU with or without V E G F for 48 hours, then fixed and stained as described. The extent of D N A synthesis was evaluated by counting the number of cells that stained positive for BrdU. Data represent the mean of four independent experiments \u00C2\u00B1 S E M . 3.2.2 V E G F is chemotactic for HFF-VEGFR-2 but not HFF-Neo cells Several reports have shown that V E G F can induce endothelial chemotaxis (Albini et al., 1996; Meyer et al , 1999). Using a modified Boyden chamber assay, we studied the 66 chemotactic response of H M E C , HFF-Neo and HFF-VEGFR-2 cells to VEGF. V E G F (20 ng/ml) induced a significant increase of H M E C and HFF-VEGFR-2 migration relative to the control (DMEM + 0.1%BSA), causing a 3.5 and a 5-fold increase, respectively, 8 hrs post-treatment (Figure 8). Again, V E G F did not induce chemotaxis in HFF-Neo cells. FGF-2, used as a positive control, was chemotactic for all three cell types. HMEC HFF-Neo HFF-VEGFR-2 Figure 8 V E G F simulates migration of H M E C and HFF-VEGFR-2 cells, but not HFF-Neo cells. Serum free-medium with or without V E G F or FGF-2 was placed in the lower chamber of a 24-well Transwell. A total of 5 x 10s cells/well were seeded in the upper chamber, and cells were allowed to migrate for 8 h through the collagen-coated polycarbonate membrane. Cells that had migrated were quantified by counting three random high power fields/well. Data represent the mean of three independent experiments + SEM. 3.2.3 V E G F activates Erkl/2 and p38 MAP Kinases in HFF-VEGFR-2, but not in HFF-Neo cells We next examined whether the MAP kinase cascade, a convergent pathway in the mitogenic action of many growth factors, including VEGF, can be mediated through the activation of VEGFR-2 alone (Kroll and Waltenberger, 1997; Mukhopadhyay et al., 1998). Serum-starved cells were stimulated with V E G F (20 ng/ml) for 0 to 30 min, and Erk was immunoprecipitated from cell lysates. Using an anti-phospho-Erk antibody, we observed that Erk phosphorylation increased in response to V E G F stimulation in HFF-67 VEGFR-2 cells, but not in HFF-Neo cells (Figure 9A). Maximum phosphorylation of Erk peaked at 15 min in HFF-VEGFR-2 cells. The kinetics of Erk phosphorylation was mirrored by Erk activity as assayed by in vitro phosphorylation of MBP (Figure 9B). We observed an increase in Erk activation after 10 minutes in HFF-VEGFR-2 cells. As with the phosphorylation, activation of Erk peaked at 15 minutes in HFF-VEGFR-2 cells. No Erk activation was detected in HFF-Neo cells following V E G F stimulation. B Time (min) 0 5 10 15 20 30 Figure 9 V E G F induces phosphorylation and activation of Erkl/2 MAP kinase in HFF-VEGFR-2 but not HFF-Neo cells. Quiescent cells were incubated with 20 ng/ml VEGF for 0 to 30 minutes as indicated. (A) Phosphorylation of Erkl/2 was determined by Western blotting using an antibody specific to phosphorylated Erkl/2 (upper panel, P-Erk-1). Membranes were reprobed with an anti-Erk antibody as a loading control. (B) Activity of Erkl/2 was determined by an in vitro kinase assay using MBP as a substrate. Expression of Erkl/2 was determined by Western blotting. Data show one experiment of four independent experiments. We also examined the effect of V E G F on p38 phosphorylation for transduced fibroblasts by Western blot using an anti-phospho-p38 antibody. We found that VEGF 68 stimulation resulted in p38 phosphorylation in HFF-VEGFR-2 cells, but had no effect in HFF-Neo cells (Figure 10). Maximum phosphorylation of p38 was detected after 15 minutes in HFF-VEGFR-2 cells. Again, V E G F had no effect in mock-transduced cells. Time (min) 0 5 10 15 20 30 P-p38 }~ HFF-Neo p38 P-p38 p38 Figure 10 V E G F induces phosphorylation of p38 MAP kinase in HFF-VEGFR-2 but not HFF-Neo cells. Quiescent cells were incubated with 20 ng/ml V E G F for 0 to 30 minutes as indicated. Phosphorylation of p38 was determined by Western blotting using an antibody specific to phosphorylated p38 ( P-p38). Data represent one experiment of three independent experiments. 3.2.4 V E G F does not induce expression of endothelial markers in primary fibroblasts transduced with VEGFR-2 A previous study has suggested that fibroblasts can transdifferentiate into endothelial cells (Kon and Fujiwara, 1994). Others have shown that VEGFR-2-expressing cells isolated from chick gastrula can differentiate into either hematopoietic or endothelial lineages and that the presence of V E G F in the culture medium is absolutely required for endothelial differentiation (Eichmann et al., 1997). To assess whether VEGFR-2 activation is sufficient for induction of expression of endothelial markers in fibroblasts, HFF-VEGFR-2 and HFF-Neo cells were incubated in the presence of 10 ng/ml V E G F for 4 days, after which expression of various endothelial markers was assayed by Western blot. We were not able to detect expression of endothelial markers such as VEGFR-1, Tie-1, Tie-2 and eNOS, as shown by Western blot (Figure 11). Expression of eNOS (Kroll and Waltenberger, 1998) and Tie-1 (McCarthy et al , 1998) } HFF-VEGFR-2 69 have been shown to be increased following V E G F stimulation of endothelial cells. However, we did not observe any increase in expression of these markers in the presence of V E G F , even in H M E C . A s seen in Figure 6 A , we did not observe any morphologic changes in H F F - V E G F R - 2 relative to HFF-Neo cells. o LU 5 X o LU > O W W \u00E2\u0080\u00A2-, Cc QL \u00E2\u0080\u00A27 LL LL O O ( S O a> a LU LU z z > > V E G F R - 1 T u b u l i n Tie-1 , T u b u l i n -Tie-2 T u b u l i n e N O S T u b u l i n Figure 11 V E G F does not induce expression of endothelial markers in H F F - V E G F R - 2 cells. Cells were incubated with V E G F for four days before cell extracts were collected and processed for western blotting. Endothelial markers examined included V E G F R - 1 , Tie -1, Tie-2 and eNOS. Membranes were reprobed with an anti-tubulin antibody as a loading control. Data represent one experiment repeated at least three times with similar results. Thus, even though V E G F / V E G F R - 2 interactions appear to be required for endothelial development from mesodermal precursors, these results suggest that V E G F R -2 alone is not sufficient to induce expression of endothelial markers in primary fibroblasts. 70 3.3 Discussion The vascular system forms through a combination of vasculogenesis and angiogenesis. In vasculogenesis, vessels form de novo via the assembly of endothelial precursors called angioblasts, whereas in angiogenesis new vessels arise by migration and proliferation of endothelial cells from preexisting vessels (Risau and Flamme, 1995). VEGFR-2 is required for the embryonic production of cells of the hemangioblastic lineage, and for vascular development in the embryo and adult (Eichmann et al., 1997; Risau and Flamme, 1995; Shalaby et al., 1997). Of the endothelial receptor tyrosine kinases identified so far, VEGFR-2 is the only receptor whose function is absolutely required for the determination ofthe endothelial lineage (Shalaby et al., 1997; Shalaby et al., 1995). Whether it acts as a switch that triggers endothelial differentiation or is only required for the migration and survival of early mesodermal precursors remains to be shown. Because it has been difficult to express VEGFR-2 in heterologous cells, to study the specific signalling pathways and biological effects triggered by VEGFR-2, we used a retroviral vector to transduce primary human foreskin fibroblasts with VEGFR-2. Two major biological activities of V E G F are to induce vascular endothelial cell proliferation, and to stimulate cell migration (Barleon et al., 1996; Connolly et al., 1989; Ferrara and Henzel, 1989; Rousseau et al., 1997). However, the mechanisms underlying these diverse processes are still not well characterized. We examined whether VEGFR-2 was able to elicit a proliferative response in the absence of other V E G F receptors. M T T and BrdU incorporation assays showed that V E G F was indeed able to cause the proliferation of HFF-VEGFR-2 cells whereas it had no effect on HFF-Neo cells. These results indicate 71 that the VEGF-induced proliferative response can be transmitted solely through the activation of VEGFR-2. This confirms the finding of another study in which NUT 3T3 cells transduced with VEGFR-2 showed a proliferative response in the presence of V E G F (Takahashi and Shibuya, 1997). A separate study showed that V E G F did not cause proliferation of VEGFR-1-transduced NIH 3T3 cells (Seetharam et al., 1995). Thus, it is likely that VEGFR-2 is the main receptor responsible for transducing VEGF-induced mitogenic signals. Erkl/2 MAP kinase is a central signalling molecule that is activated by many growth factor receptors (Waskiewicz and Cooper, 1995). Erk activation has also been associated with cell proliferation. It is possible that the mitogenic effect of VEGFR-2 may be mediated through Erk activation. We therefore investigated whether activation of VEGFR-2 by V E G F could activate the Erkl/2 MAP kinase pathway in the absence of other V E G F receptors. We found that V E G F had the capacity to induce Erkl/2 phosphorylation and activation in HFF-VEGFR-2 cells, indicating that VEGFR-2 can activate this pathway in isolation. Endothelial cell migration plays a critical role in angiogenesis. Several cytokines, such as V E G F and angiopoietin-1 have been shown to act as chemoattractants for endothelial cells (Neufeld et al., 1999; Witzenbichler et al., 1998). Furthermore, it has been hypothesized that V E G F may be critical for embryonic hematopoiesis and vasculogenesis by promoting the migration of early precursors (Traver and Zon, 2002). Using a modified Boyden chamber assay, we also demonstrated that V E G F can cause directed migration of HFF-VEGFR-2 cells, indicating that VEGFR-2 is indeed implicated in cell migration. Other studies have shown that VEGFR-1 can also direct cell migration 72 of monocytes (Barleon et al., 1996; Sawano et al., 2001). It is not known whether VEGFR-1 activates the same pathways as VEGFR-2 to induce cell migration. However, since HFF-VEGFR-2 showed a similar chemotactic response to V E G F as H M E C , it is likely that activation of both receptors is not required to induce cell migration. We then examined the p38 signal transduction pathway elicited by VEGFR-2 in response to V E G F stimulation. It has already been shown that V E G F stimulation of endothelial cells can result in phosphorylation of p38 MAP kinase (Rousseau et al., 1997). Activation of p38 results in activation of MAP