Molecular mechanisms of Angiopoietin-2-mediated destabilization of the vascular endothelium

Molecular mechanisms of Angiopoietin-2-mediated destabilization of the vascular endothelium Inaugural-Dissertation zur Erlangung der Doktorwürde der Fakultät für Biologie der Albert-Ludwigs-Universität Freiburg im Breisgau vorgelegt von Markus Thomas aus Merzig (Saar) Oktober 2008

Dekan der Fakultät für Biologie: Prof. Dr. Ralf Reski Promotionsausschussvorsitzender: Prof. Dr. Samuel Rossel Betreuer der Arbeit: Prof. Dr. Georg Fuchs Prof. Dr. Hellmut G. Augustin Referent: Koreferent: Prof. Dr. Beate Brand-Saberi Prof. Dr. Hellmut G. Augustin Tag der Verkündigung des Prüfungsergebnisses: 28.01.2009 Die vorliegende Arbeit wurde am Institut für molekulare Onkologie, Abteilung für Vaskuläre Biologie & Angiogeneseforschung an der Klinik für Tumorbiologie in Freiburg, sowie in der Abteilung Vaskläre Onkologie und Metastasierung am Deutschen Krebsforschungszentrum in Heidelberg durchgeführt.

für meine Eltern

Danksagungen Ich möchte mich ganz herzlich bei allen bedanken, die zum Gelingen meiner Doktorarbeit beigetragen haben. Herrn Professor Dr. Georg Fuchs danke ich ganz herzlich für die Übernahme der Arbeit in den Fachbereich Biologie der Alberts-Ludwigs-Universität Freiburg im Breisgau. Ein großes Dankeschön an Herrn Professor Dr. Hellmut G. Augustin für die fachliche Betreuung und für die Vorschläge und Anregungen zu einem tollen Projekt. Während der vielen Tutorials wurde ich bestens auf das harte Leben nach der Doktorarbeit vorbereitet. Mein ganz besonderer Dank gilt Ulrike Fiedler, die mich in Freiburg ganz fantastisch betreut hat und auch während meiner Zeit in Heidelberg immer ein offenes Ohr für mich hatte. Danke, dass Du immer Zeit hattest, egal wie beschäftigt Du auch warst. Ulli, ich hab echt viel von Dir gelernt (Strukturierung?)!!! Vielen Dank auch an Mélanie Héroult, die mir in Heidelberg mit vielen guten Tipps extrem weitergeholfen hat! Merci beaucoup! Ein ganz großes Dankeschön auch an all die, die nicht nur in Heidelberg sondern auch in Freiburg zu einer tollen Laboratmosphäre beigetragen haben. Da ist zum erstem die Ang- Gruppe zu erwähnen mit Arne HBIGS Bartol, Andy Benest, Eias Loos, Junhao Rudi Hu, Carleen Deppermann und Dorothee Terhardt. In weiteren Rollen: Claudia Prahst, Florence Schaffner, Simone Kutschera, Katja Fischer, Patrick Nasarre, Marion Scharpfenecker, Steffi Koidl, Dennis Pfaff, Anja Hegen, Holger Weber, Abdullah Alajati, Oliver Siedentopf, Tanju Nacak, Christine Fulda, Eva Besemfelder, Maria Riedel, Vivien Wolter, Iris Helfrich, Anna Laib, Renate Winkler, Silke Kaltenthaler, Sabine Gesierich, Susanne Bartels und Sven Christian. Ganz herzlich bedanken möchte ich mich auch bei den Kollegen in Mannheim. Vielen Dank an Jens Kroll, den Herrn Dietz und das Bübsche für die vielen tollen Stunden in der Eichbaum Brauerei. Fürs Korrekturlesen möchte ich mich bei Ulrike Fiedler, Andrew Benest und Daniel Epting bedanken.

Vielen Dank auch an Karoline Kruse, die mit mir bis zum Schluss durch Dick und Dünn gegangen ist. Der größte Dank gilt selbstverständlich meiner Schwester Kathrin und meinen Eltern, die mich in jeglicher Hinsicht unterstützt haben und immer für mich da sind. Danke!!!

1 Introduction... 1 1.1 Blood vessel formation... 1 1.2 Molecular regulation of angiogenesis... 2 1.2.1 Sprouting angiogenesis and vessel maturation... 3 1.3 Vessel formation in the adult... 6 1.4 Tumor angiogenesis... 6 1.5 VEGF/VEGFR and Delta/Notch functions during angiogenesis... 7 1.6 The Angiopoietin/Tie system... 9 1.6.1 Tie1 and Tie2 receptor: Expression, structure and function... 9 1.6.2 Angiopoietin-1 and Angiopoietin-2: Expression, structure and function... 12 1.6.3 Consequences of Tie2 activation by angiopoietins... 14 1.6.4 Angiopoietin expression in tumors and tumor-associated angiogenesis... 20 1.6.5 Angiopoietin functions during development and in the adult... 21 1.7 Endothelial barrier function and vascular permeability... 22 1.8 Cell adhesion... 24 1.8.1 Integrins and extracellular matrix... 24 1.8.2 Integrin structure... 27 1.8.3 Activation of integrins... 28 1.8.4 Composition and function of integrin-mediated cell matrix adhesions... 29 1.8.5 Integrin functions during angiogenesis... 30 1.9 Aim of the thesis... 32 2 Results... 33 2.1 Ang-2 and its role in mediating VEGF responsiveness in angiogenesis... 33 2.1.1 Ang-2 increased endothelial sprouting length in the presence of VEGF... 33 2.1.2 Overexpression of Ang-2 induced VEGF-R2 upregulation whereas regulation... of VEGF-R2 by Ang-1 overexpression was concentration dependent... 35 2.2 Ang-2 destabilizes the endothelium by an internal autocrine loop mechanism... 36 2.2.1 Ang-2 did not interfere with VE-cadherin phosphorylation... 36 2.2.2 Ang-2 did not interfere with VE-cadherin location... 37 2.2.3 Ang-2 dislocated VEGF-R2 from junctions... 38 2.2.4 The Tie receptors rapidly interacted with integrin αvβ3 after Ang-2 stimulation 40 2.2.5 Ang-2 did not phosphorylate Tie2 receptor... 41 2.2.6 Ang-2 stimulated the recruitment of focal adhesion kinase (FAK) to... αvβ3 integrin... 41 2.2.7 Ang-2 stimulated the recruitment of FAK to αvβ3 integrin in a Tie2... dependent manner... 42 2.2.8 Ang-2 still signals by Akt upon Tie2 downregulation... 44

