Hypoxia, PDGF and VEGF in Vascular Development

Transkript

1 Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 154 Hypoxia, PDGF and VEGF in Vascular Development INGRID NILSSON ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2006 ISSN ISBN urn:nbn:se:uu:diva-6894

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3 To my family

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5 List of papers This thesis is based on the following papers, referred to in the text by their roman numerals: I Nilsson I, Shibuya M, Wennström S. Differential activation of vascular genes by hypoxia in primary endothelial cells. Experimental Cell Research. 2004;299(2): II Nilsson I, Rolny C, Wu Y, Pytowski B, Hicklin D, Alitalo K, Claesson-Welsh L, Wennström S. Vascular endothelial growth factor receptor-3 in hypoxia-induced vascular development. The FASEB Journal. 2004; 18(13): III Rolny C, Nilsson I*, Magnusson P*, Armulik A, Wentzel P, Lindblom P, Norlin J, Betsholtz C, Heuchel R, Welsh, M, Claesson-Welsh L. Platelet-derived growth factor receptor- promotes early endothelial cell differentiation. In progress *Both authors contributed equally IV Kreuger J*, Nilsson I*, Kerjaschki D, Petrova T, Alitalo K, Claesson-Welsh L. Early lymph vessel development from embryonic stem cells. Arteriosclerosis, Thrombosis and Vascular Biology. 2006; 26(5): *Both authors contributed equally Reprints were made with permission from the publishers.

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7 Contents Introduction...9 The vascular system during embryogenesis and in the adult...10 Endothelial progenitors and vasculogenesis...10 Hematopoiesis...11 Angiogenesis and vascular patterning...12 Lymphatic development...13 Perivascular cells...14 Vascular effectors...15 VEGFs and VEGF receptors...15 PDGFs and PDGF receptors...22 Hypoxia...25 Hypoxia-inducible factors...25 Regulation of HIF activity...26 HIFs as transcriptional regulators...28 Hypoxia and angiogenesis...30 Hypoxia in tumor formation...32 The vascular system in health and disease...33 Present investigation...34 Aims...34 Results and Discussion...34 Paper I: Differential activation of vascular genes by hypoxia in primary endothelial cells...34 Paper II: Vascular endothelial growth factor receptor-3 in hypoxiainduced vascular development...37 Paper III: Platelet-derived growth factor receptor- promotes early endothelial cell differentiation...39 Paper IV: Early lymph vessel development from embryonic stem cells...41 Concluding remarks...45 Populärvetenskaplig sammanställning...47 Acknowledgements...49 References...51

8 Abbreviations Ang -SMA BEC E EC ECM enos ES FGF HIF HSC HRE HSPG inos LEC MAPK NO ODD PDGF PDGFR PI3-K PKC PLC PlGF py TAD VE-cadherin VEGF VEGFR VHL vsmc Angiopoietin -smooth muscle actin Blood vascular endothelial cell Embryonic day Endothelial cell Extracellular matrix Endothelial nitric oxide synthase Embryonic stem Fibroblast growth factor Hypoxia-inducible factor Hematopoietic stem cell Hypoxia-responsive element Heparan-sulfate proteoglycan Inducible nitric oxide synthase Lymphatic endothelial cell Mitogen-activated protein kinase Nitric oxide Oxygen-dependent degradation Platelet-derived growth factor Platelet-derived growth factor receptor Phosphatidylinositol 3-kinase Protein kinase C Phospholipase C Placental growth factor Phosphotyrosine Transactivation domain Vascular endothelial cadherin Vascular endothelial growth factor Vascular endothelial growth factor receptor Von Hippel Lindau Vascular smooth muscle cell

9 Introduction Most organisms are dependent on oxygen for their survival. To efficiently supply our needs we have evolved a cardiovascular system consisting of the heart circulating oxygenated blood through a specialized transportation system of blood vessels. One of the earliest reports describing this organ system in mammals came in the 17 th century 1. Different compartments in the heart account for the proper direction of blood first to the lungs where red blood cells are loaded with oxygen, and then back to the left ventricle from where the aorta distributes the oxygen-rich blood through a tree-like organization of subsequently smaller and smaller arteries. The smallest vessels, the capillaries, are only wide enough to let single blood cells through and the slow flow allow for the passive exchange of gases (oxygen and carbon dioxide), nutrients and waste products between the blood and the cells in the tissue. The capillary network is very dense due to the limitation of oxygen diffusion in the tissue. Oxygen-depleted blood is then transported to post-capillary venules that empty in subsequently bigger and bigger veins, eventually leading back to the heart. In close connection to the cardiovascular system resides the lymphatic system. One of the first descriptions of presumptive lymphatic vessels came in fact from Uppsala and Olof Rudbeck in In contrast to the closed blood vascular system, lymphatic capillaries are open in the distal end, enabling the uptake of immune cells and fluid that have escaped from the blood capillaries. The lymph, carrying e.g. immune cells that have encountered a pathogen is drained through lymph nodes that are centers for eliciting immune responses. Eventually the lymph is emptied into the subclavian vein, thereby directly interconnecting with the blood vascular system. In the adult, the vascular system normally is resting and non-expanding except for in wound healing and in the female reproductive cycle. There are however several diseases caused or characterized by increased blood vessel formation (angiogenesis) or lymphatic vessel formation (lymphangiogenesis) such as tumors, diabetic retinopathy and rheumatoid arthritis. In search of means to control vessel growth, either to prevent cancer, or to increase blood supply e.g. to ischemic myocardium after a heart infarction, an entire new research field related to vascular biology has evolved. 9

10 The vascular system during embryogenesis and in the adult In mammals, the development of the vascular system is guided both by genetic and environmental components. Spatial and temporal coordination of interactions between cells, extracellular matrix (ECM), soluble factors, oxygen and local physical forces (fluid shear stress) all have influence on this process. Endothelial progenitors and vasculogenesis The first sign of mammalian blood vascular development is evident in the extraembryonic yolk sac at mouse embryonic day, E, Morphologically distinct structures, denoted blood islands, consist of foci of so called hemangioblasts that differentiate in situ, forming a loose inner mass of hematopoietic precursors and an outer layer of endothelial precursors (angioblasts) 3-6. The presence of an hemangioblast, the presumed precursor of both endothelial cells (ECs) and hematopoietic cells, has been challenged and not until recently was the hemangioblast identified in vivo 7,8. Hemangioblasts first appear in the splanchnic (visceral) mesoderm in the posterior region of the primitive streak at E7.0. This transient cell population expresses vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2), Tal-1/SCL and brachyury and migrate to the yolk sac at E to populate the blood islands 7,9,10. Blood island angioblasts eventually differentiate into ECs and coalesce in a process denoted vasculogenesis into a honey comb-like capillary network in the yolk sac beginning at E7.5. VEGFR-2-expressing angioblasts appear at E7.5 also in the head region. These intraembryonic angioblasts are derived from splanchnic (visceral) mesoderm and develop a primitive capillary plexus at E8-8.5 similar to that in the yolk sac. The dorsal aorta and cardinal veins are formed directly from aggregating angioblasts without a capillary plexus intermediate. In contrast to the yolk sac blood islands, initial EC differentiation from angioblasts in the embryo proper thus proceeds without concomitant development of hematopoietic cells 11. The propagation of hemangioblasts/angioblasts from mesodermal precursors has been shown to be influenced by bone morphogenetic protein 4 (BMP4) and fibroblast growth factor-2 (FGF-2) 12,13, whereas VEGF-A has been shown to drive the differentiation of angioblasts into ECs 14,15. Putative hemangioblasts purified from primitive streak embryos or differentiating embryonic stem (ES) cell cultures can in the presence of platelet-derived growth factor-bb (PDGF-BB) also give rise to vascular smooth muscle cells (vsmcs) (Figure 1) 7,16,17. Whether the ability of hemangioblasts to generate SMCs has relevance in vivo remains however to be confirmed. 10