kinase activated protein kinase-2/3 and phosphorylation of the F-actin polymerization modulator, heat shock protein 27 (HSP27) (Hedges et al., 1999; Kato et al., 1999). The p38 pathway conveys the V E G F signal to microfilaments inducing rearrangements of the actin cytoskeleton that regulate cell migration (Hedges et al., 1999; Kato et al., 1999; Rousseau et al., 1997). By modulating cell migration, p38 may thus be an important regulator of angiogenesis. Here we find that the p38 pathway, as with Erkl/2 activation, can be activated by VEGFR-2 alone. Therefore, cell migration induced by V E G F in HFF-VEGFR-2 could be a consequence of the activation of the p38 pathway. It has been reported in vivo that fibroblasts have the potential to differentiate into endothelial cells, and may play a role in angiogenesis, as progenitors of endothelial cells in newly formed blood vessels (Kon and Fujiwara, 1994). However, the mechanisms by which this occurs is unknown. Moreover, fibroblasts transduced with the MyoD gene have been reported to transdifferentiate into muscle cells (Weintraub et al., 1989). Thus, it seems that, under the proper conditions, fibroblasts may have a plasticity that allows them to transdifferentiate into other cell types. Since VEGFR-2 is critical in endothelial 73 development, it is possible that activation of this receptor by V E G F could trigger induction of endothelial markers in primary fibroblasts. However, when incubated with V E G F for several days, HFF-VEGFR-2 cells did not display an endothelial phenotype, as shown by the lack of expression of endothelial markers such as VEGFR-1, tie-1, tie-2 and eNOS. HFF-VEGFR-2 also did not adopt an endothelial cobblestone morphology. This contrasts with the findings of Eichmann and colleagues, who have demonstrated that in uncommitted precursors VEGF/VEGFR-2 interactions will upregulate endothelial markers (Eichmann et al., 1997). Although HFF-VEGFR-2 cells were found to be signalling competent, other components of endothelial signalling pathways may be required for differentiation. In addition to VEGFR-2, other membrane receptors, intracellular signalling molecules or transcription factors are also likely necessary for endothelial differentiation. Alternatively, it is also possible that VEGFR-2 is not required for endothelial differentiation, but that its role is limited to drive the survival, proliferation and /or migration of early mesodermal precursors (Habeck et al., 2002). 74 Chapter 4 Effects of VEGFR -2 activation in murine bone marrow cells in vitro 75 4.1 Introduction V E G F and its receptors have been shown to play an important role in adult hematopoiesis. VEGFR-2 has been found to be expressed on a subset of hematopoietic stem cells that can differentiate into hematopoietic or vascular endothelial cells depending on the culture conditions (Gehling et al., 2000; Peichev et al., 2000; Ziegler et al., 1999). Because of the increasing amount of data that supports a role of VEGFR-2 signalling in adult hematopoietic cells, both normal and malignant, we decided to study the effects of VEGFR-2 signalling in isolation in murine hematopoietic cells. However, since subsets of hematopoietic cells express V E G F receptors, we chose not to use hematopoietic cells transduced with the full length VEGFR-2, as stimulation of those cells with V E G F may activate the endogenous V E G F receptors present on some of those cells, which in turn may mask or interfere with VEGFR-2 signalling. Moreover, it has been shown that neuropilin-1 is a receptor for V E G F and acts as a co-receptor that enhances the function of V E G F through VEGFR-2 (Zachary and Gliki, 2001). Furthermore, VEGFR-2 has been shown to heterodimerize with VEGFR-1 (Kendall et al., 1996). We therefore chose to rely on a strategy that allowed us to specifically activate VEGFR-2 with a chemical compound. The strategy we used allows us to study the unique signalling properties of VEGFR-2, without any interference from other endogenous V E G F receptors, allowing us to exclude the effects of neuropilin, or heterodimerization with VEGFR-1. The strategy we used to study VEGFR-2 signalling has recently been used to study the unique signalling effects of some hematopoietic receptors (flt-3, MPL, granulocyte-colony stimulating factor receptor, c-kit) (Jin et al., 1998a; Jin et al , 2000; 7 6 Otto et al., 2001b). The biological effects triggered by those receptors have been studied by fusing the signalling domain of these receptors to an FK506 binding protein (FKBP) that can be specifically activated using synthetic FKBP ligands (Blau, 1999; Blau et al., 1997; Jin et al., 1998a; Jin et al., 1998b; Jin et al., 2000; Otto et al., 2001b; Richard et al., 2000). This system has permitted the demonstration that the self-renewal and differentiation of hematopoietic progenitors can be influenced through distinct, receptor-initiated signalling pathways (Zeng et al., 2001). In this chapter, we used this inducible dimerization strategy to specifically study the biological effects of VEGFR-2 signalling on hematopoietic progenitors. To specifically study the unique signalling effects of VEGFR-2, we fused the cytoplasmic domain of this receptor, which contains the split tyrosine kinase domain, to a mutated FKBP 12 domain that harbours a phenylalanine to valine mutation at amino acid 36. Although other studies have shown the signalling effects of VEGFR-2 by using VEGFR-2 specific ligands, such as V E G F - E (Meyer et al., 1999), the use of a non-toxic chemical inducer of dimerization, AP20187 (Ariad Pharmaceuticals), allowed us to study with high specificity VEGFR-2 signalling pathways and biological effects in a cell autonomous manner. This strategy also allowed us to rule out any potential signalling effects that could be triggered by neuropilin-1, which acts as a co-receptor for V E G F , enhancing its binding to VEGFR-2 (Zachary and Gliki, 2001). 4.2 Results 4.2.1 Activation of VEGFR-2 delays loss of murine hematopoietic progenitors We cloned the intracellular domain of VEGFR-2 and fused it to a modified FKBP domain that can be specifically dimerized with a chemical inducer, AP20187 (Figure 77 12A). When transduced into HMEC-1 cells or murine bone marrow cells, this construct gave a 100 kDa protein (Figure 12B), which mainly localized to the cytoplasmic membrane when unstimulated (Figure 12C). Stimulation of HMEC-1 cells with 10 nM AP20187 for 0 to 30 minutes, resulted in progressive translocation of the fused VEGFR-2 construct from the cytoplasmic membrane to the cytoplasm. Phosphorylation of the construct was observed as early as 30 seconds after stimulation with 10 nM AP20187 in H M E C cells, and remained over a period of at least 30 minutes (Figure 12D). 78 A LTR j* H l R E ^ r ^ G F P l -B m FKBP 12 \u00E2\u0080\u00A2 - Myristoylation signal \u00E2\u0080\u00A2 V E G F R - 2 cytoplasmic domain \u00E2\u0080\u00A2 - HA Epitope tag $ 3 p110 HMEC-1 Bone marrow Unstimulated 2Q min Figure 12 MIG-FKBP/VEGFR-2 fusion construct. (A) The construct includes the intracellular domain of VEGFR-2 fused to a modified FKBP 12 domain. A hemagglutinin (HA) epitope tag was included at the C-terminus and a myristoylation sequence at the N-terminus. (B) Immunoblotting of HMEC-1 and bone marrow cells demonstrates expression of a 110 kDa protein, consistent with the predicted size of the VEGFR-2 construct. (C) Immunofluorescent staining of HMEC-1 cells reveals that the fusion protein localizes mainly to the cytoplasmic membrane when unstimulated. Stimulation with 10 nM AP20187 resulted over a period of 30 minutes resulted in partial translocation of the fusion protein to the cytoplasm. (D) HMEC-1 cells were incubated with 10 nM AP20187 for 0 to 30 minutes. Phosphorylation of FKBP/VEGFR-2 fusion protein was determined by immunoblotting using an antibody specific to phosphorylated VEGFR-2 (Tyr 951). The membrane was reprobed with anti-HA antibody as a loading control. The fusion protein was detected in HMEC-1 cells using an anti-HA monoclonal antibody for both immunoblotting and immunofluorescence. 79 To investigate the effect of VEGFR-2 in hematopoietic cells, bone marrow from mice treated with 5-fluorouracil to activate bone marrow precursor cells was harvested and transduced with the VEGFR-2 fusion construct. As a control, the empty MSCV-IRES-GFP (MIG) vector was used. After sorting, transduced GFP + cells were plated in EMDM medium supplemented with 10% FBS with or without 100 nM AP20187, and cells were counted at days 5, 7 and 14. For all experiments, only transduced GFP + cells were used. We found that the number of viable cells decreased rapidly in the absence of cytokines. However, in marrow cells in which the VEGFR-2 construct was dimerized by the addition of AP20187, we observed a smaller decrease in cell number (Figure 13A). After 2 weeks in culture, cell numbers in bone marrow control cultures were 2.5 fold lower than the ones in which VEGFR-2 was dimerized. This effect was not observed in VEGFR-2 transduced cells that did not receive AP20187, indicating that dimerization of VEGFR-2 is required for maintaining hematopoietic cell numbers. We next tested whether dimerization of VEGFR-2 had an additive effect on medium supplemented with hematopoietic cytokines that provide optimal growth conditions (Thorsteinsdottir et al., 1999). Transduced bone marrow cells were cultured in medium containing cytokines that are known to induce hematopoietic cell proliferation (IL-3, IL-6, SCF), with or without 100 nM AP20187 (Figure 13B). With these growth conditions, we did not observe any significant change when VEGFR-2 was dimerized in comparison to the control cells, suggesting that VEGFR-2 does not signal a proliferative or anti-apoptotic effect that is synergistic with these hematopoietic cytokines. 