2.2.9 Ang-2 directly interacted with αvβ3 integrin... 44 2.2.10 FAK was phosphorylated at Ser-910 after Ang-2 stimulation... 45 2.2.11 Focal adhesions were enlarged after Ang-2 stimulation... 47 2.2.12 Phosphorylated FAK [ps910] associated with αvβ3 integrin and with... focal adhesions... 48 2.2.13 The integrin-associated adaptor proteins talin, paxillin and... p130cas dissociated from integrin αvβ3 after Ang-2 stimulation... 49 2.2.14 Ang-2 stimulation of endothelial cells led to internalization of αvβ3 integrin.. 50 2.2.15 Co-stimulation of endothelial cells with Ang-2 and the receptor... recycling inhibitor primaquine led to a rapid loss of cell adhesion... 51 2.2.16 αvβ3 integrin was ubiquitinylated after Ang-2 stimulation and... translocated to lysosomes... 53 2.2.17 Constitutive overexpression of Ang-2 in endothelial cells resulted in... enhanced migration... 54 2.2.18 Ang-2 transduction resulted in Tie2 translocation out of the cell... junctions and enhancement of stress fiber formation... 55 2.2.19 Stimulation with Ang-2 inhibited attachment of HUVEC to fibronectin... and vitronectin... 57 3 Discussion... 59 4 Summary... 70 5 Materials and Methods... 71 5.1 Materials... 71 5.1.1 Chemicals... 71 5.1.2 Primers and sirna s... 71 5.1.3 RT-PCR and PCR reagents, buffers, nucleotides... 71 5.1.4 Kits... 72 5.1.5 Transfection reagents... 72 5.1.6 Cells... 72 5.1.7 Cell culture... 72 5.1.8 Consumables... 72 5.1.9 Proteins and chemical compounds... 73 5.1.10 Primary Antibodies... 73 5.1.11 Secondary Antibodies... 74 5.1.12 Steptavidin-Conjugates... 74 5.1.13 Nuclei and Actin staining reagents... 75 5.1.14 Immunoprecipitation... 75 5.1.15 Miscellaneous... 75

5.1.16 Solutions and buffers... 75 5.1.16.1 Preparation of Methocel... 75 5.1.16.2 RIPA cell lysis buffer... 76 5.2 Methods... 76 5.2.1 Molecular Biology... 76 5.2.1.1 RNA isolation... 76 5.2.1.2 Reverse transcription... 76 5.2.1.3 Polymerase chain reaction... 77 5.2.2 Cell culture and transfection... 77 5.2.2.1 Cell culture conditions... 77 5.2.2.2 Adenoviral transduction of primary cells with LacZ, Ang-1 and Ang-2... 77 5.2.2.3 Transfection of endothelial cells with small interfering RNA (sirna)... 78 5.2.3 Cellular assays... 78 5.2.3.1 Adhesion assay... 78 5.2.3.2 Internalization assay... 79 5.2.3.3 Recycling assay... 79 5.2.3.4 Time-lapse analysis... 80 5.2.4 In vitro angiogenesis assay... 80 5.2.4.1 Generation of endothelial cell spheroids... 80 5.2.4.2 Purification of type I collagen from rat tail tendons... 80 5.2.4.3 Collagen embedding and stimulation of endothelial cell spheroids... 80 5.2.4.4 Analysis of sprout length... 81 5.2.5 Immunocytochemistry... 81 5.2.6 Biochemical analyses... 82 5.2.6.1 Integrin immunoprecipitation/co-immunoprecipitation of focal... adhesion complex proteins and Western blot analysis... 82 5.2.6.2 Tie2-phosphorylation and co-immunoprecipitation of αvβ3 and... FAK [ps910]... 82 5.2.6.3 FAK [pt397]-, FAK [ps732]- and FAK [ps910]-phosphorylation... 83 5.2.7 Statistical analysis... 84 6 Abbreviations... 85 7 References... 89

Introduction 1 Introduction 1.1 Blood vessel formation The adult vasculature is derived from a network of blood vessels that is initially created embryonically by vasculogenesis, a process whereby vessels are formed de novo from endothelial cell precursors termed angioblasts (Risau 1997). Angioblasts derive from mesodermal cells in the early embryo. The dorsal aorta and the cardinal vein directly develop by the aggregation of angioblasts, whereas the fusion of blood islands, also derived from endothelial precursor cells, results in the formation of the honeycomb-shaped primary capillary plexus (Adams and Alitalo 2007).The primary capillary plexus further remodelled by sprouting and branching of new vessels from pre-existing ones during the process of sprouting angiogenesis (Folkman 1984). Arterioles and arteries, capillaries and venules and veins are formed (Figure 1). Vasculogenesis primarily takes place during embryonic development, whereas angiogenesis occurs both embryonically and throughout adulthood, e.g. during vessel formation in the ovary. Most physiological angiogenesis takes place embryonically where it establishes the primary vascular tree as well as an adequate vasculature for growing and developing organs (Folkman 1995). Vessels are pruned by vessel regression during subsequent steps in angiogenesis, while new vessels form. Blood flow is increased, the basal lamina is modified and smooth muscle cells (SMC) for larger calibre or pericytes (PC) for smaller vessels are recruited during blood vessel maturation (Figure 1). All these processes stabilize the vessel wall (von Tell et al. 2006). New vessels can also be formed in the heart, the lung and the allantois by splitting of already existing vessels, a process called intussuception (van Groningen et al. 1991; Patan et al. 1996; Risau 1997; Burri et al. 2004). During this process (non sprouting angiogenesis) vessels are longitudinally divided by formation and insertion of interstitial tissue into the lumen of a preexisting vessel or by bridging the vessel lumen with interstitial tissue columns. Intussusception also includes the establishment of new vessels by formation of loops in the wall of large veins. In a third process, called arteriogenesis, larger arteries are formed from smaller ones. A thick layer of SMC surrounds the vessel, giving it viscoelastic and vasomotor properties, allowing it to regulate the perfusion of the arteries (Risau 1997; Conway et al. 2001).