11 Adult vasculogenesis, in situ differentiation of circulating EC precursor cells, has been shown to play a role in the formation of new vessels in the adult. These endothelial precursor cells originate from the bone marrow and incorporate specifically into sites of active neovascularization To what extent this contribution is crucial is although disputed and there seem to be variations dependent on the model employed. Figure 1. Development of vascular cells. Mesodermally derived hemangioblasts migrate to the yolk sac where they form blood islands in which they differentiate to angioblasts or primitive hematopoietic cells. Intraembryonic angioblasts are also derived from mesodermal precursor cells. FGF is important for the propagation of hemangioblasts (angioblasts) from mesodermal precursors and VEGF-A supports differentiation into blood vascular endothelial cells (BECs). Grey arrow indicates a hemogenic potential of angioblasts/becs. Hematopoiesis Initial mammalian hematopoiesis takes place in the yolk sac 3,5. The inner cells of the blood islands differentiate to primitive nucleated erythroblasts and to a minor extent, primitive macrophages 21. These primitive hematopoietic cells downregulate expression of VEGFR-2 and maintain expression of Tal-1/SCL, whereas the EC progeny maintain expression of VEGFR-2 but downregulate Tal-1/SCL 22. Although multipotential hematopoietic precurors are present in the yolk sac 23, these cells do not contribute to hematopoiesis in the adult mammal Instead, definitive hematopoiesis and hematopoietic stem cell (HSC) development has been proposed to occur in several intra-embryonic locations. Multipotential HSCs are found at E8.5 in the para-aortic splanchnopleura 11

12 (aorta and surrounding mesoderm), an anatomical location later denoted the aorta gonad mesonephros (AGM) region Definitive HSCs capable of long-term reconstitution of adult hematopoiesis is found in the AGM region at E Definitive HSCs that are carried on to adulthood thus are not derived from the yolk sac as previously anticipated but arise in the embryo proper 24,25,31. From where do the definitive HSCs found in the AGM region arise? Clusters of cells at the ventral side of the aorta in the AGM region are proposed to represent the hematopoietic precursors. Whether these have developed in situ from a specific precursor cell, or from ECs in the aorta with so called hemogenic potential or if they have migrated from another location, is currently not fully understood At E9.5, the fetal liver is colonized by HSCs and two days later, mature erythrocytes and myeloid cells are circulating in the blood 35. Whether the HSCs colonizing the liver are derived from the AGM region is currently not known. Recently, a third site of definitive hematopoietic precursor cell and HSC production has been identified in the placenta 36,37. In conjunction with parturition, hematopoiesis and maintenance of HSCs are transferred to the bone marrow 38. Angiogenesis and vascular patterning After a primitive vascular plexus has formed through vasculogenesis, it is further expanded and remodeled by a process denoted angiogenesis. The angiogenic process involves sprouting, branching, splitting and intussusceptions of existing vessels. Subsequent maturation of vessels involves recruitment of supporting pericytes/vsmcs, and functional modifications, such as basement membrane deposition and establishment of intercellular adhesions The first step in sprouting angiogenesis comprises proteolytic degradation of the vascular basement membrane to facilitate migrating ECs to invade the surrounding stroma. Migration of the ECs is guided by angiogenic stimuli present in the surrounding mesenchyme. During subsequent maturation steps, ECs assemble into solid chords, eventually acquiring a lumen 39. The aorta and cardinal veins are assembled already during vasculogenesis. Sprouting of ECs from these large vessels in an orderly fashion between the somites (forming the intersomitic vessels) is suggested to be guided by repulsive cues. While ephrinb2, semaphorin 3A and netrins are expressed on the somites, the developing intersomitic vessels express the corresponding receptor. This allows guidance of the growing vessels in the tissue corridor between the somites 42. This illustrates one example of common mechanisms in blood vessel and nerve patterning. The proper number of branch points in the vascular patterning process might also involve Notch signaling. Loss of Notch-1 result in excessive and misdirected branching of the intersomitic vessels

13 A number of studies have demonstrated the prerequisite of a proper gradient of VEGF-A in sprouting of blood vessels Gerhardt and co-workers showed that cells in the avascular retina, e.g. astrocytes, produce a gradient of VEGF-A guiding blood vessel sprouting in the developing retina 44. Two other studies showed that a balanced expression of heparin-binding VEGF-A isoforms are required to properly shape VEGF-A gradients and vascular patterning in the developing retina and during early embryogenesis 45,46. New vessels also form via intussusception, i.e. by insertion of tissue pillars into the lumen of a pre-existing vessels 40,48. In the microvasculature, this leads to a rather fast expansion and increased complexity of the capillary network. Vascular growth and remodeling through intussuceptive growth probably play a lesser role in ectodermally derived tissues, such as the brain. Vascularization of the brain probably relies on sprouting angiogenesis due to the absence of an already established primitive vascular network formed by vasculogenesis. Angiogenesis obviously occurs frequently during embryogenesis, but it also takes place in adults during endometrial growth, ovulation and wound healing. In addition, several pathological conditions are caused or characterized by neovascularization, including responses to tissue ischemia, growth of solid tumors, arthritis and diabetic retinopathy. Lymphatic development During embryogenesis, most data support a model where lymphatic endothelial cells (LECs) evolve through trans-differentiation of a subpopulation of vein ECs at mid-gestation 49. A potential LEC-specific precursor derived from the mesoderm has also been proposed, although this far it has been described only in the chicken 50. Incorporation of circulating cells expressing the stem cell marker CD133 as well as VEGFR-3 has also indicated the contribution from adult LEC precursors in lymphangiogenesis 51. The observed induction of lymphatic cell markers in venous ECs where the first lymphatic structures will appear lends however major support for Sabin s original theory 49. At E9, ECs in the cardinal vein already expressing VEGFR-3 upregulate expression of the hyaluronan receptor LYVE-1 52,53. One day later, a subpopulation of these cells acquires the expression of the transcription factor Prox1 selectively to one side of the vessel 54. VEGF-C produced by nearby mesenchymal cells is believed to mediate migration of putative LYVE- 1/Prox1/VEGFR-3-positive LECs from the veins into the surrounding tissue 55. After budding from the veins at E10.5, LECs assemble into sac-like structures from which secondary lymphatic growth proceeds 53,56. During the LEC specification process, LECs maintain and show increased expression of LYVE-1, the glycoprotein podoplanin, Prox1 and VEGFR-3, whereas these factors are subsequently downregulated in the blood vasculature

14 The morphology of lymphatic vessels resembles that of blood vessels to a large extent. The lymphatic capillaries have however little or no basement membrane and do never attach to supporting pericytes or vsmcs. Instead they have anchoring filaments connecting to the ECM to prevent the lumen from collapsing. The bigger collecting lymphatic vessels have a vsmc layer and a basement membrane. The presence of valves in the bigger vessels prevents lymph backflow and helps to propel lymph forward 58. Perivascular cells Pericytes are mural cells embedded in the microvessel basement membrane which they share. The two cell types form specific focal contact sites with tight-, gap- and adherence junctions (Figure 2). Pericytes are related to vsmcs but the two are differently located in the vasculature and exhibit different marker expression. Pericytes are found around capillaries, arterioles, venules and around collecting venules. Larger arteries and veins have concentric layers of vsmcs 59. The acquired muscle coat functions as a flow regulator through its capacity to contract or relax. There are several markers for pericytes and vsmc; -smooth muscle actin ( -SMA), desmin, neuron-glial-2 (NG-2), PDGF receptor- (PDGFR- ) and regulator of G-protein signaling-5 (RGS-5). -SMA stains both cell types whereas antibodies directed against NG-2 stains preferentially pericytes 59. Pericytes/vSMCs developing around the trunk vessels are probably derived from mesoderm 60 whereas brain pericytes are of neural crest origin 61. Possible transdifferentiation of ECs into vsmcs have also been proposed 16. Figure 2. Schematic drawing of a blood vessel. Endothelial cells (ECs) line the lumen of blood vessels. Pericytes are embedded in the basement membrane that they share with ECs. A thicker coat of vsmcs is found around big vessels. 14

15 Initial mural cell differentiation requires transforming growth factor - (TGF- ) signaling 62,63. Genetic studies in mice have also put forward a role for angiopoietin-1 (Ang1) and its receptor Tie2 in vessel maturation and remodeling. Mice deficient for Ang1 or Tie2 show reduced vessel stabilization and coverage of pericytes 64,65. Conversely, overexpression of Ang1 leads to more stabilized and leakage-resistant vessels 66,67. Recruitment of pericytes/vsmcs to newly formed vessels are regulated by PDGF-BB and its receptor PDGFR- and prevents further proliferation and migration of ECs, thereby stabilizing the vessel 68. Vascular effectors Several growth factors regulate important aspects of blood and lymphatic vessel formation. For instance, angiopoietins, ephrins, FGFs, Notch ligands, PDGFs, TGF- and finally VEGFs, all have essential roles in these processes 11, The cognate receptors for these factors are generally expressed on ECs whereas the corresponding ligands are produced by neighboring cells, such as vsmcs, or by the ECs themselves. Coordinated signaling through these receptor-ligand complexes drives the fundamental steps in angiogenesis and lymphangiogenesis. VEGFs and VEGF receptors The VEGF family consists of five mammalian factors; placental growth factor (PlGF), vascular permeability factor (VPF)/VEGF-A, VEGF-B, VEGF-C and VEGF-D, and one parapoxvirus-encoded VEGF, VEGF-E. Recently, VEGF proteins have also been identified in snake venom, denoted svvegfs 75. The independent discovery of the founding member of the VEGF family, VPF/VEGF-A, by two groups have generated the double denotation for this factor 76,77. Differential splicing of several members gives rise to a number of isoforms that differ in biological activity. The Vegf-a gene is organized into eight exons which undergo alternative splicing giving rise to up to nine different protein isoforms in humans: 206, 189, 183, 165, 165b (alternatively spliced exon eight), 162, 148, 145 and 121 amino acids long. Corresponding isoforms in mice are one amino acid shorter. VEGF-A165 and -121 are the predominantly expressed isoforms. Exons six and seven encode heparinbinding domains which are lacking in VEGF-A121. The longer VEGF-A isoforms have an increased binding affinity to its receptors and attachment to cells and ECM through interaction with heparan-sulfate proteoglycans (HSPGs) and this will restrict long-distance diffusion of the heparin-binding isoforms. VEGF-C and D are first synthesized with long N- and C-terminal extensions that eventually are removed after proteolytic cleavage