80 80000 B 800000 * 600000 | 400000 z 200000 -i-MIG I MIG-FKBP/VEGFR-2 A MIG+AP20187 \u00E2\u0080\u00943-- MIG-FKBP/VEGFR-2 +AP20187 5 10 Time (days) 5 10 Time (days) 15 15 Figure 13 VEGFR-2 maintains hematopoietic cell number. MIG or MIG-FKBP/VEGFR-2-transduced bone marrow cells were incubated in IMDM supplemented with 10% FBS with or without 100 nM AP20187 in the absence (A) or the presence (B) of hematopoietic cytokines (IL-3, IL-6, SCF) for 0 to 14 days. Cells were harvested at specific time points as indicated and cell number was determined. Data represent the mean \u00C2\u00B1 S E M of three independent experiments. * PO.05. To test whether VEGFR-2 can preserve the viability and activity of hematopoietic progenitors in the absence of hematopoietic cytokines, VEGFR-2 and control cells were cultured in cytokine-free medium for 7 and 14 days with or without addition of dimerizer, after which cells were plated in methylcellulose medium to assay for hematopoietic 81 progenitors. We found that dimerization of VEGFR-2 maintained hematopoietic progenitor potential in liquid culture to a certain extent. Over a 2-week period in culture, we observed an 8-fold decrease in the number of progenitors in control bone marrow cultures. In contrast, when the FKBP-VEGFR-2 construct was dimerized with AP20187, we observed a 3-fold increase in the maintenance of progenitors over control cultures, consistent with the findings in Figure 13 (Figure 14A). Although VEGFR-2 dimerization maintained the hematopoietic progenitor population for a period of two weeks in the absence of other cytokines, we did not observe a significant change in the proportion of different hematopoietic progenitors as measured by the CFC assay (Figure 14B). This result suggests that VEGFR-2 promotes hematopoietic cell survival and/or proliferation, but does not affect differentiation of hematopoietic progenitors. 82 O LL. o n \u00E2\u0080\u0094 O i \u00E2\u0080\u0094 GJ - O E 50 45 40 35 30 25 20 15 10 5 0 B 100% 80% 60% 40% 20% 0% CMIG \u00E2\u0080\u00A2 MIG-FKBP/VEGFR-2 \u00E2\u0080\u00A2 MIG+AP20187 \u00E2\u0080\u00A2 MIG-FKBP/VEGFR-2 +AP20187 DayO Hi \u00E2\u0080\u00A2 CFU-Mix \u00E2\u0080\u00A2 BFU-E \u00E2\u0080\u00A2 \u00E2\u0080\u00A2 CFU-GM \u00E2\u0080\u00A2 CFU-M \u00E2\u0080\u00A2 CFU-G I III MIG MIG-FKBPA/EGFR-2 D a y 7 MIG+AP20187 MIG- MIG-FKBP/VEGFR-2 FKBP/VEGFR-2 \u00E2\u0080\u00A2AP20187 100% 80% 60% 40% 20% 0% D a y 14 MMM MIG+AP20187 MIG- MIG-FKBP/VEGFR-2 FKBP/VEGFR-2 \u00E2\u0080\u00A2AP20187 Figure 14 VEGFR-2 delays the loss of CFCs. Hematopoietic progenitors transduced with MIG- or MIG-FKBP/VEGFR-2 were grown in IMDM containing 10% FBS for 7 or 14 days, and then plated in complete methylcellulose medium to test for progenitor activity as measured by CFC number after 10 days (A). Dimerization of VEGFR-2 did not affect the proportion of different progenitors over time as measured by scoring for the type of colonies formed in the CFC assay (B). Data represent the mean \u00C2\u00B1 SEM of three independent experiments. * PO.05. S3 To confirm that VEGFR-2 can independently maintain the multipotent hematopoietic progenitor population, we utilized the CFU-Spleen (CFU-S12) assay following liquid culture of bone marrow cells for 7 days in cytokine-free medium. Colonies were enumerated in each of the spleens harvested 12 days following injection of bone marrow cells (Figure 15A). As seen in Figure 15B, VEGFR-2 dimerization resulted in a 5-fold increase in the proportion of CFU -S12 cells, compared to bone control marrow cultures. These results suggest that VEGFR-2 can maintain the activity and viability of primitive hematopoietic progenitors in the absence of other exogenous cytokines. 84 A MIG MIG-FKBP/VEGFR-2 Figure 15 VEGFR-2 dimerization maintains the number of multipotential bone marrow progenitors. Hematopoietic primitive myeloid progenitors cultured for 7 days in IMDM + 10%FBS, with or without AP20187, were injected into lethally irradiated B6C3 mice, and spleen colonies were counted after 12 days (A, B ) . Data represent the mean \u00C2\u00B1 SEM of three independent experiments. * P<0.05. 4.2.2 VEGFR-2 does not increase S-phase entry in hematopoietic precursors It is known that, in endothelial cells, V E G F can induce cell proliferation. It has been suggested that this effect is mainly mediated through VEGFR-2 (Ferrara, 1999a). Moreover, we have demonstrated in the previous chapter that VEGFR-2 can induce proliferation of primary fibroblasts. We tested whether dimerization of VEGFR-2 also resulted in bone marrow cell proliferation, which could account in part for the delay in 8 5 the loss of hematopoietic progenitors that we observed. Bone marrow cells were grown in cytokine-free medium for 2 days, then exposed to BrdU with or without AP20187 for 2 hours. Cytospin preparations of cells were then labelled with an anti-BrdU antibody (Figure 16A). We found that dimerization of VEGFR-2 did not result in a greater proportion of cells which incorporated BrdU, indicating that VEGFR-2 signalling alone may not be sufficient to induce proliferation of hematopoietic progenitors (Figure 16B). A Figure 16 VEGFR-2 dimerization does not increase BrdU uptake in hematopoietic progenitors. MIG- or MIG-FKBP/VEGFR-2-transduced bone marrow cells cultured for 2 days in IMDM supplemented with 10% FBS were treated for 2 hours with 10 nM BrdU with or without 100 nM AP20187 and cytospin preparations were stained with an anti-BrdU antibody conjugated with Alexa 594. DAPI staining was used to identify nuclei (A). The number of BrdU* cells were quantitated and expressed as a percent of total nuclei counted (B). 200-300 cells were counted per cytospin preparation. Data represent the mean \u00C2\u00B1 SEM of three independent experiments. 8 6 4.2.3 VEGFR-2 activation reduces the number of apoptotic cells in hematopoietic precursors It has been shown that V E G F can induce anti-apoptotic signalling through PI3-kinase in endothelial cells subjected to serum deprivation (Gerber et al., 1998b). Since we observed a delay in loss of progenitors when VEGFR-2 is dimerized, we postulated that this effect was caused by an inhibition of apoptosis since VEGFR-2 dimerization alone did not affect proliferation of hematopoietic progenitors. It has been shown that caspase 3 is present in hematopoietic precursor cells and is activated during apoptosis (Nicholson and Thornberry, 1997; Zermati et al., 2001). To test whether VEGFR-2 inhibits hematopoietic cell apoptosis, transduced bone marrow cells were subjected to cytokine deprivation, and incubated with or without AP20187 for 14 days. At this point, cytospins were made and stained for the activated form of caspase-3 (Figure 17A). We found that the proportion of apoptotic cells was 2-fold lower in bone marrow cells in which VEGFR-2 was dimerized compared to bone marrow control cultures (Figure 17B). Hence, inhibition of apoptosis through VEGFR-2 signalling would explain in part the maintenance of hematopoietic progenitors observed. 87 B jfl 15 TJ > \u00C2\u00ABJ S 10 ffl \"(fl > 0 a o \u00E2\u0080\u00A2 < - + + - - + \u00E2\u0080\u00A2 pp59 -p59 MIG FKBP/VEGFR2 '- + + - - + t i pp44 pp42 Up44 r p 4 2 Figure 18 VEGFR-2 dimerization activates Akt and Erkl/2 MAP kinases in hematopoietic progenitors. Quiescent HMEC-1 cells were incubated with 10 nM AP20187 for 0 to 60 minutes as indicated (A, B). Cytokine-starved MIG- or MIG-FKBP/VEGFR-2-transduced bone marrow progenitors were treated for 20 minutes with 100 nM AP20187 (C, D). Phosphorylation of Akt (A, C) or Erkl/2 (B, D) was determined by immunoblotting using antibodies specific to phosphorylated Akt or Erkl/2. Lanes: 1,4: untreated; lanes 2,5: 100 nM AP20187 for 20 min; lanes 3,6: pre-treatment with 20 LUM LY294002 (Akt) or 10 uM U0126 (Erk) for 90 min, then treated with 100 nM AP20187 for 20 min. Membranes were reprobed with anti-Akt or anti-Erkl/2 antibodies as loading controls. Data represent one experiment of three independent experiments showing similar findings. 90 To specifically study the above signalling pathways in mediating hematopoietic progenitor survival, we used specific inhibitors of each signalling pathway. LY294002 is an inhibitor of PI3-kinase, which activates Akt, while U0126 has been shown to block Erk 1/2 MAP kinase phosphorylation by specific inhibition of M E K (Davies et al., 2000). To determine whether VEGFR-2 mediated survival was mediated through Akt and/or Erkl/2, transduced GFP + bone marrow cells were incubated with or without 100 nM AP20187 in the presence of the PI3-kinase inhibitor LY294002, or the M E K inhibitor U0126 at concentrations that blocked each of these kinases (Figure 18C, D). Cell number was monitored over a period of 14 days. We found that inhibition of PI3-kinase blocked the anti-apoptotic effect of VEGFR-2 dimerization induced by cytokine deprivation, indicating the essential role of this pathway in VEGFR-2-mediated survival in bone marrow cells (Figure 19A). Blockade of PI3-kinase also inhibited the survival of hematopoietic progenitors induced by VEGFR-2 dimerization (Figure 19B), further demonstrating the critical role of this pathway in survival of hematopoietic progenitors. In contrast, blockade of the MAP kinase pathway with U0126 did not inhibit VEGFR-2 induced cell survival (Figure 19C). Interestingly, despite minimal effect on cell survival, inhibition of Erkl/2 partially inhibited hematopoietic progenitor activity mediated by VEGFR-2 (Figure 19D). This discrepancy suggests that hematopoietic progenitors are more dependent on the Erkl/2 MAP kinase pathway for survival than more mature/differentiated cells such as macrophages, which constitute the majority of cells present after 14 days in liquid culture (data not shown). 91 \" 80000 -| 70000 \u00E2\u0080\u00A2J 60000 a . 50000 O <- 40000 V g 30000 ^ 20000 10000 0 5 10 Time (days) Day 7 Day 14 -MIG -MIG+LY294002 -MIGMP20167 -MIGMP20187HY294002 -MIG-FKBP/VEGFR-2 - MIG-FKBP/VEGFR-2 H Y 2 94002 MIG-FKBP/VEGFR-2 \u00E2\u0080\u00A2AP20187 - MIG-FKBP/VEGFR-2 \u00E2\u0080\u00A2AP20187+LY294002 \u00E2\u0080\u00A2 MIG \u00E2\u0080\u00A2 MIG-H.Y294002 \u00E2\u0080\u00A2 MIG\u00C2\u00BBAP20187 B MIG+AP20187+LY294002 DI MIG-FKBP/VEGFR-2 D MIG-FKBP/VEGFR-2 \u00E2\u0080\u00A2LY294002 B MIG-FKBP/VEGFR-2 +AP20187 B MIG-FKBP/VEGFR-2 \u00E2\u0080\u00A2AP20187*LY294002 - A - M I G -A -M IGHJ0126 -6-MIG+AP20187 - t-MIG+AP20187*U0126l - \u00E2\u0080\u00A2 - MIG-FKBP/VEGFR-2 -o-MIG-FKBP/VEGFR-2 \u00E2\u0080\u00A2U0126 -B-MIG-FKBPA/EGFR-2 \u00E2\u0080\u00A2AP20187 - \u00C2\u00AB - MIG-FKBP/VEGFR-2 \u00E2\u0080\u00A2AP20187*110126 \u00E2\u0080\u00A2 MIG \u00E2\u0080\u00A2 MIG*U0126 \u00E2\u0080\u00A2 MIG+AP20187 \u00E2\u0080\u00A2 MIG+AP20187+U0126 El MIG-FKBP/VEGFR-2 El MIG-FKBP/VEGFR-2 \u00E2\u0080\u00A2U0126 Fl MIG-FKBP/VEGFR-2 \u00E2\u0080\u00A2AP20187 n MIG-FKBP/VEGFR-2 \u00E2\u0080\u00A2AP20187HJ0126 Figure 19 Effect of PI3-kinase or M E K inhibition on hematopoietic progenitor survival. MIG- or MIG-FKBP/VEGFR-2-transduced hematopoietic progenitors were cultured in IMDM supplemented with 10% FBS with or without 100 nM AP20187 in the presence or in the absence of 20 uM of the PI 3-kinase inhibitor, LY294002 (A, B), or the M E K inhibitor, U0126 (C, D), for 0 to 14 days. Cells were harvested at specific time points and cell number was determined (A, C). Cells were collected after 7 and 14 days and plated in methylcellulose medium to assay for hematopoietic progenitor activity (B, D). Data represent the mean \u00C2\u00B1 SEM of three independent experiments. * P<0.05. 