Introduction Figure 1: Schematic overview of vasculogenesis and angiogenesis. The figure shows the processes (red) and the appearances (black) during vasculogenesis and angiogenesis. SMC/PCT = smooth muscle cell/pericyte; A = artery. Adapted from (Risau 1997). 1.2 Molecular regulation of angiogenesis The formation of new blood vessels is a highly complex process that can be divided in an activation phase and a resolution phase (Folkman and D'Amore 1996; Carmeliet 2003). Both phases are rate-limited by transcription factors, different growth factors, chemokines and adhesion molecules. Vessels are dilated and leaky during the activation phase. The matrix is degraded by the activation of matrix metalloproteases (MMP s) and endothelial cells proliferate and migrate to form new vessel sprouts. Newly formed vessels are stabilized during the resolution phase by the recruitment of pericytes (PC) or smooth muscle cells (SMC). The process is completed by the assembly of a basement membrane that is separating the endothelial cells with the SMCs (Pepper 1997).

Introduction 1.2.1 Sprouting angiogenesis and vessel maturation Hypoxia, inflammatory processes or cytokines all affect vascular homeostasis (Pugh and Ratcliffe 2003). Various signaling cascades and genetic programs are activated during the initiation of angiogenesis that lead to the upregulation of regulators of angiogenesis such as endothelium nitric oxide synthase (enos), vascular endothelial growth factor (VEGF), Angiopoietin-2 (Ang-2) and/or different proteases. Within this activation phase (Figure 2), VEGF stimulates the production and the release of nitrite oxide (NO), a molecule whose production is catalyzed by enos. Vessels dilate as a response to NO and become leaky by VEGF (Ku et al. 1993; Ziche et al. 1994; Ziche et al. 1997; Ferrara 1999; Dvorak 2000). The basal lamina and the extracellular matrix are degraded by matrix metalloproteases, such as MMP2, MMP3, and MMP9, or by inhibition of corresponding protease inhibitors, like PAI1 or TIMP (Kalluri 2003). ECM degradation liberates heparin sulphate bound growth factors like VEGF from the extracellular matrix (Lee et al. 2005). Highly polarized cells at the front of the sprout, so-called tip cells, sense the VEGF gradient in the tissue which results in the formation of numerous filopodia (Gerhardt et al. 2003; Ruhrberg 2003). Tip cells are followed by stalk cells which in turn are followed by phalanx cells. The stalk cell is the first cell located behind the tip cell whereas the phalanx cell is the first cell in the sprout which is covered with perivascular cells. Newly formed vessels are stabilized during the resolution phase (Figure 2). There are four signaling pathways activated during this phase (PDGF/PDGFR, S1P/EDG1, Ang/Tie and TGFβ). Platelet derived growth factor (PDGF) B is secreted primarily by the activation of endothelial cells via VEGF, thereby facilitating the recruitment of perivascular cells (Hellstrom et al. 2001). Higher levels of PDGF-B mrna are detected in stalk cells, supporting the notion that PDGF mediates pericyte recruitment. This step is further supported by Sphingosine-1- phosphate (S1P) and its receptor (EDG1) which is highly expressed by endothelial cells. The phenotypes of EDG1- and PDGF-B-deficient mice are very similar which suggests that both pathways are closely linked to each other (Kluk and Hla 2002). It is controversially discussed if the EDG1 pathway also affects matrix production, the interaction of endothelial cells with their surrounding cells or the vessel maturation process (Cho et al. 2003). The angiopoietins (Ang-1 and Ang-2) and their receptors Tie1 and Tie2 are a third pathway, involved in the formation and maturation of new vessels. Ang-1 is constitutively expressed by numerous cell types and controls vascular quiescence. In contrast, the contextually agonistic and antagonistic ligand Ang-2 is almost exclusively produced by endothelial cells (EC) themselves and thereby controls vascular homeostasis through an autocrine loop mechanism. Context dependency of Ang-2 functions is reflected by the finding that Ang-2 mediates vessel regression in the absence of angiogenic activity, but it has a pro-angiogenic function in the presence of angiogenic growth factors (Maisonpierre et al. 1997; Lobov et al.

Introduction 2002; Visconti et al. 2002). However, the signaling pathways of the angiopoietins during vessel maturation are incompletely understood. One possible molecule involved in the recruitment of mural cells is the EC-derived heparin binding EGF-like growth factor (HB- EGF). Its expression in endothelial cells is upregulated by Ang-1 (Iivanainen et al. 2003), but only when they are in contact with mural cells. HB-EGF-mediated receptor (ErbB1 and ErbB2) activation is sufficient, but not essential to induce SMC migration. However, there is evidence that hepatocyte growth factor is also involved in Ang-1-mediated SMC recruitment. Stimulation of endothelial cells with Ang-1-induced SMC migration towards endothelial cells in a co-culture assay. This effect could be reversed by the addition of a neutralizing anti-hgf antibody, indicating that Ang-1 is regulating HGF expression (Kobayashi et al. 2006). The TGF-β pathway is the fourth pathway influencing vessel maturation. TGF-β first induces mural cell differentiation and stimulates the production of extracellular matrix components (Dickson et al. 1995; Hirschi et al. 1998; Oh et al. 2000). Later, it inhibits the proliferation and migration of recruited mural cells and thereby stabilizes the vasculature (Sato and Rifkin 1989). Furthermore, it is expressed in almost all cells of the tissue and can have concentration dependent pro- or anti-angiogenic functions (Pepper 1997; Gohongi et al. 1999).

Introduction Activation phase VEGF-A: Vascular leakage, endothelial cell proliferation, migration and enhanced protease production Endothelial cell Permeability Detachment Pericyte/SMC Ang-2 Degradation Integrin Migration Basal membrane Notch Dll4 Tip cell Phalanx cell Ang-1 TGF-β HB-EGF Stalk cell PDGF-B Stabilization Differentiation Recruitment Resolution phase Figure 2: Regulation of angiogenesis: The activation phase is induced by VEGF signaling which results in vascular leakage, followed by a higher expression of proteases, endothelial cell migration and proliferation. Vessel disintegration is supported by Angiopoietin-2 which interacts with integrin signaling and thereby enhances detachment and migration. Tip cells migrate towards a VEGF gradient, a mechanism that is highly regulated by the Delta/Notch (Chapter 1.5) system, thereby guiding the sprouts in the correct direction. Blood vessels are stabilized by platelet-derived growth factor B (PDGF-B) during the resolution phase which recruits pericytes. Ang-1 and TGF-β signaling further support vessel maturation by pericyte differentiation and stabilization.