16 Figure 3. VEGF ligands and receptors. PlGF, VEGF-A and VEGF-B homodimers bind full length VEGFR-1 as well as the secreted variant svegfr-1. VEGF-A, proteolytically processed VEGF-C and human VEGF-D bind VEGFR-2. VEGF-C and VEGF-D bind and activate VEGFR-3. Heterodimers of PlGF/VEGF-A bind also the VEGFR-1/2 receptor heterodimer whereas VEGF-C binds to the VEGFR- 2/3 heterodimer. * denotes the human isoform. VEGFs are secreted as disulfide bonded homodimers (although heterodimers of VEGF-A with PlGF or VEGF-B have also been identified) Fully processed VEGF-C and D are however secreted as non-covalently bound dimers 82. VEGFs bind in an overlapping manner to three plasma membrane-bound receptor tyrosine kinases, VEGFR-1, -2 and -3 (denoted Flt-1, Flk-1 and Flt-4, respectively, in mice). VEGF-A, -B and PlGF bind VEGFR-1, VEGF-A and -E bind to VEGFR-2 and VEGF-C and -D bind to VEGFR-3. Proteolytically processed VEGF-C and human VEGF-D gain affinity also to VEGFR-2, whereas mouse VEGF-D only binds and activates VEGFR-3 (for mammalian ligand/receptor interactions, see Figure 3) The VEGFRs exhibit a typical tyrosine kinase receptor structure, consisting of an extracellular domain composed of seven immunoglobulin-like (Ig)-like folds and an intracellular split tyrosine kinase domain. In VEGFR-3, the fifth Ig-domain is replaced by a disulfide bridge. Alternative splicing of the Vegfr-1 gene gives rise to a soluble isoform, svegfr-1, lacking the kinase motif 85. In addition, a secreted isoform of VEGFR-2 and a truncated human VEGFR-3 have also been identified 86,87. In addition to binding to HSPGs, the longer isoforms of VEGF-A (containing exon seven) can also interact with neuropilin-1 and -2, of which the latter also interacts with VEGF-C 88,89. Neuropilins lack obvious intracellular catalytic activity and were first identified as receptors for the col- 16

17 lapsin/semaphorin family of axon guiding proteins 90. In ECs, neuropilins enhance binding of VEGF to its receptor and may thereby modulate VEGFR activation. VEGFR-2 can also interact with platelet-endothelial cell adhesion molecule-1/cd31 and vascular-endothelial (VE)-cadherin forming a mechanosensory complex mediating integrin-regulated responses to fluid shear stress, and complex formation between VEGFR-2 and VE-cadherin regulates vascular permeability 91,92. Expression of VEGF receptors VEGFR-2 is expressed on vascular precursors, hemangioblasts/angioblasts, already at E7.0 in mice whereas VEGFR-1 is expressed one day later (E8.0) 93. Later on, VEGFR-1 is primarily expressed in blood vascular ECs (BECs), but also on HSCs, monocytes/macrophages and vsmcs VEGFR-2 expression is retained in BECs but downregulated in hematopoietic cells. In addition, VEGFR-2 is found on LECs and neuronal cells 98. VEGFR-3 expression is first detected in angioblasts in the head mesenchyme and in the cardinal vein ECs at E At E12.5, expression is more restricted to venous ECs, developing LECs, monocytes/macrophages as well as a weak expression in capillary BECs 52,99. Later in development (E14.5), expression of VEGFR-3 is downregulated in blood endothelium with the exception of subpopulations of capillary and high endothelial venules in the adult However, up-regulation of VEGFR-3 in BECs also occurs in blood vessels actively engaged in angiogenesis during e.g. tumor growth 103. Biological function of VEGFs and VEGF receptors The absolute requirement for VEGF-A in cardiovascular development has been demonstrated by gene inactivation studies. Mice lacking only a single VEGF-A allele die around E11-12 due to impaired cardiac development and compromised intra- and extraembryonic vessel formation (Table 1) 104,105. In contrast, PlGF-deficient mice revealed only minor abnormalities under normal physiological conditions, but when challenged with ischemia, inflammation, wound healing or implantation of tumors postnatally, these mice exhibited decreased capacity to induce angiogenesis and arteriogenesis 106. In monocytes, PlGF has been shown to mediate migration via VEGFR VEGF-B has been reported to promote angiogenesis in matrigel plug assays using the recombinant protein or when the assay is performed in transgenic mice overexpressing VEGF-B in ECs 108,109. VEGF-B knockout mice appear healthy and fertile but exhibit a reduced heart size and an impaired recovery from myocardial ischemia 110. VEGF-B-transduced effects through VEGFR- 1 in ECs increase activity and expression of urokinase type plasminogen activator (upa) and plasminogen activator inhibitor-1 (PAI-1) 111. These proteins are involved in cellular adhesiveness and proteolysis of the ECM, 17

18 implying a role for VEGF-B in ECM degradation, cell adhesion and migration. Table 1. Mutations of genes involved in blood vascular- or lymphatic development. Genotype Phenotype Reference VEGF-A +/- E10-11 Compromised cardiovascular development 104,105 VEGF-C -/- E15.5- Tissue edema due to lack of lymphatic vasculature. 55 VEGF-D -/- Minor lymphatic hypoplasia in the lung 112 VEGFR-1 -/- E Vascular disorganization due to EC overgrowth. 113,114 VEGFR-1 TK -/- Normal embryonic development. Decreased tumor angiogenesis and monocyte migration in vitro. 115,116 VEGFR-1 TM-TK -/- 50% die due to vascular defects 117 VEGFR-2 -/- E8.5 Impaired development of ECs and hematopoietic cells. 118,119 VEGFR-3 -/- E Impaired remodeling of blood vessels. 120 Neuropilin-1 -/- E14 Defective neural patterning and blood vessel formation 121,122 Neuropilin-2 -/- Defective neural patterning and reduction of lymphatic capillaries Neuropilin-1 -/- /2 -/- E8.5 Impaired vascular development 126 Angiopoietin-1 -/- E12.5 Heart defects, impaired vascular remodeling and hematopoiesis 65 Angiopoietin-2 -/- Lymphatic hypoplasia 127 Tie-2 -/- E9.5 Heart defects, impaired vascular remodeling and hematopoiesis 64 TGF- 1 -/- E10.5 Defective vasculogenesis 128 Podoplanin -/- At birth. Respiratory failure, defect lymphatic patterning 129 Prox1 -/- E14.5 No lymphatic vessels 53,54 PDGF-B -/- Perinatal Microvascular hemorrhage, lack of pericytes 68,130,131 PDGFR- -/- Perinatal Microvascular hemorrhage, lack of pericytes 130,132 HIF-1 -/- E11 Abnormal cardiac development, mesenchymal cell death, impaired vascular remodeling HIF-1 -/- E10.5 Growth retardation, defective vascularization and hematopoiesis 136,137 HIF-2 -/- E9.5- Defective vascular remodeling, lung maturation or catecholamine production In VEGFR-1-deficient mice, ECs, which initially develop and migrate normally, fail to assemble into functional vessels leading to embryonic death at E An apparent overgrowth of ECs, caused by an increased proliferation of endothelial precursors were observed in these mice 113,114,141. In contrast, animals lacking the kinase domain (VEGFR-1 TK -/-, Table 1) were 18