4.3 Discussion Although many studies postulate that V E G F mediates most of its effects through VEGFR-2, it is difficult to make a clear statement about this because of the complexity of the V E G F - V E G F R system. To specifically study the effects of VEGFR-2 signalling, we used a strategy that allowed the specific activation of the VEGFR-2 signalling domain by using a chemical inducer of dimerization. A similar strategy has previously shown that 92 the thrombopoietin (Tpo) receptor c-mpl can induce long term proliferation of murine hematopoietic progenitors, whereas the effects of flt-3 and G-CSFR are much more modest (Zeng et al., 2001). In this study, the VEGFR-2 construct was localized to the cytoplasmic membrane in unstimulated HMEC-1 cells, and translocated into the cytosol with concomitant phosphorylation following stimulation with AP20187, a phenomenon observed with many endogenous hematopoietic receptors. Interestingly, however, Otto et al. (Otto et al., 2001a) have shown that membrane localization of the thrombopoietin receptor, mpl, is not required for the full range of c-mpl function in hematopoietic cells. We were interested in the role of VEGFR-2 in hematopoietic progenitors since little is known about the effects of this receptor on hematopoiesis. In murine bone marrow, our results indicate that VEGFR-2 can maintain hematopoietic progenitor potential following dimerization, since cells fail to survive in the absence of AP20187. It is interesting to note that when hematopoietic progenitors were cultured in the presence of hematopoietic cytokines (IL-3, IL-6, SCF), there was no effect of VEGFR-2 dimerization on cell number. This would suggest that VEGFR-2 does not induce a proliferative signal in hematopoietic progenitors that is distinct from IL-3, IL-6 and SCF. Although Erkl/2 MAP kinase was activated, VEGFR-2 dimerization failed to induce proliferation of bone marrow progenitors, indicating that signals provided by other factors may be necessary for the proliferation of hematopoietic cells. It is likely then that the delay in cell loss observed through VEGFR-2 dimerization reflects an effect on cell survival. This was verified when we found that VEGFR-2 signalling decreased the fraction of apoptotic cells, when hematopoietic progenitors were cultured in the absence of exogenous cytokines. 93 The importance of cell survival in the maintenance of hematopoietic progenitors was further demonstrated by blocking the PI3-kinase pathway. This signalling pathway, through Akt, has been shown to play a crucial role in cytokine-mediated survival (Cantley, 2002; Gerber et al., 1998b), as well as in the self-renewal of primary multipotential hematopoietic progenitors (Zhao et al., 2002). Blockade of PI3-kinase completely abolished the maintenance of hematopoietic progenitors mediated by VEGFR-2, indicating that this pathway is critical for VEGFR-2 mediated survival in bone marrow progenitor cells. In contrast, we did not observe a significant effect on cell survival mediated by VEGFR-2 when the Erkl/2 MAP kinase pathway was blocked using the M E K inhibitor, U0126. This would imply that, although the Erkl/2 MAP kinase pathway has been shown to play a role in apoptosis prevention (Franklin and McCubrey, 2000), the PI3-kinase pathway through Akt is the main regulator of VEGFR-2-induced survival signalling in hematopoietic progenitors. However, it is noteworthy that inhibition of Erkl/2 activation did reduce the number of hematopoietic progenitors as measured by the CFC assay. This effect may be explained by the fact that most cells remaining after 14 days in culture in the absence of exogenous cytokines are differentiated, and studies have shown that the cytokine-induced survival of differentiated cells, such as macrophages, is mediated mainly by the PI3 -kinase pathway and not the Erkl/2 M A P kinase pathway (Jaworowski et al., 1999; Xaus et al., 2001). Thus, this may indicate that hematopoietic progenitors are more dependent on the Erkl/2 MAP kinase pathway for VEGFR-2-induced survival than more mature cells. Although VEGFR-2 signalling promoted survival of hematopoietic progenitors and maintained their progenitor potential, it did not seem to affect the differentiation of 94 those progenitors. In contrast, other hematopoietic cytokine receptors, such as m p l , induce a dramatic expansion o f multipotential progenitors and megakaryocytes (Richard et al. , 2000; Z e n g et al. , 2001). A recent study has demonstrated that a combinat ion o f signals, J A K 2 plus either c-kit or flt-3 together can support extensive hematopoietic progenitor cell self-renewal even though neither o f these receptors can sustain the growth o f bone marrow cells alone (Zhao et al. , 2002). Whether V E G F R - 2 requires additional signals to induce cel l proliferation in hematopoietic cells remains unknown and further studies w o u l d be needed to assess this issue. T h e strategy used in the present study allowed us to demonstrate that V E G F R - 2 can activate the PI3-kinase and E r k l / 2 pathways, without interaction with other V E G F receptors such as the neuropilins or V E G F R - 1 , in hematopoietic progenitors. These results show that V E G F R - 2 can induce maintenance o f hematopoietic progenitors in the absence o f exogenous hematopoietic cytokines. T h i s m a y help to explain, at least in part, the critical role o f V E G F R - 2 not only in embryonic hematopoiesis, but also in adult hematopoiesis, both normal and malignant. 95 Chapter 5 Activation of VEGFR-2 in bone marrow cells leads to accumulation of myeloid cells in vivo: role of GM-CSF 96 5.1 Introduction V E G F may play an important role in the development of haemopoietic cells, but available data do not provide a definitive answer to this question. For example, although murine embryos with inactivating VEGFR-2 mutations remain 'bloodless' it is unclear if this is due to a primary stem cell defect or defective vascularization and blood island formation (Shalaby et al., 1997). In addition, it has been reported that V E G F and SCF co-stimulate the development of common precursors for primitive and definitive murine haemopoiesis (Kennedy et al., 1997a) and that V E G F is essential for the reconstitution of hematopoiesis in lethally irradiated animals (Gerber et al., 2002). VEGFR-2 is expressed on a significant proportion of normal and malignant hematopoietic cells. Moreover, it has been shown that pluripotent hematopoietic stem cells are restricted to the CD34 + VEGFR-2 + cell fraction in human cells (Ziegler et al., 1999). However, it has also been reported that neither VEGFR-2 + CD34 l o w / \" cells nor VEGFR-2 + CD34 + cells have long-term reconstitution capacity in mice (Haruta et al., 2001). Hematopoietic malignancies often result from impaired control of early hematopoietic stem cell survival, proliferation and self-renewal. V E G F is expressed in cell lines derived from various hematological malignancies (Bellamy et al., 1999). RNA for both V E G F receptors have been detected in some but not all hematopoietic malignancies (Bellamy et al., 2001; Fiedler et al., 1997; Padro et al., 2002). In addition, several reports have documented inhibitory effects by various classes of small molecule inhibitors targeting VEGFR-1 and VEGFR-2 on the growth of human myeloid leukaemia cell lines and in acute myeloid leukaemia blasts independently of their effects on 97 angiogenesis (Mesters et al., 2001). Moreover, Dias et al. have shown that neutralisation of human VEGFR-2 with specific monoclonal antibodies prolonged survival in mice xenotransplanted with human VEGFR-2 + leukemic cell lines (Dias et al., 2001). Therefore, understanding the effects of V E G F and its receptors on hematopoietic cells will prove essential for the development of new therapies targeting the growth of hematopoietic malignancies. Most groups studying the effects of V E G F signalling in normal and malignant hematopoietic cells have used strategies to block V E G F signalling by using either blocking antibodies or small molecule kinase inhibitors. In the present study, we have used the opposite approach by activating VEGFR-2 in normal bone marrow cells, and investigating its effects on hematopoiesis. By using the VEGFR-2 fusion protein mentioned in the previous chapter, we studied the hematopoietic effects that arise when VEGFR-2 is expressed and activated in a relatively large proportion of hematopoietic cells in vivo. AP20187 is well tolerated in vivo, which allows its use in studying specific signalling pathways, and evaluate its potential use in therapeutic strategies. We examined bone marrow progenitors and the proportion of mature hematopoietic cells in the blood and bone marrow of mice following VEGFR-2 activation. Furthermore, we also looked at the expression of some hematopoietic growth factors triggered by VEGFR-2 and their potential role in mediating VEGFR-2 effects. 98 5.2 Results 5.2.1 Activation of VEGFR-2 induces expansion of bone marrow myeloid cells Although VEGFR-2 plays a critical role in the formation of the hematopoietic system, its role in adult hematopoiesis remains unclear (Gerber and Ferrara, 2003). V E G F and its receptors have been reported to be expressed in a variety of hematopoietic malignancies, and such expression is usually associated with poor prognosis (Verstovsek et al., 2003). We have demonstrated in the previous chapter that VEGFR-2 dimerization in murine bone marrow results in hematopoietic progenitor survival in vitro. To investigate the role of VEGFR-2 activation in in vivo, murine bone marrow was transduced with the MIG-FKBP-VEGFR-2 construct, which can be dimerized with the chemical inducer of dimerization AP20187. The empty vector MIG was used as control. We have previously demonstrated that the MIG-FKBP-VEGFR-2 construct is signalling-competent when stimulated with AP20187 in vitro, and can induce biological responses in murine bone marrow cells. Transduced bone marrow was used to transplant lethally irradiated B6C3 mice. Four to six weeks after transplantation, bone marrow and peripheral blood were obtained from mice to determine baseline engraftment of transduced cells (based on GFP expression). Following this, mice were injected in the peritoneal cavity with 10 mg/kg/day AP20187 or vehicle (4% EtOH, 10% PEG-400, and 1.7% Tween 20 in water) for 10 days, after which they were sacrificed. Peripheral blood and bone marrow were harvested and the proportion of GFP + cells was determined by flow cytometry. To confirm that the cells which express the FKBP-VEGFR-2 construct were indeed GFP + , cells from the bone marrow were stained with LY5.1 and LY5.2 to confirm the donor/recipient origin of the bone marrow cells. We found in a typical 99 experiment that >90% of GFP\" cells were LY5.1\", and therefore recipient-derived, which therefore implies that the vast majority of GFP\" cells do not express the FKBP-VEGFR-2 construct in the FKBP-VEGFR-2-transplanted animals. We observed, on average, a 3.5-fold increase in the proportion of GFP + cells in the bone marrow of the mice in which the VEGFR-2 construct was dimerized with AP20187 (Figure 20). By contrast, no significant changes were observed in the proportion of GFP + cells in the peripheral blood of these mice. Mice in which VEGFR-2 was not dimerized (mice that were transplanted with empty vector bone marrow or received vehicle) did not show any changes in the proportion of GFP + cells in either bone marrow or peripheral blood. 