Introduction 1.3 Vessel formation in the adult Generally, the formation of new blood vessels is completed in the adult. However, angiogenesis can be induced during wound healing and during the ovarian cycle (Klagsbrun 1991; Klagsbrun and D'Amore 1991; Risau 1997) by the activation of the quiescent endothelium. Proliferation rates of endothelial cells range from several months to several years in the healthy adult yet a suddent increase in EC proliferation is a cardinal feature of angiogenic growth. This process when new capillaries are formed by activation, proliferation and migration of endothelial cells and the formation of a lumen afterwards is called physiological angiogenesis (Folkman 1984; Breier et al. 1997). Disorders of the equilibrium of the cytokine milieu can cause enhanced but insufficient capillary growth (pathological angiogenesis) which may lead to diseases, such as psoriasis, rheumatoid arthritis, as well as tumor growth and metastasis (Folkman 1971; Breier et al. 1997; Plendl et al. 1999; Carmeliet 2003). Angiogenesis was thought to be the only mechanism of adult vessel formation, whereas vasculogenesis was thought to be restricted to embryonic development. Recent studies suggest that bone marrow-derived and in the blood circulating endothelial progenitor cells can contribute to mechanisms similar to vasculogenesis that occur in the adult (Asahara et al. 1999; Lyden et al. 2001; Rafii et al. 2002; Chavakis et al. 2007). 1.4 Tumor angiogenesis Highly proliferating tissues such as tumors need an adequate supply with nutrients and oxygen for sustained growth mediated by energy and nutrition consumption. A tumor is able to grow to a size of 1-3 mm 3 without blood perfusion (Folkman 1971; Folkman 1990; Hanahan and Folkman 1996). To further maintain a continuous supply of blood, the tumor initiates angiogenesis or utilizes host vessels, a process that is called vessel cooption (Pezzella et al. 1997; Holash et al. 1999). Prior to the initiation of angiogenesis, the tumor is an avascular mass that can stay in a state called dormancy (Holmgren et al. 1995). The transmission from avascular to the angiogenic phenotype is called the angiogenic switch (Bergers and Benjamin 2003). The tumor secrets different cytokines (e.g. VEGF) and the angiogenic switch may occur when the balance of pro- to anti-angiogenic factors is tipped in favor of the pro-angiogenic molecules. Pro-angiogenic gene expression is further enhanced by metabolic or mechanical stress, immune cells or genetic mutations (Carmeliet and Jain 2000). The blood vessels in the tumor are qualitatively different from quiescent blood vessels. The expression of growth factors in tumors is dysregulated, leading to a tortuous vasculature

Introduction which may also contain blind ending capillary sprouts. There is little differentiation of vessels to veins, arteries and capillaries. Additionally, the high expression of VEGF often leads to haemorrhages (Dvorak et al. 1988). Functional perivascular cells like pericytes are largely missing (Morikawa et al. 2002; Ozawa et al. 2005) or show lower density and looser connection with endothelial cells (Abramsson et al. 2002; Morikawa et al. 2002). Furthermore, the endothelial cell lining is defective and composed of disorganized, loosely connected and branched, overlapping or sprouting endothelial cells (Yuan et al. 1995; Hashizume et al. 2000). 1.5 VEGF/VEGFR and Delta/Notch functions during angiogenesis Vascular endothelial growth factor-a (VEGF) belongs to a growth factor family of seven members (VEGF-A to F and PlGF, placenta growth factor). VEGF signaling is mediated by VEGF-receptor 2 (VEGF-R2) which is vascular specific (Ferrara et al. 2003). The importance of the VEGF/VEGFR system as a critical regulator of vessel formation was demonstrated in knockout mice where the loss of one allele of VEGF is embryonically lethal due to of impaired vasculogenesis and angiogenesis (Carmeliet et al. 1996; Ferrara et al. 1996). The system is comprised of three receptors VEGF-R1 to VEGF-R3. VEGF-R1 and -R2 are mostly expressed by blood endothelial cells (Terman et al. 1991; Peters et al. 1993; Quinn et al. 1993). VEGF-R2 signaling regulates vascular tone, protease production, cell proliferation, migration and survival (Ferrara et al. 2003) and increases permeability (Senger et al. 1983). VEGF-R3 is primarily expressed by lymphatic endothelial cells in the healthy adult (Kaipainen et al. 1995). However, VEGF-R3 is upregulated in the microvasculature of tumors and wounds (Valtola et al. 1999; Paavonen et al. 2000; Petrova et al. 2008). Recent studies demonstrated that VEGF-R3 is also expressed by blood endothelial cells in angiogenic sprouts. VEGF-R3 blockade resulted in decreased sprouting, vascular density, vessel branching and endothelial cell proliferation in mouse angiogenesis models (Tammela et al. 2008). Several groups have reported that VEGF-R2 not only modulates endothelial cell functions during angiogenesis but also stimulates the interaction with adhesion molecules like VEcadherin (Carmeliet and Collen 1999; Grazia Lampugnani et al. 2003) or the integrins αvβ3 and αvβ5 (Friedlander et al. 1995; Soldi et al. 1999). Inhibition of these integrins with blocking antibodies led to inhibition of tumor angiogenesis in vivo (Kumar et al. 2000; Kumar et al. 2001). Additionally, VEGF induces an increase in permeability which can be reversed by Angiopoietin (Ang)-1. Ang-1, an anti-permeability factor, is the agonistic ligand of Tie2 receptor and leads to its phosphorylation thereby maintaining vascular quiescence (Gamble et al. 2000; Thurston et al. 2000). EC quiescence has largely been attributed to stimulating