19 viable with no obvious vascular defects, apart from impaired monocyte migration in vitro and decreased tumor angiogenesis 115,116. Based on these studies, VEGFR-1 seems to function as a negative regulator of EC division during development 141. Also, since the intracellular signaling regulated by VEGFR-1 seems to be of little importance, the binding and sequestering of VEGF-A away from VEGFR-2 could be a mechanism whereby VEGFR-1 controls the bioavailability of VEGF-A during development. The prerequisite for VEGFR-1 to trap VEGF-A at the cell surface was illustrated by further deletion of the transmembrane domain in VEGFR-1 TK -/- mice (VEGFR-1 TM-TK -/- ) that generates only non-anchored receptors. Here, 50% of the embryos died due to poor development of blood vessels. These mice exhibited reduced amounts of VEGF-A bound to the cell surface and decreased VEGFR-2 activation 117. However, in the adult, VEGFR-1 appears to play an important role in for instance pathological angiogenesis. Treatment with PlGF induces revascularization of ischemic heart and development of collateral vessels in ischemic limbs. In addition, in the presence of neutralizing antibodies to VEGFR-1, blood vessel formation in tumors, ischemic retinas and inflammatory conditions like autoimmune arthritis, were all reduced, suggesting an important role for VEGFR-1 in these processes 142. The anti-inflammatory effects of neutralizing VEGFR-1 were attributable to reduced recruitment of HSCs and migration of leukocytes to inflamed areas 142,143. Targeted inactivation of VEGFR-2 interferes with hemangioblast/angioblast differentiation and vasculogenesis. Essentially no ECs or hematopoietic cells develop and the mice die around E ,119. The fundamental importance of VEGF-A and VEGFR-2 in early vascular development is apparent during embryogenesis. Also maintenance of the adult vasculature is dependent on these factors as exemplified by the decreased vascular density and capillary regression in various organs after inhibiting VEGF-A signaling 144,145 VEGF-A also induces lymphangiogenesis The different experimental models used, wounded or unharmed skin and inflamed cornea in which VEGF-A was administered, may account for different mechanisms by which VEGF-A induce lymphangiogenesis. The effect could be transduced through VEGFR-2 expressed on LECs, or the effect could be indirect by inducing blood vascular leakage and recruitment of macrophages and dendritic cells that secrete VEGF-C and D 147. VEGF-C is important for the budding and migration of early LECs to form the primary lymph sacs and VEGF-C -/- mice consequently lack a lymphatic vascular system and die perinatally (E15.5-) from fluid accumulation in the tissues 55. Loss of VEGF-C thus seems not essential for blood vessel formation during development although administration of VEGF-C to the mouse cornea or in ischemic skin has been shown to potently induce angiogenesis Adenovirus expressing full-length VEGF-C in the non-harmed skin or rabbit hindleg muscle primarily induces lymphangiogenesis with 19

20 only minor effects on blood vessel formation Although human VEGF- D has been shown to be a strong inducer of lymphangiogenesis (as well as angiogenesis) in over-expression studies in adult animals 152,155,156, the mouse homologue seems dispensable during development, suggesting that VEGF-C or another, as-yet-unknown ligand compensates for the loss of VEGF-D 112. Fully processed human VEGF-D injected into the cremaster muscle or administered by adenovirus to the skin, rabbit hindleg muscle or intracardially in pig primarily induces angiogenesis 152,157,158. The pro-angiogenic effect of VEGF-C and -D are probably mediated via VEGFR-2-activation on BECs and whether VEGF-C and D elicit lymphangiogenic or angiogenic responses appear to depend on the relative expression of VEGFR-2 and -3 on BECs and LECs. Mice deficient for VEGFR-3 die at E9.5 due to defective remodeling and maturation of the primitive blood vascular plexus into larger vessels 120. The early lethality (prior to the emergence of lymphatic vessels) suggests an important role of VEGFR-3 in blood vascular development. The necessity of VEGFR-3 in lymphatic development has instead come from patients with congenital lymphedema exhibiting missense mutations in the Vegfr-3 gene and in an animal model overexpressing a soluble VEGFR-3, sequestering VEGFR-3-specific ligands 159,160. That inactivation of the two known ligands to VEGFR-3 (although not yet evaluated as a double-knockout) do not add up to the more dramatic effect of loosing VEGFR-3 may suggests alternative, VEGF-C/D-independent signaling via VEGFR-3 in early blood vessel formation, possibly transduced via heterodimerization with VEGFR-2 (see below). VEGF receptor tyrosine kinase signaling The main functions of the VEGFs are to promote survival, induce proliferation and enhance migration and permeability of ECs, all of which contribute to angiogenesis and lymphangiogenesis. These biological effects are regulated by the VEGFs through interactions with tyrosine kinase receptors. The VEGF ligands bind and induce dimerization of two VEGF receptor molecules. The close proximity of the intracellular tyrosine kinase domains of the receptors induces transphosphorylation of tyrosine residues in the juxtaposed domain. Dimerization between two receptors of the same kind has received most attention whereas heterodimerization between different VEGFRs has been reported more sporadically. Examples are heterodimers between VEGFR-1 and -2 or VEGFR-2 and -3 in response to a VEGF-A/PlGF ligand, VEGF-A or VEGF-C The signaling properties of VEGFR heterodimers compared to homodimers remain to be elucidated in more detail. Tyrosine phosphorylation sites in VEGFR-1 mediate binding to e.g. phosphatidylinositol-3-kinase (PI3-K), phospholipase C- (PLC ), SHP-2, growth-factor-receptor-bound-2 (Grb2) and Nck. However, the downstream 20

21 signal transduction events leading to e.g. migration are poorly characterized 75. Figure 4. VEGFR-2 phosphorylation sites and signal transduction. The intracellular domains of dimerized VEGFR-2 are shown with tyrosine phosphorylation sites indicated by numbers. Binding of signaling molecules to certain phosphorylation sites initiates signaling cascades which leads to specific biological responses. For details, see text. Reproduced with permission 75. The affinity of VEGF-A to VEGFR-1 is 10-fold higher than for VEGFR- 2. Nevertheless, signaling through VEGFR-2 appears to play a more important functional role in mediating EC survival, proliferation and permeability by VEGF-A (Figure 4). PLC binds to phosphorylated tyrosine (py) 1175 in VEGFR-2 and mediates activation of protein kinase C (PKC) and the p42/p44 mitogen-activated protein kinase (MAPK) pathway leading to proliferation of ECs 75. VEGF-A-induced activation of PI3-K/Akt and EC survival may be mediated by binding of the adaptor protein Shb to py ,165. Activation of the serine/threonine kinase Akt or PKC also regulates nitric oxide (NO) production and permeability through activation of endothelial NO synthase (enos) Increased NO production can also be mediated by increased intracellular Ca 2+ levels after PLC and diacylglycerol (DAG) activation 169. The small GTP-binding protein Rac, as well as p38 MAPK, heat-shock protein-27 (HSP-27) and focal adhesion kinase (FAK) are involved in cytoskeletal and migratory responses to VEGF-A

22 IQGAP1 is a recently identified binding partner of VEGFR-2, mediating migration via Rac 175. Binding of T-cell specific adaptor (TSAd) to py951 in VEGFR-2 has also been shown to mediate migration of ECs 176,177. Several signaling pathways have been delineated for VEGFR-2 whereas much less is known for VEGFR-3. Binding of Shc and Grb-2 to py1337 in VEGFR-3 has been shown so far 178. Proliferation, migration and survival of cultured adult human LECs involve p42/p44 MAPK, PKC and PI3-K/Akt signaling 179. PDGFs and PDGF receptors The PDGF family of growth factors consists of PDGF-AA, -BB, -CC, -DD and the heterodimer PDGF-AB 180. The PDGF-A and B isoforms are denoted classical PDGFs and were identified almost three decades ago , whereas the PDGF-C and D isoforms were fairly recently identified Like the VEGFs, PDGFs are synthesized as homo- or heterodimers, covalently linked by disulfide bonds. Both PDGF-A and B polypeptides are produced as pro-proteins with N-terminal extensions. These are removed during the transit through the trans-golgi 188. A C-terminal extension of the PDGF-A and B polypeptide chains, called the retention motif, mediate interaction with HSPGs. Thereby, the proteins are retained at the cell surface or in the ECM, allowing local, paracrine effects Further proteolytic processing outside the cells allows the release of freely diffusible PDGFs. PDGF-C and D polypeptides are secreted with an attached N-terminal CUB (complement subcomponent Clr/Cls sea Urchin EGF-like BMP-1) domain that sterically prevents interaction with the receptors To gain biological activity, the CUB domains must be cleaved off by extracellular proteases, such as plasmin or tissue plasminogen activator (tpa) 185,186,193. PDGFs bind in an overlapping manner to two plasma membrane-bound receptor tyrosine kinases, PDGFR- and -. PDGF-AA, -BB, -AB and CC display high affinity for binding to PDGFR-, and PDGF-BB and -DD to PDGFR-. As for the VEGFRs, binding of ligands induces homo- or heterodimerization of receptors. PDGF-AB, -BB and CC are capable of binding and activating also the PDGFR- / heterodimer (Figure 5) 180. Expression of PDGF receptors PDGF-A/PDGFR- and PDGF-B/PDGFR- are generally expressed in a defined paracrine setting in vivo. The pair-wise distribution is seen in various locations where epithelium connects to the mesenchyme (expressing PDGF- A and PDGFR-, respectively) or in the endothelium and surrounding pericytes/vsmcs (expressing PDGF-B and PDGFR-, respectively) 68,198. Cells dependent on PDGFR- expression include alveolar SMCs, oligodendrocytes, chondrocytes, placental spongiotrophoblasts and intestinal-, kid- 22