0 4.5 1 * \u00C2\u00A3 3.5 a S 3 \" 2 \u00E2\u0080\u00A2 1.5 -d T 1 1 i ^ 5 t o 14 0. u-0 1 3 1 2 f 1 \u00E2\u0080\u00A2! o 0. 0 j Peripheral blood m m \u00E2\u0080\u00A2 Vector FKEP/VEGFR2 FKBPWEGFR2 \u00E2\u0080\u00A2ARM 187 Figure 20 VEGFR-2 induces expansion of retrovirally transduced hematopoietic cells in vivo. Ratios of the proportion of GFP + cells post-treatment relative to pre-treatment are shown for bone marrow (top panel) and peripheral blood (lower panel). Data \u00C2\u00B1 SEM shows average of 8 to 10 mice per group. * P< 0.01. This result indicates that VEGFR-2 activation can elicit a marked expansion of transduced hematopoietic cells in the bone marrow, but interestingly, this expansion of i marrow \u00E2\u0080\u00A2 \u00E2\u0080\u00A2 Vocflw +AP2O107 FKBIW8GFR2 FK0FA/EGFR2 \u00E2\u0080\u00A2AP2Q!fl7 100 GFP + cells in the bone marrow does not translate into an increase in the proportion of GFP + cells in the peripheral blood during the time frame of the experiment. We next determined the phenotype of cells responding to AP20187 in mice transplanted with MIG-FKBP-VEGFR-2-transduced marrow cells. AP20187 treatment did not result in significantly increased numbers of leukocytes or erythrocytes in the peripheral blood of mice transplanted with MIG-FKBP-VEGFR-2 (Table V). Table V Peripheral blood counts Mice Red blood cell counts (xlO 1 2 cells/L) White blood cell counts (xl09cells/L) Vector 6.77 \u00C2\u00B1 1.08 5.42 \u00C2\u00B1 0.90 Vector+ AP20187 7.93 \u00C2\u00B1 1.88 3.82 \u00C2\u00B1 0 . 4 6 FFBP/VEGFR-2 7.31 \u00C2\u00B1 1.24 4.77 \u00C2\u00B1 0 . 9 1 FKBP/VEGFR-2 + AP20187 9.26 \u00C2\u00B1 1.83 5.67 \u00C2\u00B1 0 . 3 4 Cells from bone marrow (Figure 21) and peripheral blood (Figure 22) of MIG or MIG-FKBP-VEGFR-2 transplanted mice injected with AP20187 or vehicle for 10 days were stained for the myeloid markers CD l i b (monocyte/macrophage) and Gr-1 (granulocyte), lymphoid markers CD5 (T-cells) and B220 (B-cells) or an erythroid marker (Terll9) to determine whether VEGFR-2 activation affects the proportion of specific hematopoietic populations. VEGFR-2 dimerization induced a significant increase in the proportion of GFP + myeloid cells ( C D l l b + and Gr-1 +; 1.5-fold-increase on average) in the bone marrow of transplanted mice (Figure 21). It is interesting to note that when the percentage of cells of different subsets of the bone marrow (lymphoid, myeloid and erythroid) are added, the total exceeds 100% in the FKBP-VEGFR-2 transplanted mice stimulated with AP20187. This did not apply for any of the other subgroups tested. One possible explanation for this phenomenon lies in the fact that expression of some of the markers used is not absolutely restricted for the specific lineage for which it was tested. 1 0 1 For example, C D l l b is expressed on subsets of activated B and T lymphocytes, eosinophils and Natural Killer (NK) cells in addition to being expressed in mature and immature myeloid cells (Cabanas and Sanchez-Madrid, 1999). Moreover, B220 is expressed on dendritic cell precursors (Asselin-Paturel et al., 2001; del Hoyo et al., 2002) and monocyte precursors (Dannaeus et al., 1999) in addition to B lymphocytes. This could indicate that VEGFR-2 activation can affect, to a certain extent, the differentiation and expansion of immature cells, which have been shown to co-express lymphoid and myeloid markers (Lu et al., 2002). We also observed a moderate increase in the GFP + proportion of myeloid cells compared to the GFP\" population in the peripheral blood (Figure 22), but this increase was not as marked as observed in the bone marrow. These results suggest that VEGF-elicited myeloid expansion can be mediated solely through VEGFR-2 (Gabrilovich et al., 1998; Melani et al., 2003). However, VEGFR-2 activation did not significantly affect the levels of lymphoid or erythroid cells in the bone marrow or peripheral blood. 102 A CDIIb Vector Vector + AP20187 FKBP/VEGFR2 FKBP/VEGFR2 +AP20187 Gr-1 CD5 Ter119 10 \u00E2\u0080\u00A2 \u00E2\u0084\u00A2 10 4 HI id i r j B 2 2 0 i ' : f *r0 \" KSSff 17 10 r0 1?.0\u00C2\u00A3>% id id1 id iri trf sif ,J ,rf ,^ VE-Cadherin ^ k CD31 1 - S VE-Cadherin V h ft \ - \"Six, Jn^. >*'\" m \u00E2\u0080\u00A2 B VE-Cadherln+CDilb VE-CadheriiHCDIIb . If \u00E2\u0080\u00A2 y \ \u00E2\u0080\u00A2 % JtJ \u00E2\u0080\u00A2 , 4 * / * P * \ i VE-CadherirnCDIIb '\u00E2\u0080\u00A2-4 * ' \ . VE-Cadherin-MZDIIb Figure 27 Bone marrow derived endothelial cells incorporate into tumour vasculature. (A) Tumour sections (~8 urn) were stained with either CD31 or VE-cadherin (red) and were examined for the presence of bone marrow-derived cells (GFP +, green) present as part of the blood vessels. DAPI was used to counterstain nuclei (blue). (B) To confirm the phenotype of bone marrow-derived endothelial cells, B6RV2 tumour sections were stained with CD1 lb labeled with PE (red) and VE-cadherin-Alexa 350 (blue). We then quantified the relative contribution of bone marrow cells to the formation of B6RV2 blood vessels. As a source of GFP + bone marrow cells in transplanted animals, 124 we either used cells obtained from GFP + transgenic mice (Okabe et al., 1997) that were injected into lethally irradiated recipients immediately after harvesting (Figure 28A), or by harvesting bone marrow cells from C3Pep mice that were expanded ex vivo (2 days) before transduction with a retroviral vector encoding GFP (MIG) (Figure 28B). A CJ u > '55 o a. \u00E2\u0080\u00A2 D L LL. O JZ w a c n c n a> > TJ O O 03 2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 CD31 VE-Cadherin B 8 2 > 1-8 1 1 6 a. a. LL. o s 85 o.6 i n 5 0.4 T3 2 0.2 o OQ 0 1.4 1.2 1 0.8 CD31 VE-Cadherin Figure 28 Bone marrow-derived endothelial cells have a relatively small contribution to the formation of blood vessels in B6RV2 lymphomas. B6RV2 tumours were implanted sub-cutaneously in the dorsal area of B6C3 mice previously transplanted with GFP + bone marrow from GFP transgenic donors (A) or with bone marrow transduced ex vivo with a GFP-encoding vector (MIG) (B). Data \u00C2\u00B1 SEM represents the average of the quantification of 4 tumours per group. 125 This approach allowed us to determine whether ex vivo culture of bone marrow cells can have deleterious effects on the endothelial progenitor pool potentially present in bone marrow cells. Contribution of bone marrow cells to the formation of tumour blood vessels was minimal. In both cases, less than 1% of B6RV2 blood vessels were found to have incorporated GFP + bone marrow-derived endothelial cells. Ex vivo transduction of bone marrow cells prior to transplantation did not result in significant differences in the levels of tumour vasculature bone marrow-derived endothelial cells compared to mice transplanted with unmanipulated bone marrow cells, indicating that in vitro culture of bone marrow cells does not result in loss of endothelial progenitor activity within the time frame of this experiment. 6.2.2 Determination of the existence of an adult hemangioblast Bone marrow cells have been shown to be able to fuse with other cell types such as hepatocytes (Grompe, 2003) and this has lead some authors to believe that hematopoietic cells may not be as multipotent as once thought, since cell fusion could in fact be responsible for some of the reported plasticity of stem cells (Terada et al., 2002). Analysis of D N A content of bone marrow-derived endothelial cells in liver tissue has previously shown that they can arise by differentiation of bone marrow cells, and not by cell fusion (Bailey et al., 2004). However, DNA content analysis can be misleading, as fused cells have been shown to lose cellular DNA, which could potentially lead to inaccurate interpretation of results (Nowak, 1985; Pratt et al., 1992; Zheng et al., 1995). To determine whether the bone marrow-derived endothelial cells observed in B6RV2 tumours arise by differentiation or fusion, we used a strategy that allowed us to visually 126 determine cell fusion in mice. The following strategy was used: bone marrow cells were harvested from Z/AP double reporter transgenic mice. These mice express a transgene consisting of the lacZ gene flanked by two loxP sites (Lobe et al , 1999). Therefore, bone marrow from these animals express the lacZ reporter gene before Cre-mediated excision occurs. Cre excision, however, removes the lacZ gene, allowing expression of the second reporter, the human alkaline phosphatase gene (hPLAP). To determine whether bone marrow-derived endothelial cells arise by fusion or differentiation, we transplanted bone marrow harvested from Z/AP mice into mice that constitutively express the Cre recombinase (pCX-NLS-Cre). Therefore, if bone marrow-derived endothelial cells result from differentiation of bone marrow cells, they will express the lacZ gene, and stain blue in the presence of X-gal reagent. However, if bone marrow cells fuse with endothelial cells from the recipient mice (PCX-NLS-Cre), the Cre recombinase will excise the lacZ gene, therefore allowing expression of the human alkaline phosphatase. Fused cells will therefore appear purple when stained for alkaline phosphatase with the NBT/BCEP reagent. B6RV2 tumours harvested from PCX-NLS-Cre mice transplanted with bone marrow harvested from Z/AP mice were cyosectioned and stained with X-gal (lacZ) and NBT/BCLP (hPLAP), followed by CD31 staining with diamino-benzidine (DAB) (Figure 29). 