Introduction the interaction between endothelial and peri-endothelial cells (Suri et al. 1996) and by strengthening endothelial-endothelial contacts (Gamble et al. 2000). VEGF, which is upregulated during hypoxia, is known to regulate Ang-2 expression (Mandriota and Pepper 1998; Oh et al. 1999), a key player during the process of remodelling. Ang-2 supports matrix degradation, endothelial cell migration (Hu et al. 2006) and the assembly of new blood vessels (Yancopoulos et al. 2000). Another important system, involved in angiogenesis is the Delta/Notch system. Studies performed in the mouse retina and zebrafish embryo demonstrate that this system is a key regulator of sprouting angiogenesis by regulating tip cell versus stalk cell communication. Notch signaling (in the stalk cell) induces a quiescent and non-sprouting phenotype in endothelial cells whereas adjacent cells (the tip cells) express Dll4, therefore promoting sprouting activity. This model demonstrates a very attractive mechanism for the highly mosaic patterns of Notch activation and Dll4 expression in the endothelium of the retina (Claxton and Fruttiger 2004; Hellstrom et al. 2007; Hofmann and Iruela-Arispe 2007; Lobov et al. 2007). The ability of Notch to regulate sprouting is partially coupled to the VEGF/VEGFR pathway. Dll4 expression is induced by VEGF signaling and hypoxia (Diez et al. 2007; Lobov et al. 2007). This could be an explanation for the high expression of Dll4 at leading tip cells. VEGF-R2 is also highly expressed in these cells. It has been hypothesized that Dll4 expression can increase Notch activation in adjacent cells (stalk cells) which in turn downregulates VEGF-R2 levels (Williams et al. 2006). Additionally, Notch signaling is also involved in arteriovenous differentiation, in arteriogenesis in the adult and in sprouting angiogenesis (Shutter et al. 2000; Hellstrom et al. 2007; Limbourg et al. 2007). Artery specific expression has been reported for several Notch receptors and ligands, such as Dll4 in mouse and zebrafish (Shutter et al. 2000; Siekmann and Lawson 2007). Studies with the corresponding knockout mice have been carried out and have demonstrated the significiance of these molecules during arteriovenous differentiation. The results demonstrate that disruption of Notch signaling due to mutations in the zebrafish results in the loss of other artery specific markers like ephrin B2 (Lawson et al. 2001). The Eph/ephrin system further regulates arterial-venous differentiation of the vasculature. EphB4- and ephrinb2-deficient mice die at E10.5 because of severe structural defects of the cardiovascular system (Gerety et al. 1999).

Introduction 1.6 The Angiopoietin/Tie system Davis and co-workers identified a new angiogenic growth factor by secretion trap cloning in 1996 which was named Angiopoietin-1. Ang-1 binds Tie2, a receptor tyrosine kinase, and induces Tie2 phosphorylation. This process does not induce endothelial cell proliferation (Davis et al. 1996) but stabilizes newly formed vessels and acts as a vessel sealing (Thurston et al. 2000) and as an anti-inflammatory molecule (Gamble et al. 2000). Angiopoietin-2 was found in 1997 by homologue cloning, which is closely related to Ang-1 and also binds to Tie2, but acts as an antagonist and is involved in the process of vessel destabilization (Maisonpierre et al. 1997; Scharpfenecker et al. 2005). 1.6.1 Tie1 and Tie2 receptor: Expression, structure and function Tie1 and Tie2 are endothelial cell specific receptors that have almost the same molecular weight of approximately 135 and 150kDa respectively. Both receptors are very similar in the cytoplasmic region (76% sequence identity), but show only 33% similarity in the extracellular part (Schnurch and Risau 1993). They are tyrosine kinases with Ig and EGF homology domains. The extracellular domain consists of two immunoglobulin (Ig)-like domains that are flanked by three EGF (epidermal growth factor)-like cysteine repeats followed by three fibronectin type III domains (Figure 3). Ig Ig EGF Ig FN III extracellular intracellular cell membrane TK I TK II Tie1 Tie2 Figure 3: Schematic overview of both Tie receptors. green: immunoglobulin (Ig)-like domains, orange: EGF-like domains, yellow: fibrinogen-like domains, red: kinase domains.

Introduction The smaller intracellular domains of both receptors consist of two kinase domains which can bind different molecules after autophosphorylation. Tie1 is exclusively expressed by endothelial cells, whereas Tie2 is also expressed by hematopoetic cells, endothelial precursor cells (Dumont et al. 1992; Sato et al. 1995) and tumor cells such as Kaposi sarcoma tumor cells (Brown et al. 2000). During early development, Tie1 can be detected from E8.5 in differentiating angioblasts of the head mesenchyme, in the splanchnopleure and in the dorsal aorta but also in migrating endothelial cells of the developing heart (Korhonen et al. 1994). Tie1 has major functions during development. Mice lacking the Tie1 gene die between E13.5 and P1 due to a loss of structural integrity of vascular endothelial cells, resulting in oedema and haemorrhage (Sato et al. 1995). In conclusion, Tie1 plays an important role during endothelial cell differentiation and during the regulation of vessel integrity. Full length Tie1 is thought to heterodimerize with Tie2 (Marron et al. 2000). Other studies have shown that Tie1 can be cleaved by γ- secretases following VEGF stimulation. The cleaved form, remaining in the membrane, interacts with Tie2 and is supposed to support Tie2 signaling (Marron et al. 2000; Tsiamis et al. 2002; Marron et al. 2007). Receptor shedding also occurs after stimulation with the phorbol ester PMA, stimulation with tumor necrosis factor alpha (TNF-α), and by shear stress (Yabkowitz et al. 1997; Yabkowitz et al. 1999; Chen-Konak et al. 2003). Furthermore, Tie1 has been identified as an orphan receptor. Recent evidence suggests that Comp-Ang-1, a special designed form of Ang-1, can bind to Tie1 under certain conditions (Saharinen et al. 2005). The second Angiopoietin receptor, Tie2, is also expressed by endothelial cells. Yet, the expression is downregulated in already established/quiescent EC. Endothelial cells in larger vessels express Tie2 more abundantly in comparison to smaller vessels (Dumont et al. 1992; Schnurch and Risau 1993). It could be shown that Tie2 is upregulated during tumor angiogenesis (Wong et al. 1997; Peters et al. 1998; Takahama et al. 1999). The receptor dimerizes by ligand binding. Thereby, Tie2 can also form heterotetramers with Tie1 (Marron et al. 2000). Following binding of the activating ligand Ang-1, Tie2 is autophosphorylated and intracellular signaling pathways are activated. Tie2 has been shown to co-localize with endothelial caveolae (Yoon et al. 2003). Caveolae are membrane invaginations enriched with signaling molecules. They play a key role in signal transduction and protein trafficking. Tie2-deficient mouse embryos die at E10.5 due to vessel remodelling defects in the plexus of the yolk sac, of the brain and severe heart defects. The mice have 30% and 75% less endothelial cells at E8.5 and E9, respectively (Dumont et al. 1994; Sato et al. 1995; Suri et al. 1996). The importance of Tie2 for vascular integrity was further analyzed by transgenic expression of Tie2 that could be switched off by the oral application of tetracycline. Loss of Tie2 function leads to endothelial cell apoptosis and haemorrhage (Jones et al. 2001b).