23 ney- and skin mesenchymal cells, while pericytes/vsmcs are the main PDGFR- -expressing cells 199. Figure 5. Receptor binding specificity of the different PDGF isoforms. PDGF-AA, - BB, -CC and AB bind to the PDGFR- homodimer. PDGF-BB and DD bind the PDGFR- homodimer. Finally, PDGF-BB, -AB and CC bind and activate also the PDGFR- / heterodimer. Biological function of PDGFs and PDGF receptors The classical PDGFs were reported long ago to be strong mitogens for mesenchymal cells such as fibroblasts and SMCs, as well as for cells of neurocrest origin, such as oligodendrocytes 200. Since then, a more detailed picture of which types of cells that are dependent on PDGFs has evolved, much thanks to gene inactivation studies in mice. Surviving pups lacking PDGF-A or PDGFR- knockout mice partially rescued by reintroduction of a PDGFR- transgene fail to form the epithelial septa between lung alvaeoli, leading to lung emphysema-like symptoms 201,202. The failure of compartmentalization of the alveolar sacs correlates with a lack of alveolar SMCs 195. Another example of folding of epithelium (expressing PDGF-A) over mesenchymal tissue (expressing PDGFR- ) is found in intestinal villi formation, which is also impaired in PDGF-A and PDGFR- knockout mice 196. Defective oligodendrocyte development causing hypomyelination is evident in the PDGF-A knockout and in mice expressing a Src signaling-deficient PDGFR- 203,204. The PDGF-B/PDGFR- pathway is critical in recruiting perivascular cells to vessels. PDGF-BB is strongly expressed by ECs located at the distal end (tip) of growing vessel sprouts in the retina 44. This will create a gradient of PDGF-BB, which allows for proliferation, directed migration and attachment 23

24 of pericytes/vsmcs to the growing vessel. To obtain a proper gradient, PDGF-BB needs to be ECM- or cell-bound and mice lacking the retention motif in PDGF-B consequently fail to mediate the proper investment of perivascular cells around vessels 205. PDGF-B or PDGFR- knockout mice die at late gestation from extensive microvascular bleedings 131,132. The hemorrhaging stems from a lack of pericyte/vsmc coverage of vessels especially in the brain, but also in the skin, lung, kidney and heart 68,198. Initial development of pericytes/vsmcs seems however not affected as the amount of pericytes/vsmcs around e.g. the aorta is normal. Only the expansion and migration of perivascular cells during later vascular progression seems dependent on PDGF-B/PDGFR This may explain why loss of PDGF- B/PDGFR- is most critical in organs lacking a primary vascular network established by vasculogenesis but that completely depend on sprouting and migration of new vessels. Apart from the pericyte/vsmc defect, the naked vessels appear distorted and hyperplastic, possibly due to the increased expression of VEGF-A in the knockouts 130. Mice lacking the Pdgf-b and Pdgfr- genes display anemia and thrombocytopenia 131,132. This hematopoietic defect has been reevaluated by Kaminski and co-workers 206. They showed that hematopoietic cells from PDGF-B -/- or PDGFR- -/- mice has the ability to completely reconstitute an irradiated recipient and seems not to be dependent on PDGF-B or PDGFR- expression. The compromised liver development (the main site of embryonic hematopoiesis) in these animals has instead been proposed as the major cause of the hematopoietic defect 206. Although PDGF-BB can bind and activate both PDGFR- and -, single and double mutant studies of PDGF-A and B or PDGF-A and -C in vivo indicate that PDGF-AA and -CC are the principle ligands of PDGFR-, whereas PDGF-BB probably plays a minor role as a PDGFR- ligand 185,207. This observation is also strengthened by the restricted tissue distributions of the different PDGF ligand/receptors in vivo. Future research will further answer the question in what tissues PDGF-CC and -DD may influence and overlap with the function of the classical PDGFs. PDGF receptor tyrosine signaling PDGFs transduce a diverse set of biological responses, such as proliferation, migration, differentiation and cell survival 208. PDGFRs are activated in a similar fashion as the VEGFRs and signaling molecules such as Src, PI3-K, PLC and Ras are engaged. Most of these signaling molecules are responsible for cell proliferation in response to PDGFs whereas PDGF-induced cell migration involves the PI3-K and PLC /PKC signaling modules 199. Biochemical studies in vitro have revealed that both PDGFR- and - can mediate similar biological responses although they differentially bind Crk and RasGAP 209. In vivo however, gene inactivation studies strongly imply that vessel-associated pericytes/vsmcs are the primary PDGFR- responsive cell types, whereas several different mesenchymal/neural crest- 24

25 derived cell types depend on PDGFR- expression. In mice carrying a receptor chimera where the intracellular part of PDGFR- is exchanged for PDGFR- shows no obvious phenotype. The opposite approach of fusing the PDGFR- cytoplasmic domain onto the PDGFR- extracellular domain however leads to multiple abnormalities in the pericyte/vsmc population 210. This may suggest that PDGFR- has a unique signaling capacity, at least in perivascular cells. PI3-K signaling has been shown to be the primary pathway downstream of PDGFR- in vivo 203. In PDGFR-, PI3K signaling is also of major importance 211. Activation of RasGAP in PDGFR- signaling appears to result in negative regulation of pericyte/vsmc formation possibly by keeping Ras in an inactivated state 212. Hypoxia Oxygen is an essential factor required for metabolic processes including ATP (energy) production. Inadequate supply of oxygen, denoted hypoxia, will change the metabolic state, as well as many other important functions in affected cells. Hypoxia is defined as the threshold where the oxygen concentration becomes limiting for normal cellular processes. The atmosphere consists of 20.9% (normoxia, or ambient oxygen pressure), but cells in tissues normally experience much lower concentrations, typically around 5% 213. In this respect, 3-5% oxygen still represents an appropriate environment for cells although they are close to the hypoxic range. At this stage, oxygen is not yet limiting for the cell, but further decrease in oxygen levels will trigger hypoxia-induced responses 214. The oxygen level is closely monitored by intracellular mechanisms. Under hypoxic conditions, an oxygen sensing machinery activates a family of transcription factors known as hypoxia-inducible factors (HIFs). HIF-1, the founding member of this family, activates a series of genes participating in compensatory mechanisms that support cell survival in a potentially lethal microenvironment. HIFs thus function as master regulators of adaptive responses to hypoxia 215. During embryogenesis, the increasing need for oxygen drives the development of a cardiovascular system. In addition, hypoxia is also considered to promote pathological formation of blood vessels in solid cancers, diabetic retinopathy and rheumatoid arthritis. Finally, hypoxia constitutes a complicating factor in many modern disease states, such as heart infarction. Hypoxia-inducible factors The HIF transcription factors are composed of one of three alpha subunits (1, 2 or 3 ), and beta ( ) subunits (HIF-1 is also denoted aryl hydrocar- 25