127 Figure 29 Bone marrow-derived endothelial cells arise by differentiation and not by cell fusion. (A) B6RV2 tumours were harvested from PCX-NLS-Cre mice transplanted with Z/AP bone marrow. Tumour sections were stained with X-gal (blue), NBT/BCIP (purple) and CD31 (brown). Arrows highlight stained bone marrow-derived endothelial cells. No NBT/BCIP + cells were detected in the tumour sections examined. (B) As control for cell fusion, spleen sections were stained with NBT/BCIP. Since DAB produces a brown color that could potentially mask the purple color generated by NBT/BCIP, tumour sections were carefully examined after NBT/BCIP staining and before CD31 staining. In tumour sections obtained from 5 different animals, we were able to observe a rare proportion of blood vessels containing lacZ+ cells (Figure 29A), indicating that differentiation of bone marrow-derived endothelial precursors contributes to the formation of tumour blood vessels. By contrast, no hPLAP + endothelial cells were observed, allowing us to rule out cell fusion or engulfment of hematopoietic cells as a mechanism implicated in the generation of bone marrow-derived endothelial 128 cells. Since no hPLAP + cells could be detected in tumour tissues, spleen sections from the same mice were stained with the NBT/BCIP reagent and used as a positive control (Figure 29B). Studies have suggested that, in the embryo, a single cell known as the hemangioblast can give rise to cells of both the hematopoietic and endothelial lineages. In adult bone marrow, however, the existence of the hemangioblast remains controversial. To determine whether adult bone marrow contains a hemangioblast, mice were transplanted with a single GFP + hematopoietic stem cell as previously decribed (Corbel et al., 2003), and we examined whether it could give rise to GFP + tumour endothelial cells. To confirm that single cells injected were indeed hematopoietic stem cells, their ability to reconstitute all blood lineages was tested. At various times after transplantation, 150 JX\ of peripheral blood was collected from the tails of recipients and red blood cells were lysed. Nucleated cells were stained with^ biotin-conjugated rat antibodies to IgG2a, IgG2b, B220, Gr-1 and Mac-1, followed by phycoerythrin-conjugated streptavidin (Caltag), and analyzed by flow cytometry. Animals with >15% GFP + blood leukocytes (35%, 38% and 48% GFP + blood leukocytes) and showing contribution of GFP + cells to all blood lineages were chosen for further analysis and were implanted with either 5 x 106 L L C and B6RV2 tumour cells. In extensive analyses of B6RV2 and L L C tumours from single cell transplanted mice, we found low numbers of blood vessels (around 1%) from either B6RV2 or L L C tumours that incorporated bone marrow-derived cells that stained positive for either CD31 or VE-cadherin (Figure 30). 129 B6RV2 \"3 O 0 ^ $ 1.8 1 1.6 9-1.4 CD31 VE-Cadherin Lewislungcarcinoma 1 2 % 1.8 I 1.6 I 1.4 T VE-Cadher in Figure 30 A single hematopoietic stem cell can give rise to endothelial progenitor cells that incorporate into tumour blood vessels. Tumours (B6RV2-upper panel; LLC-bottom panel) were implanted in the dorsal area of mice transplanted with a single GFP + primitive hematopoietic stem cell. Tumour sections were quantified for the presence of GFP + bone marrow-derived cells present into blood vessels stained with either CD31 or VE-cadherin. Data \u00C2\u00B1 SEM represents the average of the quantification of 3 tumours per group. Interestingly, the proportion of bone marrow-derived endothelial cells incorporating into tumour blood vessels was similar to that of whole bone marrow transplants. The presence of bone marrow-derived endothelial progenitors that incorporated into the vasculature of 130 animals transplanted with a single hematopoietic stem cell indicates that primitive hematopoietic stem cells have the potential to give rise to endothelial cells in addition to hematopoietic cells, and may therefore represent a population of adult hemangioblasts. 6.2.3 Role of V E G F and VEGFR-2 in the mobilization and differentiation of bone marrow-derived endothelial cells V E G F is a potent inducer of both angiogenesis and vasculogenesis, and has been reported to mobilize VEGFR-2 + endothelial progenitors from the bone marrow (Hattori et al., 2001). To determine whether increased V E G F secretion by tumour cells could lead to an increase in the contribution of bone marrow-derived endothelial progenitors to the tumour vasculature, B6RV2 cells were transduced with a retroviral vector encoding the human V E G F i 6 5 cDNA ( M S C V n e o - V E G F i 6 5 ) . Human V E G F i 6 5 has previously been shown to be biologically active in mice (Redaelli et al., 2004; Yang et al., 2003). Tumour cells were implanted in the dorsal area of transplanted mice, and after 10 days were harvested, cryosectioned, stained with either CD31 or VE-cadherin and quantified. Although B 6 R V 2 - V E G F tumours had an increased growth rate and displayed increased vascularity compared to B6RV2 wild-type tumours (Figure 31C), we did not observe an increase in the contribution of bone marrow cells to blood vessel formation compared to wild-type B6RV2 tumours (Figure 31 A, B). 131 CD31 VE-Cadherin B6RV2-VEGF B6RV2 Figure 31 V E G F secretion by tumour cells does not increase the contribution of bone marrow-derived cells to the formation of tumour blood vessels. B6RV2-VEGF tumours were implanted sub-cutaneously in the dorsal area of C3Pep mice previously transplanted with GFP + bone marrow from GFP transgenic donors (A) or with bone marrow transduced ex vivo with a GFP-encoding vector (MIG) (B). (C) Photograph displaying the increase tumour mass of B6RV2-VEGF tumours (left) compared to B6RV2 tumours (right). Data \u00C2\u00B1 SEM represents the average of the quantification of 4 tumours per group. 132 To ensure that the V E G F secreted by tumour cells was able to perfuse the bone marrow, V E G F ELISA was performed on the serum of mice implanted with B6RV2-VEGF tumours 10 days previously. Human V E G F 1 6 5 was detected in the serum of mice implanted with B6RV2-VEGF tumours (28.0 \u00C2\u00B1 4.7 pg/ml). By comparison, no detectable levels of V E G F 1 6 5 were detected in the serum of mice implanted with wild-type B6RV2 tumours. These results indicate that, in our model, increased V E G F secretion by tumour cells does not result in increased proportion of bone marrow-derived cells incorporating into tumour blood vessels, even though angiogenesis was markedly increased and tumours grew faster. Since serum V E G F levels produced by B6RV2-VEGF tumours may not have been sufficient to mobilize bone marrow endothelial progenitors, we used the strategy described in the previous chapters to selectively activate VEGFR-2 with a chemical inducer of dimerization in bone marrow cells, and examine whether activation of this receptor was sufficient to mobilize endothelial progenitors from the bone marrow and recruit them into B6RV2 vasculature. Briefly, bone marrow cells from C3Pep mice were transduced with a MIG-FKBP/VEGFR-2 construct and used to transplant lethally irradiated B6C3 mice. Four to six weeks after transplant, mice were implanted with B6RV2 tumours and were injected daily for 10 days with 10 mg/kg AP20187 or vehicle in order to dimerize the FKBP/VEGFR-2 construct. We have shown in the previous chapter that this strategy increases myeloid progenitor activity and expands the myeloid cell population (CD1 lb + , Gr-1+) in the bone marrow and peripheral blood of transplanted mice. However, VEGFR-2 activation in hematopoietic cells did not result in increased levels of VEGFR-2 + VE-cadherin+ endothelial progenitors in the bone marrow or 133 peripheral blood of transplanted mice (Figure 32A, B), nor did it result in increased contribution of bone marrow-derived endothelial cells to the vasculature of B6RV2 tumours (Figure 32C). It therefore appears that the VEGF/VEGFR-2 pathway may not be sufficient for the recruitment and/or expansion of endothelial progenitor cells to the tumour vasculature. 134 A FKBP-VEGFR2 FKBP-VEGFR2 +AP20187 VEGFR2 3 VE-Cadherin;; B VE-Cadherin id id -GFP -FKBP-VEGFR2 id id id id FKBP-VEGFR2 +AP20187 GFP CD31 * 0 8 \u00E2\u0080\u00A2 \u00E2\u0080\u00A2 FKBPA/EGFR2 FKBP/VEGFR2*AP20187 s 2 VE-Cadherin S 1,8 FKBP/VEGFR2 FKBP/VEGFR2*AP20187 Figure 32 VEGFR-2 activation in bone marrow cells does not result in endothelial progenitor mobilization or recruitment into tumour vasculature. Mice transplanted with FKBP/VEGFR-2 were injected with or without AP20187 for 10 consecutive days. Following this, bone marrow cells (A) and peripheral blood (B) were harvested and stained with VEGFR-2 or VE-cadherin to detect endothelial progenitors. (C) B6RV2 tumour sections from the same mice were stained with CD31 or VE-cadherin to detect GFP + bone marrow cells incorporated into tumour vasculature. Data \u00C2\u00B1 SEM represents the average of the quantification of 4 tumours per group. 135 6.2.4 Determination of the proportion of endothelial progenitors in human umbilical cord blood Since incorporation of bone marrow-derived endothelial progenitors into tumour vasculature is such a rare event, it is likely that endothelial progenitors are present in exceedingly low numbers in the circulation. To determine the proportion of circulating endothelial progenitors in humans using a rich source of hematopoietic stem cells, we examined umbilical cord blood using a functional assay. CD133 + cells, Lin\" cells and total mononuclear cells from umbilical cord blood were cultured for up to 6 weeks following plating of non-adherent cells over 3 days in endothelial cell medium supplemented with angiogenic cytokines (VEGF, bFGF). Serial plating of non-adherent cells ensured that we measured only transplantable endothelial progenitors, and not mature endothelial cells (Lin et al., 2000). After 3 to 4 weeks, colonies of adherent cells appeared and were quantified by counting the total number of colonies. The number of endothelial colonies was normalized to the total number of cells originally plated. Endothelial colonies adopted a cobblestone morphology and stained for endothelial markers such as vWF and the antigen marker P1H12 (Figure 33A). Analysis of endothelial colonies following expansion revealed that the cells express endothelial specific nitric oxide synthase (eNOS), Tie-1, Tie-2 and VEGFR-2 (Figure 33B, C), therefore confirming their endothelial phenotype. We observed a 16-fold enrichment of endothelial colonies in the CD133-purified cells over total mononuclear cells and a 2.7-fold increase over Lin\" cells (Figure 3 3D), indicating that the CD133+ cells are markedly enriched for endothelial progenitors, which is consistent with the observation that CD133 + VEGFR-2 + cells are associated with a population of endothelial progenitors 136 (Peichev et al., 2000). Nevertheless, the frequency of endothelial progenitors was less than 1 in 107 cells of the total mononuclear cell population, highlighting the rarity of this cell population. Mononuclear cells Lineage negative AC133-positive Figure 33 Rarity of endothelial progenitors in post-natal human mononuclear cells. Cells from umbilical cord blood (CD133+, Lin\", total mononuclear cells) were plated in medium supplemented with V E G F and bFGF. Endothelial colonies, which appeared 3 to 4 weeks after plating, stained positive for the endothelial markers vWF and PI HI 2 (immunostaining) (A) expressed eNOS, Tie-1 and Tie-2 by immunoblotting (B) and VEGFR-2 as determined by flow cytometry (C). Four weeks after plating, colonies were quantified by counting the total number of PI HI2 + colonies, and expressed as the total number of endothelial progenitors normalized to the original number of cells plated for each subset of mononuclear cells. Data represents the average of 7 to 12 independent experiments for each subset of mononuclear cells. * indicates a significant difference with # (P < 0.05). 