Introduction These results suggest that the Ang-Tie system plays a key role during vessel remodelling, maturation and stabilization of the cardiovascular system. Furthermore injection of soluble Tie2 (stie2-fc) inhibits ischemia-induced retinal neovascularization in a mouse model (Takagi et al. 2003). Yet, this soluble form is also present under physiological conditions in the serum. This is due to Tie2 cleavage which had been shown after PMA stimulation (Reusch et al. 2001). Soluble Tie2 is further increased during coronary artery disease (Chung et al. 2003). Abnormal vessel structures are not only caused by Tie2-deficency. Constitutively active Tie2 mutants in human patients lead to veins with a higher diameter by enhanced proliferation of endothelial cells. The endothelial cells are surrounded by a layer of smooth muscle cells that varies in thickness. The range is between areas with normal coverage to areas completely devoid of SMC (Vikkula et al. 1996). Double knockout mice for Tie1 and Tie2 have been created to shed further light to the signaling pathways of both receptors during vascular development. These mice die like Tie2- deficient mice around E10.5 due to cardiovascular defects but also as a consequence of severe defects in the vascular system. Vasculogenesis proceeds normally in these mice. The authors concluded from their results that Tie1 and Tie2 are essential for maintaining the integrity of mature vessels but that they are dispensable for early angiogenic sprouting (Puri et al. 1999).

Introduction 1.6.2 Angiopoietin-1 and Angiopoietin-2: Expression, structure and function The Angiopoietins are a family of growth factors known to be essential for blood vessel formation. There are four angiopoietins, Angiopoietin-1 (Ang-1), Angiopoietin-2 (Ang-2), Angiopoietin-3 (Ang-3), and Angiopoietin-4 (Ang-4). The best characterised Angiopoietins are Ang-1 and Ang-2. Ang-3 is mouse specific and Ang-4 is its homologue in humans. They are all ligands for the Tie2 receptor (Davis et al. 1996; Maisonpierre et al. 1997; Kim et al. 1999; Nishimura et al. 1999; Valenzuela et al. 1999). Figure 4: Schematic representation of the angiopoietin ligands with their coiled-coil and fibrinogen-like domains. Opposing effects the similar ligands have been associated with the receptor-binding fibrinogen-like domains and the coiled-coil domains which mediate different multimerization patterns of the ligands in vitro. Human Ang-1 has three different splice forms. Splice variants of Ang-1 (Ang-1-1.3 kb, Ang-1-0.9 kb, Ang-1-0.7 kb) and Ang-2 (Ang-2 443 ) are indicated as a black line with smaller font size under the particular molecule. Numbers indicate the similarity of the domain in comparison to the Ang-1 molecule. The figure also shows an avian form of Ang-2 (Ang-2B) which is partially truncated at the amino-terminal coiled-coil domain as a result of alternative splicing, mouse Ang-3 and the human orthologue Ang-4 (Jones et al. 2001a).

Introduction All Angiopoietins are composed of two domains (Figure 4). There is a coiled-coil domain at the N-terminus which is responsible for homo-oligomerization of the ligands. Ang-1 is a tetramer whereas Ang-2 is a dimer (Davis et al. 2003; Kim et al. 2005). Oligomerization is necessary for receptor activation but not for receptor binding. This is mediated by the fibrinogen-like domain which is located in the C-terminus (Davis et al. 1996; Procopio et al. 1999). The angiopoietins are secreted glycoproteins with a molecular weight of 75 kda. Ang-1 has 498 aa and is located on chromosome 8q22. Ang-2 has 496 aa and is located on chromosome 8q23. Both molecules show a sequence homology of about 60 % (Davis et al. 1996; Maisonpierre et al. 1997). Angiopoietin-1 is expressed by smooth muscle cells and by the endocardium in the adult. It binds Tie2 like Ang-2 with an affinity of about 3 nm (Maisonpierre et al. 1997) to the IgG domain and the EGF domain of Tie2 (Fiedler et al. 2003). Ang-1 is produced in four different splice variants. The splice variants with 1,5 kb (full length Ang-1) and 1,3 kb bind the receptor and induce its autophosphorylation. The proteins with 0,9 kb and 0,7 kb also bind Tie2 but do not induce autophosphorylation (Huang et al. 2000). A novel Ang-2 splice variant, Ang-2B, with a truncated amino-terminal domain has been detected in chicks (Mezquita et al. 1999). Another splice variant (Ang-2(443)) in endothelial cells has been identified which lacks parts of the coiled-coil domain and can not stimulate Tie2 phosphorylation (Kim et al. 2000d). Ang-1 acts as an agonist of the Tie2 receptor, whereas Ang-2 is the antagonist (Maisonpierre et al. 1997). However, Ang-2 is also able to context-dependently induce receptor phosphorylation depending on the cell type, cell confluence, stimulation time, or ligand dosage (Kim et al. 2000c; Teichert-Kuliszewska et al. 2001; Daly et al. 2006). Ang-2 is primarily expressed by endothelial cells and is stored in Weibel-Palade bodies (WPB) (Fiedler et al. 2004). Following activation of the endothelium by cytokines (e.g. Histamine or Thrombin), Ang-2 is rapidly released from WPB (Fiedler et al. 2004). It acts in an autocrine manner on the Tie2 receptor by binding as homodimers or multimers (Procopio et al. 1999). Recent studies have demonstrated that endogenous Ang-2 acts through an internal autocrine loop mechanism. The experiments suggested that endogenously released Ang-2 cannot be inhibited by exogenous soluble Tie2 receptor (Scharpfenecker et al. 2005). Ang-2 levels are upregulated under hypoxic conditions (Oh et al. 1999; Kim et al. 2000e; Huang et al. 2002; Pichiule et al. 2004). Under physiological conditions, Ang-2 is expressed in regions where vessel remodelling occurs; for example during vascularization of the retina or during vessel regression in the ovary (Maisonpierre et al. 1997; Goede et al. 1998). Yet, its expression is also upregulated under pathological condition e.g. in the endothelium of tumors (Zagzag et al. 1999; Zhang et al. 2003; Oliner et al. 2004) and in tumor cells (Tanaka