26 bon receptor nuclear translocator, ARNT). Both the HIF- and HIF- subunits are members of the basic helix-loop-helix (bhlh) family of transcription factors. The N-terminal parts of the HIF protein, the bhlh and the Per- Arnt-Sim (PAS) domains are required for HIF dimerization and DNA binding (Figure 6) HIF-1 and HIF-2 proteins also contain two transactivation domains (TADs) in the C-terminal half 219. Within the N-terminal TAD there is an oxygen-dependent degradation (ODD) domain that is responsible for degradation of the subunit under normoxic conditions 220,221. The main function of the C-terminal TAD is to recruit transcriptional coactivators such as CBP, p300, SRC1 and TIF-2 217,222,223. In hypoxic conditions, the HIF heterodimer (named HIF-1, -2 or -3) translocates to the nucleus where it binds to a core DNA sequence (5 -ACGTG- 3 ), denoted the hypoxia-responsive element (HRE), located in the promoter/enhancer regions of hypoxia-regulated genes 224. Definition of this DNA motif and discovery of the prototypic HIF factor, HIF-1, can be attributed to Gregg Semenza and co-workers 225,226. Although the HIF-1 and -2 subunits can bind to the same DNA motifs, they appear to control rather distinct biological functions, especially during development. Whereas HIF-1 is expressed in virtually all cell types, HIF- 2 exhibit a more restricted expression pattern in endothelial cells and in catecholamine producing cells in the organs of Zuckerkandl 227. The biological function of HIF-3 is under investigation 228, but a HIF-3 splice variant, denoted inhibitory PAS (IPAS), appears to function as a negative regulator of hypoxia-inducible responses 229. Figure 6. Functional domains of the HIF- subunit. The basic helix-loop-helix (bhlh) and the Per Arnt Sim (PAS) domains in the N-terminus are important for dimerization and DNA binding. The oxygen-dependent degradation domain (ODDD), situated within the N-terminal transactivation domain (N-TAD), is responsible for the degradation of HIF- in normoxia. The main function of the C-TAD is to recruit transcriptional co-activators. Regulation of HIF activity HIF- protein levels, which are extremely low under normoxic conditions, dramatically increase in response to hypoxia, along with the transcriptional activity. The -subunits are, on the other hand, present in similar levels in both normoxia and hypoxia 230. The presence of HIF- subunits only in conditions of low oxygen tension or after treatment with iron chelators had for a 26

27 long time puzzled researchers until a group of oxygen-dependent enzymes essential for regulating HIF- protein levels was discovered. Figure 7. Dual regulation of HIF- stability and activity by prolyl and asparaginyl hydroxylations. In normoxia, hydroxylation on two proline residues within the ODD domain serves as recognition sites for pvhl and targets the protein to polyubiquitylation and degradation in the proteasome. An additional hydroxylation of an asparagine residue in the C-terminal TAD inhibits the binding of transcriptional coactivators. Under hypoxic conditions, HIF- is phosphorylated and translocated into the nucleus where it dimerizes with HIF-. After binding to HRE DNA motifs and co-factor recruitment, HIF induces gene transcription. Reproduced with permission 231. Hydroxylation of two proline residues in the HIF- ODD domain, executed by a family of prolyl hydroxylase domain proteins (PHDs), serves as a recognition/binding site for the von Hippel-Lindau (pvhl) E3 ubiquitin ligase complex 232,233. Binding of pvhl targets HIF- for polyubiquitylation and subsequent degradation by the proteasome 234,235. The PHD enzymes are members of the 2-oxoglutarate-dependent hydroxylase superfamily 236. To obtain full transcriptional activity, HIFs must bind to the HRE DNA sequence and recruit transcriptional co-factors. This event is also regulated by oxygen levels. In the presence of oxygen, an asparagine residue in the C- terminal TAD gets hydroxylated by a HIF asparaginyl hydroxylase called factor-inhibiting HIF (FIH-1) 237,238. This will silence the TAD domain by 27

28 preventing the binding of transcriptional co-activators CBP/p300. The enzymatic modifications executed by the prolyl and the asparaginyl hydroxylases are dependent on oxygen and iron (Fe 2+ ), explaining the fact that HIF- subunits escape degradation in the absence of oxygen or iron (Figure 7) Additional pathways for silencing HIF activity in normoxia include acetylation of a lysine residue in HIF-1 ODD domain by the ARD1 acetylase. This modification increases the interaction with pvhl, resulting in enhanced degradation of HIF The stability and activity of HIF- subunits may also be influenced by reactive oxygen species, hydrogen peroxide (H 2 O 2 ), as well as by growth factor- and cytokine-induced phosphorylation 217, HIFs as transcriptional regulators When the protein levels of HIF- increase, e.g. in response to hypoxia, it translocates to the nucleus, dimerizes with the subunit and activates the transcription of a number of target genes displaying an HRE motif. Nuclear localization signal (NLS) domains in the - and subunit confer autonomous translocation into the nucleus 248. One group of HIF-1 target genes is involved in the adaptive response facilitating oxygen delivery to oxygen-deprived tissues. It includes e.g. genes coding for erythropoietin, VEGF-A and inducible NOS (inos) (Table 2, edited from 224 ). The erythropoietin (Epo) gene, encoding a kidney hormone, was discovered as the first true hypoxia-inducible gene in EPO stimulates red blood cell production (erythropoiesis), thereby increasing oxygen delivery. Hypoxia also promotes iron uptake and transport by increasing the expression of transferrin and the transferrin receptor 249,250. Another well-known hypoxia-regulated gene is Vegf-a, which plays a crucial role in development and growth of blood vessels 251,252. One of the VEGFRs, encoded by the Vegfr-1 gene, is also a direct HIF target, harboring an HRE motif 253. The Vegfr-2 gene, which at first was reported to lack HIF binding sites, has now been shown to be upregulated by HIF Hypoxia also affects vascular tone and local blood flow by induction of vasoconstrictors, such as endothelin-1 (ET-1) 255, or by increased expression of genes regulating vasodilation, such as heme oxygenase-1 (Ho-1) and inos 256,

29 Table 2. Hypoxia-inducible genes harboring HRE sequences Gene Function Oxygen supply 1 -adrenergic receptor Adrenomedullin Atrial natriuretic peptide (ANP) Beast cancer resistance protein (BCRP) Endothelial nitric oxide synthase (enos) Endothelin-1 Erythropoietin Ferrochelatase Heme oxygenase 1 Inducible nitric oxide synthase (inos) Leptin Transferrin Transferrin receptor Plasminogen activator inhibitor-1 (PAI-1) Vascular endothelial growth factor-a (VEGF-A) VEGF-D VEGF receptor-1 (VEGFR-1) VEGFR-2 Vessel diameter Vessel diameter Blood volume Heme binding Vessel diameter Vessel diameter Erythropoiesis Heme synthesis Vessel diameter Vessel diameter Metabolism/Angiogenesis Iron transport Iron transport Blood flow Angiogenesis (Lymph)angiogenesis Angiogenesis Angiogenesis Cellular metabolism Aldolase A (ALDA) Carbonic anhydrase-9 (CA-9) Cytochrome P450 2C11 (CYP2C11) CYP3A6 CYP4B Enolase 1 Glucose transporter 1 (Glut1) Glucokinase Glutathione peroxidase-3 (GPx-3) Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) Lactate dehydrogenase A Multidrug resistance gene 1 (MDR1) Phosphoenolpyruvate carboxykinase (PEPCK) Phosphofructokinase L (PFKL) 6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase-3 6-Phosphofructo-2-kinase/fructose-2,6-biphosphatase-4 Phosphoglycerate kinase 1 (PGK1) Glycolysis ph regulation Xenobiotic metabolism Xenobiotic metabolism Eicosanoid synthesis Glycolysis Glucose uptake Glycolysis Glutathione peroxidase Glycolysis Glycolysis Xenobiotic transporter Gluconeogenesis Glycolysis Glycolysis Glycolysis Glycolysis Cell growth and metabolism Connective tissue growth factor (CTGF) Ecto-5 -nucleotidase (CD73) Endoglin Insulin growth factor binding protein-1 (IGFBP-1) Intestinal trefoil factor Transforming growth factor- 3 (TGF- 3) Growth factor Intestinal barrier function TGF- coreceptor Growth factor Intestinal barrier function Placenta development 29

30 Cell growth and apoptosis CXCR4 Bcl-2/E1B 19kDa interacting protein (BNip3) Met Myeloid cell factor-1 (Mcl-1) Nip3 Noxa Nucleophosmin Nur77 Serine/threonine protein phosphatase 5 (PP5) Stromal cell-derived factor-1 (SDF-1 or CXCL12) Telomerase reverse transcriptase (TERT) Wilms tumor suppressor (Wt1) Chemokine receptor Pro-apoptotic Proto-oncogene Anti-apoptotic Pro-apoptotic Pro-apoptotic p53 inhibition Orphan steroid receptor Anti-apoptotic Chemokine Telomere extension Tumor suppressor gene Others CD18 Cited2/p35srj Collagen prolyl 4-hydroxylase I DEC1 and DEC2 Ets-1 Furin Glucose-regulated protein 94 (GRP94) Inhibitor of differentiation/dna binding protein 2 (ID2) Membrane type-1 matrix metalloproteinase (MT-1) Prolyl hydroxylase domain protein 2 and 3 (PHD2/PHD3) Retrotransposon VL30 Leukocyte adhesion Transcription cofactor Hydroxylase Transcription factors Transcription factor Pro-protein convertase Chaperone Transcriptional repressor Matrix metalloproteinase Oxygen sensing Retrotransposon Another group of genes upregulated by HIF-1 acts to compensate for the loss of oxygen-dependent metabolism in hypoxia. The increased expression of various glucose transporters and glycolytic enzymes under hypoxic conditions, enumerated in Table 2, allows for oxygen-independent generation of ATP (glycolysis). When oxygen levels fall to a critical point, metabolic switches turn off oxidative phosphorylation and mitochondrial electron transport and instead oxygen-independent energy production (glycolysis) is induced. In the glycolytic pathway, four ATP molecules are produced when glucose is metabolized to two molecules of pyruvate. As two ATP molecules are consumed during this process, this leaves a net yield of two ATP. Compared to aerobic conditions where pyruvate is further oxidized in the Kreb s cycle and the net yield is 31 molecules of ATP, anaerobic glycolysis is much less efficient. Recently, a chemokine receptor involved in migration, CXCR4, was found to be induced by hypoxia 258. Its expression on certain tumor cells has been shown to allow for homing of metastatic tumor cells to specific organs expressing the corresponding ligand, stromal cell-derived factor-1 259,