137 6.3 Discussion Classically, tumour neovascularization has been thought to be limited to angiogenesis. Prior research has demonstrated this process to be mediated through the release of angiogenic factors such as VEGF, bFGF, PDGF and NO (Folkman and Shing, 1992). However, recent advances in vascular biology have led scientists to revisit this conventional concept. In vitro studies have demonstrated that subsets of cells isolated from bone marrow, peripheral blood or umbilical cord blood have the potential to give rise to mature endothelial cells (Lin et al., 2000). These endothelial progenitors, or angioblasts, have also been detected in vivo in bone marrow transplant models (Bailey et al., 2004; Lyden et al., 2001). Studies have shown that bone marrow-derived endothelial progenitors may contribute to neovascularization during ischemic conditions or tumour angiogenesis (Isner and Asahara, 1999; Lyden et al., 2001; Murayama et al., 2002). However, the relative levels of contribution of endothelial progenitors remain unclear, as some studies have argued that it is essential to tumour neovascularization (Asahara et al., 1999; Lyden et al., 2001), while other groups failed to detect any significant contribution (De Palma et al., 2003; Gothert et al., 2004b). The purpose of this paper was to definitively demonstrate the existence endothelial progenitors in vivo and examine their potential link to primitive hematopoietic stem cells. Using mice transplanted with bone marrow expressing GFP, we demonstrated that bone marrow-derived endothelial cells can contribute to the formation of new blood vessels in B6RV2 tumours. The incorporation of bone marrow-derived endothelial cells into tumour blood vessels remained a rare event however, as, on average, less than 1% of CD31- or VE-cadherin-stained blood vessels exhibited 138 incorporation of bone marrow-derived endothelial cells. These results contrast with those of Lyden et al., who have reported that around 90% of blood vessels in B6RV2 tumours are composed of bone marrow-derived endothelial cells (Lyden et al., 2001). This discrepancy may be due to different analytical methods such as the use of /3-galactosidase instead of GFP as a marker for bone marrow-derived cells and different antibodies used to identify endothelial cells. Furthermore, the close proximity of leukocytes, such as platelets, to the tumour endothelium could have led to false identification of bone marrow-derived endothelial cells, since vWF was used to identify bone marrow-derived endothelial cells. Recent studies have demonstrated that transplanted bone marrow cells can turn into unexpected lineages including myocytes, hepatocytes, neurons and many others. A potential problem, however, is that reports discussing such differentiation in vivo tend to conclude donor origin of transdifferentiated cells on the basis of the existence of donor-specific markers such as lacZ, GFP or the Y chromosome (Asari et al., 2004; Trotman et al., 2004). In recent years, studies have shown that bone marrow cells have the potential to fuse with different cell types such as hepatocytes and myocytes. It has therefore been argued that some of the transdifferentiation events reported for bone marrow cells may in fact be caused by cell fusion (Terada et al., 2002). In this study, we demonstrate definitively that bone marrow-derived endothelial cells originate through cell differentiation of a progenitor. The origin of those progenitors, their place in the hematopoietic hierarchy and their link to primitive hematopoietic stem cells remained unclear however. It has been previously demonstrated that single adult human hematopoietic stem/progenitor cells give rise to endothelial cells in vitro following serial 139 passage of these cells in long-term culture (Pelosi et al., 2002). Moreover, Bailey et al. have shown that they can detect vWF + and CD31 + bone marrow-derived cells in the vasculature of mice transplanted with a single hematopoietic stem cell (Bailey et al., 2004). In our study, we show that tumours implanted into mice transplanted with a single hematopoietic stem cell showed rare bone marrow-derived VE-cadherin+ and CD31 + cells incorporating into the vasculature. The use of VE-cadherin as a specific marker for endothelial cells allowed us to demonstrate definitively that these bone marrow-derived cells were of endothelial origin, therefore confirming the existence of an adult hemangioblast in vivo. Moreover, bone marrow-derived endothelial cells were detected in B6RV2 tumours implanted in serially transplanted animals, confirming the long term repopulating capacity of endothelial progenitors (data not shown). Even though the percentage of endothelial progenitor incorporation was very low, these results confirm the existence of cells with hemangioblastic activity in post-natal bone marrow. It is interesting to note that hematopoietic stem cells in the single cell-transplanted animals used in this study could also participate to the regeneration of damaged skeletal myofibers (Corbel et al., 2003), therefore confirming the multilineage potential of those primitive hematopoietic stem cells. Since the levels of incorporation of bone marrow-derived cells into tumour vasculature are low, it is likely that the differentiation of cells with hemangioblastic activity towards the endothelial lineage is a relatively rare event, which would make endothelial progenitors a rare cell population in comparison to other hematopoietic mononuclear cells. Indeed, we show here that endothelial progenitors are extremely rare among umbilical cord blood mononuclear cells, consisting of less than 1 in 10 cells. The 140 proportion of endothelial progenitors we observed here was much lower than those previously reported (Peichev et al., 2000; Quirici et al., 2001), which could be due to the fact that we determined this proportion using a functional differentiation assay to determine the proportion of endothelial progenitors in contrast to an assay that examines expression of cell surface markers. The cell surface markers CD34, CD 133 and VEGFR-2, which are used to detect endothelial precursors, are also present on other cell subsets in blood and bone marrow cells and could therefore explain the higher proportion of endothelial progenitors observed in those papers. Endothelial progenitors are enriched in the CD133+ population of mononuclear cells however, which also marks a population of primitive hematopoietic stem cells (Bhatia, 2001). However, whether CD133+ cells constitute a population of adult hemangioblast remains to be demonstrated at the clonal level. V E G F and its receptor VEGFR-2 have been shown to be critical in the differentiation and proliferation of endothelial progenitors in vitro (Fons et al., 2004). Moreover, increased levels of V E G F in mice in vivo have been associated with mobilization of endothelial progenitors through activation of VEGFR-2 (Hattori et al., 2001). Since endothelial progenitor incorporation into tumour vasculature is such a rare event, we were interested in determining whether V E G F was able to increase levels of incorporation of VE-cadherin+ and CD31 + cells into the tumour vasculature. However, when we examined the role of V E G F in the in vivo recruitment of bone marrow-derived endothelial progenitors, we found that overexpression of V E G F by tumour cells, even though it resulted in increased angiogenesis and tumour growth, did not cause any increase in the levels of incorporation of bone marrow-derived endothelial cells into the 141 tumour vasculature. These results do not correlate with those of Hattori et al. (Hattori et al., 2001), which reported rapid mobilization of VEGFR-2 + circulating endothelial precursor cells in response to increased serum levels of VEGF. However, the VEGF serum levels that they observed in their model were much higher than the ones we measured in our system. It is therefore possible that higher V E G F serum concentrations are required in order to mobilize bone marrow-derived endothelial cells, even though those levels we measured are sufficient to promote tumour angiogenesis. Alternatively, it is possible that local V E G F secretion by tumour cells may increase endothelial progenitor recruitment and local angiogenesis to equivalent proportion, in contrast to intravenous V E G F injection, which produces increased concentration of V E G F in the serum, which may be more potent to mobilize endothelial progenitors. Furthermore, even though V E G F may be important for the mobilization of endothelial progenitors, other cytokines such as angiopoietin-1 and bFGF may be important in the subsequent differentiation of those progenitors (Young, 2004). However, activation of VEGFR-2 in bone marrow cells did not expand or mobilize VEGFR-2 +VE-cadherin + endothelial progenitors, nor did it increase the levels of incorporation of endothelial progenitors into the tumour vasculature, even though we observed that VEGFR-2 was able to expand and partially mobilize a population of bone marrow Gr-1 + C D l l b + myeloid cells. It is therefore possible that VEGFR-2 activation alone is not sufficient to induce