Introduction et al. 1999; Koga et al. 2001; Torimura et al. 2004). Moreover, retinal neurons (Hackett et al. 2000) and Müller cells (Yao et al. 2007) are a source of Ang-2. Ang-1, in contrast, is primarily expressed by mesenchymal cells and acts in a paracrine manner on the endothelium. It is abundantly expressed by the myocardium during early development and by perivascular cells later in development and in adult tissue (Suri et al. 1996; Maisonpierre et al. 1997; Gale et al. 2002). Yet, it is also expressed by tumor cells (Stratmann et al. 1998; Zagzag et al. 1999) and neuronal cells of the brain (Stratmann et al. 1998). 1.6.3 Consequences of Tie2 activation by angiopoietins Ang-1 and Ang-2 can both bind to the Tie2 receptor, but only Ang-1 induces its phosphorylation and thereby the activation of the receptor (Davis et al. 1996). Ang-2 does not induce receptor phosphorylation. It can compete with Ang-1 to act thereby as an antagonist (Maisonpierre et al. 1997). Yet, some studies also identified Ang-2 as an agonist of the Tie2 receptor (Kim et al. 2000c; Daly et al. 2006). Ang-1 binding to Tie2 leads to an activation of different pathways inside the cell by recruiting different adaptor proteins to the receptor. Signaling is related to many different processes including cell survival, migration, inflammation and permeability. Activation of Tie2 leads to phosphorylation of the subunit p85 of phosphatidylinositol 3- kinase (PI3K) and thereby to its activation. PI3K activates Akt which in turn phosphorylates and activates the Forkhead transcription factor FOXO-1 (FKHR-1). FKHR-1 is a strong inducer of Ang-2 expression and inhibits Ang-2 liberation (Dumont et al. 1993; Kim et al. 2000b; Jones et al. 2001a; Hodous et al. 2007; Semones et al. 2007). Activation of Akt also stimulates the phosphorylation and thereby the inhibition of proapoptotic proteins, like BAD and procaspase-9 (Cardone et al. 1998; Kim et al. 2000b). Additionally, Akt upregulates survivin, a classical apoptosis inhibitor (Figure 5), and thereby supports cell survival (Papapetropoulos et al. 2000; Harfouche et al. 2002).

Introduction Figure 5: Schematic representation of angiopoietin signaling in regulating the quiescent and the activated phenotype of the endothelium. (A) Ang-1 is produced in non-endothelial cells and binds to Tie2 which leads to Tie2 autophosphorylation. In a next step, PI3K and Akt are activated which in turn promotes survival or antiapoptotic signals through proteins like, Survivin, Caspase-9, enos and Bad. Inactivated FAK in the cell further supports survival of endothelial cell through Akt. On the other hand, Rho GTPases are activated by Ang-1 which reduces endothelial cell permeability by sequestering Src through mdia. Thereby, VEGF-R2-mediated Src phosphorylation and subsequent VE-cadherin internalization is inhibited. VE-PTP interacts with Tie2 in the presence, but not in the absence of cell-cell contacts. VE-PTP inhibition in endothelial cells is associated with increased permeability. Furthermore, several proteins like Dok-R or Grb14 associate with phosphorylated Tie2 and thereby inhibit endothelial cell proliferation. Ang-1/Tie signaling is required for vessel stabilization. Ang-2 acts as an antagonistic regulator on endothelial cells and thereby leads to vessel destabilization and pericyte drop out. The exact molecular mechanisms of how this process is regulated are not known. Potential molecules that are involved in this process are mentioned in the scheme. FOXO transcription factors are also involved in Ang/Tie signaling by regulating protein synthesis. Their phosphorylation leads to an inactive from which promotes endothelial cell survival, quiescence and vascular stabilization whereas the activated form supports vascular destabilization and apoptosis. (B) Tie2 activation under certain conditions results in cell migration, inflammation and vascular leakage. Cell migration is mediated via activation of FAK by PI3K, adaptor proteins of Dok-R, e.g. Nck and PAK and maybe by Shp-2, which is thought to dephosphorylate autophosphorylation sites of Tie2. Translocation of Tie2 to cell-matrix attachment sides in subconfluent cells is discussed to promote endothelial cell migration by the activation of Dok-R and its adaptor proteins. The interaction of ABIN-2 with Tie2 is thought to inactivate NFκB via the IKK complex and thereby induces destabilization and inflammation. Rho activation is blocked during Ang/Tie-mediated vascular leakage, which liberates Src from mdia. VEGF promotes VEGF-R2 activation which in turn activates Src and induces VE-cadherin internalization. Abbreviations: Ang, angiopoietin; SMC, smooth muscle cell; HB-EGF, heparin-binding epidermal growth factor-like growth factor; PDGF, platelet-derived growth factor; TGF, transforming growth factor; BMP, bone morphogenetic protein pathway; Dok-R, docking protein R; MAPK, mitogenactivated protein kinase; PAK, p21-activated kinase; PI3-K, phosphatidylinositol 3 kinase; Akt, protein kinase B; FAK, focal adhesion kinase; enos, endothelial nitric oxide synthase; FKHR, forkhead transcription factor; VE-PTP, vascular endothelial tyrosine phosphatase.