31 Hypoxia influences in addition many other transcription factor pathways, such as those mediated by AP-1, ets-1, fos and jun, NF- B and p53. It is therefore possible that a variety of cellular responses to hypoxia are mediated by HIF-independent mechanisms. Hypoxia and angiogenesis The cardiovascular system is the first organ to become functional during embryogenesis and its proper function is critical for continued growth of the embryo. Before the onset of heart and blood vessel development, the embryo resides in a fairly hypoxic environment and this stimulus is considered to constitute the major driving force for blood vascular development 213. It has also been shown that the number of hematopoietic precursors increases under hypoxic conditions implying that the expansion of precursor cells may be driven by hypoxia 261. The observation that hypoxia can inhibit differentiation but instead keep precursor cells proliferating has recently been described for a number of cells, such as neuronal stem cells 262. According to this report, inhibition of differentiation by hypoxia is mediated by HIF-1 binding to the Notch-1 intracellular domain, thereby building a transcriptional complex enhancing downstream Notch signaling. One of the key players in regulating the extent of vascularization in a given tissue is the oxygen level. Many genes involved in angiogenesis, including the Vegf-a gene, are upregulated by hypoxia 251,252,263. The induction of VEGF-A by hypoxia has therefore been considered to be the main driving force for development of new vessels under ischemic conditions. Several pieces of evidence suggest a role for hypoxia and HIFs in development of the vascular system. The lethal phenotype of targeted inactivation of the major mediator of hypoxia, Hif-1, demonstrated defects in both the intraembryonic and extraembryonic vasculature, in addition to neural defects, cephalic mesenchymal cell death and cardiac defects ,264. Deletion of HIF-1 selectively in ECs does not affect vasculogenesis or angiogenesis during development. However, in the absence of HIF-1, there is decreased EC proliferation and migration in response to VEGF-A in vitro as well as in vivo, in the matrigel plug assay 265. Wound healing and tumor angiogenesis are also delayed or decreased in these mice. Another recent study showed that inactivation of both HIF-1 and HIF-2 -induced responses in ECs due to the introduction of a dominant-negative HIF construct, leads to embryonic death at E The mice were growth retarded, exhibited heart defects and impaired vascular remodeling. These studies thus illustrate the importance of EC responses to hypoxia. Embryos deficient for the dimerization partner HIF-1 also exhibit lethality coupled to defects in vascular remodeling 137,267. A decrease in the number of hematopoietic progenitors is also observed in these mice 136. Although initial EC differentiation and early vasculogenesis were unaffected in HIF- 31

32 1 - and HIF-1 -deficient mice, these embryos were unable to form properly branched vessel networks, suggesting a role for hypoxia sensing during normal vascular patterning. Inactivation of HIF-2 exhibited diverse phenotypes, depending on mouse strain. One study found HIF-2 to be important for remodeling of the primary vascular network, whereas another study presented decreased production of catecholamines as the main phenotype 138,139. A third study showed that inactivation of HIF-2 obstructed lung maturation 140. By selective breeding, viable HIF-2 mice have been generated which exhibit a defect in hematopoiesis 268,269. While the role of HIF-2 in embryogenesis is still somewhat unclear, HIF-1 seems however not to fully compensate for HIF-2 deficiency in any of these reports. Overall, more studies are needed to determine how, and to what extent, the vascular patterning during development, or in adult tissues, are driven by local hypoxia versus e.g. hypoxia-independent genetic cues. Hypoxia in tumor formation Solid tumors must be sufficiently perfused by blood to be able to grow beyond 2-3 cubic millimeters 270. The current dogma is that tumors rapidly outgrow their blood supply, leading to emergence of hypoxic regions, which in turn stimulate the release of angiogenic growth factors such as VEGF-A 252. HIF-1 and HIF-2 are overexpressed in many tumors, presumably as a result of hypoxia 271. One example of connection between tumorigenesis and increased HIF levels is the von Hippel-Lindau (VHL) disease caused by germline or somatic mutation in the VHL gene. Loss of VHL results in the stabilization of HIFs in normoxic cells. The patients suffer from highly vascularised tumors, such as hemangioblastomas and renal cell carcinomas. Furthermore, HIF-2 has been suggested to have a tumor enhancing role in renal cell carcinomas lacking VHL 272. Knockdown of HIF-2, but not HIF- 1, by sirna in a mouse tumor model blocks tumorigenesis. HIF-2 has also been shown to specifically induce tumor-promoting genes in the same tumor model 273. Therefore, there have been high expectations on the possibility to inhibit tumor formation and pathological vascularization by interfering with hypoxia-mediated transcription. Early hopeful findings showed that blood vessel formation was reduced in tumors lacking HIF-1 274,275. However, the great complexity in treatment regimens, using HIFs as targets, has been shown by Gabriele Bergers lab 276. Implantation of HIF-1 -deficient astrocytoma cells intracranially or subcutaneously resulted in completely different responses. Tumors in the vesselpoor subcutis exhibited reduced growth and vessel density. In contrast, when cells were implanted into highly vascularized brain tissue, HIF-1 -/- tumour cells grew more aggressively. Using the same experimental setup, VEGF-A -/- cells showed tumor regression and decreased vessel formation in both sites. 32

33 Together, these findings suggest that tumor responses elicited by HIFs depend on the microenvironment. The vascular system in health and disease Apart from being a conduit for blood, the vascular system takes part in the exchange of gases and nutrients with the nearby tissue as well as regulating blood clotting, blood flow, inflammation and wound healing. Normal, healthy blood vessel growth occurs throughout development as well as during wound healing and in the female reproductive cycle. There are also numerous diseases caused or characterized by angiogenesis. Rheumatoid arthritis (RA) is an autoimmune disease which eventually destroys the peripheral joints. The inflammation leads to increased fluid secretion in the joints as well as the appearance of hypoxic areas due to the increasing distance between blood vessels in the swollen tissue. Local hypoxic areas probably contribute to the reported increased VEGF-A serum levels in these patients and for the increased blood vessel growth in the joints. In an animal model of RA, inhibition of VEGF-A revealed the symptoms, suggesting that therapies aiming at attenuating angiogenesis may be beneficial 277. Psoriasis is also characterized by inflammation and induction of angiogenesis. The red rashes are signs of the highly permeable blood vessels formed in this condition 278. Patients with diabetes exhibit elevated VEGF-A plasma levels. In the retina, this leads to growth of highly permeable blood vessels. The overgrowth of blood vessels leads to scarring and cell death resulting in visual impairment or even blindness 279. VEGF-A has been established as a key angiogenic player in cancer. The expression of VEGF-A in tumor cells and stroma correlates with tumor progression and vascularization. The tumor vasculature can also provide a route by which tumor cells get into the circulation and form metastasis. Tumor metastases also spread with lymphatic vessels that are formed in or around growing tumors 78,279. Impaired lymphatic vessel capacity leads to lymphedema. The increase in protein-rich interstitial fluid leads to swelling, increased pressure and inflammation. Lymphedema can be inherited (mutation in the Vegfr-3 gene) or acquired by surgical removal of lymphatic vessels or by a parasitic infection (filiarisis) 58. There are thus many diseases where inhibition of angiogenesis could be a therapeutic goal. In contrast, there are also conditions where an increased angiogenesis would be beneficial, e.g. after a stroke or heart infarction. 33