recruitment of endothelial progenitors into the tumour vasculature. The lack of VEGFR-2 induced mobilization or expansion of endothelial progenitors could suggest a requirement for the activation of other receptors in VEGF-induced expansion and/or mobilization of endothelial progenitors. This lack of mobilization could also be a result of the 142 experimental settings used in this experiment, since we only examined the activation of the kinase activity of VEGFR-2. Furthermore, it is possible that cells will respond differently to AP20187, which can penetrate inside the cells, compared to V E G F which will bind the extracellular portion of the receptor. Therefore, the pharmacokinetics of AP20187 would prevent the formation of an extracellular ligand gradient, which may be necessary in order to induce cell migration. Thus, it is possible that the extracellular domain of VEGFR-2 is required in order for endothelial progenitor cells to migrate in response to a V E G F gradient. Alternatively, this may represent a requirement for VEGFR-1 signaling, or VEGFR-1 /VEGFR-2 heterodimerization, in order to recruit endothelial progenitors. Our findings demonstrate that there is an adult hemangioblast, but the differentiation of a marrow stem cell towards the endothelial lineage is a rare event, and V E G F stimulation does not enhance this process. Understanding the factors that regulate contributions from primitive hematopoietic stem cells and their circulating progenitors to new vessel formation may ultimately provide additional ways to influence the process of neovascularization, which may prove beneficial in treating conditions such as diabetic retinopathy and cancer. 143 Chapter 7 Conclusions and future prospectives V E G F and its receptors, particularly VEGFR-2, have been implicated in neovascularisation processes. The fact that most tumours secrete V E G F suggests that its importance may be greatest in the context of tumour angiogenesis. Moreover, tumour endothelium is believed to express increased levels of VEGFR-2 (Takahashi et al., 1995). Experimental data also shows that V E G F acts in a paracrine manner on endothelial cells, resulting in the expansion of the tumour vasculature and the tumour mass. By contrast to the relatively large amount of information on the role of V E G F in solid tumours, little is known of its effects in \"liquid tumours\" such as leukemias and lymphomas. V E G F and its receptors have been shown to be present on a variety of hematopoietic malignancies. Moreover, VEGFR-2 expression in some hematological disorders has been shown to be associated with poor prognosis (Verstovsek et al., 2002). Before the significance of V E G F and VEGFR-2 expression in malignancies can be fully appreciated however, it is important to have a better understanding of the signalling pathways and the biological effects triggered by VEGFR-2 when it is activated by VEGF. The main goal of the research presented in this thesis was to acquire a better understanding of VEGFR-2 signalling effects. However, it has proven hard to study VEGFR-2 signalling in isolation due to the numerous V E G F receptors and ligands. To overcome this problem, we relied on various strategies to study VEGFR-2 signalling, without having to worry about the possible interference of other V E G F receptors. In the first part of this study, we used retroviral transduction to express VEGFR-2 in fibroblasts, which do not express other endogenous V E G F receptors. This model has allowed us to elucidate some of the signalling pathways induced following VEGFR-2 activation. In particular, we have shown that VEGFR-2 can activate the Erk 1/2 and p38 MAP kinase 145 pathways, Moreover, we also demonstrated that VEGFR-2 activation resulted in cell proliferation and migration. Even though it was previously known that V E G F could induce these signalling pathways and biological effects in endothelial cells, this study allowed us to confirm that VEGFR-2 activation alone was sufficient to induce them. However, even though VEGFR-2 is critical in the establishment of the endothelial lineage, we were not able to detect expression of endothelial markers in fibroblasts following its activation. These results could be interpreted in different ways: it could mean that either VEGFR-2 does not act as a switch that can promote the appearance of an endothelial phenotype, or that fibroblasts are too differentiated to be able to adopt an endothelial phenotype, even though they have been reported to transdifferentiate to other cell types such as endothelial cells (Kon and Fujiwara, 1994) and myocytes (Lattanzi et al., 1998). It is interesting however that VEGFR-2 was able to induce cell migration in response to VEGF. It was already known that VEGFR-1 can induce cell migration in response to VEGF, particularly in monocytes (Barleon et al., 1996), but here we show that VEGFR-2 activation can also trigger migration. This VEGFR-2 migration might play an important role in endothelial migration during angiogenesis. Moreover, it could be critical in the establishment of the endothelial and hematopoietic lineages by promoting the migration of early mesodermal precursors in response to V E G F gradients during early embryonic stages. Even though studying VEGFR-2 signalling in fibroblasts provided us with some answers concerning some of the functions of this receptor, we were interested in studying its function in compartments in which it is endogenously expressed, particularly hematopoietic cells. Other than the fact that it is expressed on subsets of normal and 146 malignant hematopoietic cells, little is known about the potential role of VEGFR-2 in these cells. However, studying VEGFR-2 signalling in hematopoietic cells in isolation would prove difficult, since subsets of hematopoietic cells have been shown to express VEGFR-1, and therefore stimulating VEGFR-2-transduced hematopoietic cells with V E G F would result in a certain degree of endogenous VEGFR-1 signalling \"contamination\". To overcome this problem, we used a VEGFR-2 construct that could be selectively activated with a chemical inducer of dimerization, which prevented interference from endogenous V E G F receptors. This VEGFR-2 construct was phosphorylated in the presence of AP20187, and could induce a signalling cascade that resulted in Erk 1/2 MAP kinase and Akt phosphorylation in vitro. More importantly, we were able to demonstrate that VEGFR-2 activation could induce survival of hematopoietic cells though the PI3-kinase pathway, a mechanism that may be important in some hematopoietic malignancies. In this part of the study, we only used sorted, transduced GFP + bone marrow cells for all experiments, and therefore, we were not able to identify a potential paracrine mechanism that could be triggered by VEGFR-2 activation. To have a better overview of the role of VEGFR-2 in hematopoietic cells, we used bone marrow transduced with the same VEGFR-2 construct to transplant mice. This approach allowed us to simulate a situation in which a relatively large proportion of hematopoietic cells express VEGFR-2, which has been shown to occur in some hematological malignancies. We observed that VEGFR-2 was able to promote the expansion of myeloid progenitors, not only among the cells that expressed the actual receptor, but also in untransduced cells, which led us to hypothesize that VEGFR-2 could promote myeloid cell expansion in a cell autonomous fashion and a paracrine fashion. 147 We confirmed the induction of a paracrine mechanism by showing that VEGFR-2 was able to up-regulate GM-CSF. Based on our in vitro and in vivo observations, we were able to propose a model for the effects of VEGFR-2 in hematopoietic cells (Figure 34). Figure 34 Hypothetical model for the possible effects induced by VEGFR-2 activation in hematopoietic cells. According to this model, autocrine or paracrine stimulation of VEGFR-2 by V E G F would result in activation of signalling pathways such as Erk 1/2 MAP kinase and PI3-kinase, which would promote cell survival in a cell autonomous manner. Moreover, VEGFR-2 activation would induce expression of GM-CSF. The secreted GM-CSF could in turn stimulate VEGFR-2 + GM-CSFR + cells via an autocrine loop. It is therefore possible that the signalling pathways induced by VEGFR-2 and GM-CSF would 148 synergise, which would result in the rapid expansion of myeloid cells expressing VEGFR-2. Moreover, the GM-CSF secreted by VEGFR-2 + cells could in turn stimulate GM-CSFR cells in a paracrine manner. It is likely that this mechanism may be important in promoting the expansion and survival of malignant hematopoietic cells that express VEGFR-2, since blocking VEGFR-2 can inhibit leukemic cell growth (Dias et al., 2000). However, further studies will be required in order to determine the relative importance of VEGFR-2 signalling in physiological hematopoiesis. More specifically, due to its expression on primitive hematopoietic stem cells, it will be interesting to study the effects of VEGFR-2 on the self-renewal, proliferation and differentiation of hematopoietic stem cells. Finally, we examined the contribution of bone marrow cells in the formation of the vascular network of tumours. Due to the conflicting reports published on this topic in the recent years, it is difficult to evaluate the specific role of bone marrow-derived cells in the formation of tumour blood vessels. Even though we were able to detect bone marrow-derived endothelial cells in the vascular network of tumours, their contribution was minimal. Furthermore, we were able to demonstrate that bone marrow-derived endothelial cells arise by differentiation, and not by fusion of bone marrow-derived cells and endothelial cells. We were able to show that primitive hematopoietic stem cells have the potential to give rise to endothelial progenitors, therefore confirming the existence of an adult hemangioblast. The contribution of bone marrow-derived endothelial cells to the tumour vascular network was so minimal however that it is unlikely that these cells play an important role in the elaboration of tumour blood vessels. We therefore examined whether V E G F could increase the contribution of bone marrow-derived endothelial cells 149 to tumour blood vessel formation, since it has been reported that V E G F can recruit endothelial progenitors from the bone marrow. 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"Graduate"@en . "Effects of VEGFR-2 signalling in post-natal hematopoiesis and vasculogenesis"@en . "Text"@en . "http://hdl.handle.net/2429/17216"@en .