34 Present investigation Aims The mechanisms behind many important aspects of blood- and lymphatic vessel formation have yet not been elucidated in detail. The general aim of this thesis was therefore to characterize the effect of hypoxia, PDGF and VEGF on vascular development and function. More specifically, we wanted to investigate: Responses to hypoxia by ECs from different vascular beds Mechanisms behind hypoxia-induced vascularization Contribution of PDGF-BB and PDGFR- in EC differentiation Lymphatic specification during development Results and Discussion Paper I: Differential activation of vascular genes by hypoxia in primary endothelial cells Although ECs display many common features, they also exhibit remarkable heterogeneity in vivo. Differences may be manifested morphologically in shape and size. The different organization of the EC plasma membrane in gaps or pores (fenestrations) is another morphological hallmark of EC subpopulations. Endothelial regulation of permeability, vascular tone, coagulation, inflammation and angiogenesis also differ between vascular beds 280. Differential gene expression analysis on arteries and veins, and on normal vs. tumor vessels, has also confirmed that there exist molecular differences among ECs The endothelium lines the inner lumen of blood vessels and can readily receive and respond to systemic mediators and flow dynamics, as well as factors produced by cells in the underlying tissue, such as vsmcs and pericytes. In conditions of insufficient perfusion, such as in ischemic diseases or in a tumor, hypoxia may activate ECs to express and release vasoactive substances, inflammatory mediators and growth factors. These stimuli can act in an autocrine manner stimulating for instance cell survival and vessel permeability and growth, and/or in a paracrine setting regulating the behavior of vsmc and pericytes, as well as certain blood cells 285,286. The most evident 34

35 response to acute hypoxia in ECs, as in most cell types, is transcriptional activation of specific genes, coordinated by HIF transcription factors. HIF- subunits are induced by hypoxia and when heterodimerized with the subunit, mediate upregulation of genes of importance for e.g. vascular growth and function. In an effort to characterize differential responses to hypoxia in ECs derived from different vascular beds, we employed primary ECs from capillary vessels derived from foreskin dermis and adrenal cortex, arterial ECs from the pulmonary artery and vein ECs from the umbilical and saphenous veins. Cells were grown under normoxic (20.9% O 2 ) or hypoxic (0.5% O 2 ) conditions for up to 24 hours. Transcript and protein levels were then analyzed using real-time PCR, fluorescence-activated cell sorting (FACS) analysis and western blotting techniques. While HIF-1 was induced by hypoxia in all cells tested, the related HIF-2 displayed a more restricted expression pattern. HIF-2 showed strong induction in capillary ECs, but weak expression in saphenous vein ECs, and no expression in umbilical vein or arterial ECs. The effect of hypoxia on HIF expression was also kinetically different. For instance, while hypoxia induced a rapid and transient expression of HIF-1 in capillary ECs, expression of HIF-2 was slightly delayed and instead sustained for up to 24 hours. These differences indicate that HIF-1 and HIF-2 may have complementary rather than strict redundant roles in regulating gene expression in these cells. In vein ECs, induction of HIF-1 was sustained up to 24 hours, whereas in arterial cells, only a weak, transient induction was recorded. We next investigated expression of genes regulating vascular growth and remodeling. We found that capillary ECs to a greater extent than venous/arterial cells respond to hypoxia with increased expression of VEGF-A which appeared to correlate with expression of HIF-2. Several other studies have implicated HIF-2 in VEGF-A expression Vegfr-1, a well characterized hypoxia-regulated gene, was induced in all cell types except for HUVECs. The major signaling receptor for VEGF-A, VEGFR-2, the angiopoietin receptor Tie2 and PDGF-BB were induced in capillary, but not vein or arterial ECs. Hypoxia has previously been shown to activate the Tie2 and Pdgf-b gene , whereas the effect on VEGFR-2 expression is less clear. HIF-2 has been implicated in transcriptional regulation of the Vegfr-2 and Tie2 genes 227,254,288,299 and our data lend further support for these findings. The effect on VEGF signaling components and the Tie2 receptor shows that hypoxic capillary ECs have the capacity to regulate blood vessel growth by inducing potent autocrine signaling, as well as to receive paracrine survival and/or remodeling signals from perivascular cells expressing angiopoietins. In contrast to the increased transcript levels, the amount of VEGFR-1 protein on the cell surface of dermal capillary ECs was reduced in response to hypoxia. Addition of neutralizing antibodies against VEGF-A inhibited this effect, indicating a VEGF-A-mediated internalization/downregulation of 35

36 VEGFR-1 in response to hypoxia. The activity state of VEGFR-2 in response to hypoxia was also investigated. VEGFR-2 phosphorylation after VEGF-A binding was reduced by hypoxia, again indicating that VEGF-A produced by ECs mediate VEGFR activation and subsequent downregulation in an autocrine fashion. The function of VEGFR-1 in ECs is not entirely understood, and why hypoxia is a strong inducer of VEGFR-1 expression remains to be determined. Perhaps VEGFR-1 has an important function for blood vessel formation rather than solely acting as an inert decoy receptor for VEGF-A 114,141,300,301. Alternatively, increased expression of the soluble form of VEGFR-1 (capable of binding VEGF-A, but lacking intrinsic kinase activity) could also be important for fine-tuning the VEGF-A response mediated through VEGFR- 2. The lymphatic vascular system arises from vein endothelium during development. It was therefore interesting to note that the Vegfr-3 gene is expressed to a considerable extent in vein ECs, in contrast to the very low levels found in capillary and arterial ECs. Furthermore, only vein ECs responded to hypoxia with up-regulation of VEGFR-3. Expression of VEGF-C and D, ligands to VEGFR-3, were activated in both hypoxic vein and arterial cells, but not in capillary ECs. VEGF-C, D and VEGFR-3 has been reported to be activated by hypoxia However, it is not known whether the Vegfr-3 or Vegf-c promoter/enhancer regions contain functional HIFbinding sites or not. In contrast, the Vegf-d promoter has been found to contain a hypoxia-responsive sequence and our data supports the notion that Vegf-d is a hypoxia-regulated gene 305. The Et-1, inos and Ho-1 genes all harbor HIF-binding sites in their promoter/enhancer regions, and we found these genes to be activated by hypoxia. Expression of the vasoconstrictor ET-1 was increased in all cells but adrenal cortex capillary ECs, whereas HO-1 (the enzyme responsible for generation of the vasodilator carbon monoxide by degradation of heme) was induced in all cells but saphenous vein ECs. In addition, HO-1 also converts heme to biliverdin and subsequently bilirubin, that functions as an antioxidant reducing oxidant-induced cellular injury 306. Finally, inos was most potently induced in capillary ECs, indicating that ECs within the microcirculation may be a site for production of the vasodilator NO. In summary, our data show that endothelial cells from different vascular beds manifest different transcriptional responses to hypoxia. This is likely to reflect not only diverse functions in the vasculature, but also differences in expression of oxygen-sensing components, such as HIF-1 and HIF-2. Our data demonstrate the specific activation of genes encoding pro-angiogenic factors in capillary ECs and lymphangiogenic factors in vein ECs. 36

37 Paper II: Vascular endothelial growth factor receptor-3 in hypoxia-induced vascular development Mature BECs express a set of markers including CD31, VE-cadherin and VEGFR-2. Most embryonic blood vessels co-express VEGFR-2 and VEGFR-3 up to mid gestation; in fact, 71% of the CD31+ cells in E9.5 mice co-express VEGFR-2 and VEGFR-3 suggesting that both receptors have essential roles in angiogenesis 307. Later during development, concomitant with the emergence of the lymphatic system, VEGFR-3 expression becomes restricted to LECs 52. Notably, both VEGF-C and VEGF-D has been shown to exert angiogenic properties in certain experimental settings 149,152,308. Embryonic stem (ES) cells are pluripotent cells isolated from the inner cell mass of the mouse blastocyst. In culture, ES cells self-renew without differentiation in the presence of leukemia inhibitory factor (LIF). After withdrawal of LIF, ES cells undergo successive commitment and terminal differentiation. Hematopoietic and vascular cells, as well as neuronal and glial cells, cardiomyocytes and adipocytes, are examples of differentiated cell types derived from ES cell cultures. As a model system for studies of early mouse development, ES cells are allowed to differentiate and aggregate into structures called embryoid bodies (Figure 8). Embryoid bodies have the ability to form all three germ layers: endoderm, mesoderm and ectoderm, thereby recapitulating post-implantation embryonic tissues 309. In particular, early stages of vasculogenesis and initial vascular maturation can be studied using this model Figure 8. Schematic illustration of the embryoid body model. After removal of leukemia inhibitory factor (LIF), ES cells are allowed to aggregate and differentiate into embryoid bodies in hanging drops. After 4 days, embryoid bodies are transferred to chamber glass slides and cultured in normoxia or hypoxia, or treated with VEGF-A for the periods indicated. At day 8 or 12, embryoid bodies may be analyzed by immunohistochemistry, real-time PCR or western blot. 37