Introduction A EC Ang-2 EC VE-PTP p110 p85 P nucleus FOXO-1 Ang-1 Tie2 P P Dok-R Grb2 Grb14 P Akt Survivin Caspase-9 Ras MAPK cascade Cell proliferation Rho enos Bad VEGF VEGFR2 P P Src Src mdia p110 p85 FAK Paxillin Cas VE-cadherin α v β 3 extracellular matrix Vessel stabilization Pericyte/SMC recruitment Serotonin HB-EGF PDGF-B TGFβ BMP pathway nucleus PC B Cell migration RasGAP P Dok-R P P Inflammation, thrombosis EC αvβ3 FAK Nck Pak P Shp-2 P Abin-2 NFκB NFκB inactive active IKK complex p110 p85 Grb7 P P Rho mdia Src-P VEGFR2-P Tie2 VE-cadherin-P Vascular leakage extracellular matrix

Introduction Tie2 activation has also been shown to be related to endothelial migration. This seems to be dependent on the receptor presentation on the cell surface (Fukuhara et al. 2008; Saharinen et al. 2008). Tie2 is polarized in activated endothelial cells and translocated to the extracellular matrix where it binds to matrix immobilized Ang-1. The Akt pathway is blocked, whereas Dok-R (docking protein R) is phosphorylated. Activated Dok-R interacts with rasgap, Nck and Crk. All these molecules are involved in cell migration, proliferation, cytoskeletal reorganization and the regulation of the ras signaling cascade (Jones and Dumont 1998). Tie2 is translocated to cell-cell junctions in quiescent endothelial cells, where it engages via trans complexes with Tie2 molecules of opposing cells. In this context, Tie2 interacts with VE-PTP, a molecule which is strongly associated with an intact barrier function, thereby inhibiting paracellular permeability. Additionally, Akt is activated and thereby induces endothelial cell survival and stability of the endothelium via the phosphorylation of enos (Figure 5) (Fukuhara et al. 2008; Saharinen et al. 2008). Src is activated during VEGFmediated angiogenesis. This leads to the activation of VAV, a guanine-nucleotide-exchange factor (GEF) for Rac. Rac further activates VE-cadherin at Ser665. Beta-arrestin-2 is recruited to VE-cadherin which leads to its internalization in a clathrin-dependent manner (Gavard and Gutkind 2006; Dejana et al. 2008). This process supports permeability and migration. Ang-1-mediated Tie2 signaling inhibits this pathway by activation of mdia through Rho. This leads to an association of Src and mdia. Src is not longer available for VEcadherin activation and internalization (Gavard et al. 2008). Other molecules like Grb2, Grb7, Grb14, ShcA, the protein tyrosine kinase SHP2 and the previously mentioned p85 subunit interact with Tie2 via SH2 domains. These molecules seem to be involved in cell migration, proliferation, differentiation and apoptosis (Huang et al. 1995; Kontos et al. 1998). Ang-1 is also able to activate focal adhesion kinase (FAK) via Tie2 (Kim et al. 2000a). This in turn leads to the phosphorylation of paxillin. The MAP kinase ERK is activated in further steps (Tournaire et al. 2004) which supports migration. In turn, when blocking Tie2 activation, Ang-1-induced migration via ERK is inhibited. Endothelial cell sprouting is mediated by the secretion of plasminogen and metalloproteinase following Ang-1 stimulation (Kim et al. 2000a). All these in vitro activation phenotypes of Ang-1 are supported by in vivo studies in mice which have shown that Ang-1 overexpression promotes vessel formation in the heart (Suri et al. 1998). Ang-1 acts as an anti-inflammatory cytokine. It protects against endotoxic shock-induced by LPS and thereby prevents from microvascular leakage (Witzenbichler et al. 2005). It further blocks cell surface activity and the expression of tissue factor (TF), an initiator of blood coagulation, which is involved in thrombosis and inflammation. It reduces VEGF-stimulated leukocyte adhesion to endothelial cells (Kim et al. 2001). Cardiac allograft atherosclerosis (Nykanen et al. 2003) and radiation-induced cell damage (Cho et al. 2004) are protected by

Introduction Ang-1 and by a specifically designed pentameric Ang-1, named Comp-Ang-1. Furthermore, Tie2 activation also leads to ABIN-2 recruitment which interferes with NF-κB signaling (Hughes et al. 2003; Jeon et al. 2003; Tadros et al. 2003). This prevents endothelial cells from undergoing apoptosis and the induction of inflammation. Subsequent signaling is likely mediated by the PI3K/Akt pathway because blocking PI3K results in suppression of ABIN-2- induced inhibition of cell death (Tadros et al. 2003). Ang-1 may also be involved in inflammatory diseases, like rheumatoid arthritis (RA). Synovial fibroblasts are a key player during RA and a major source of Ang-1. Ang-1 is even upregulated during this disease by inflammation promoting cytokines, like TNF-α (Gravallese et al. 2003; Scott et al. 2005). TNF-α but also IL-1β are further capable to induce the expression of the transcription factor epithelium-specific Ets-like factor (ESE-1) which is also detectable in the synovium of RA patients (Grall et al. 2003). ESE-1 has been shown to upregulate Ang-1 indicating that this transcription factor regulates the high Ang-1 mrna levels during RA (Brown et al. 2004). Ang-1- and Tie2-deficient mice show severe defects in the recruitment of pericytes and in their interaction with endothelial cells (Dumont et al. 1994; Sato et al. 1995; Suri et al. 1996). These findings have been supported by venous malformations in patients which are caused by a constitutively active form of Tie2. These mutants show reduced SMC coating (Vikkula et al. 1996). In a rat model of diabetic retinopathy, Ang-2 expression is highly increased which causes pericyte dropout (Hammes et al. 2004). However, the mechanisms involved in Ang/Tie-mediated SMC recruitment are poorly understood. One possible molecule involved in the recruitment of mural cells is the EC-derived heparin binding EGF-like growth factor (HB-EGF). Its expression in endothelial cells is upregulated by Ang-1 (Iivanainen et al. 2003), but only when they are in contact with mural cells. HB-EGF-mediated receptor (ErbB1 and ErbB2) activation thereby induces SMC migration. Besides HB-EGF, PDGF-B is also expressed by endothelial cells and is involved in pericyte recruitment (Lindahl et al. 1997). PDGF-B signals through its receptor PDGFRβ which is expressed by endothelial cells. It is a very potent chemoattractant which promotes the proliferation of SMCs and pericytes during their recruitment to the endothelium (Hellstrom et al. 1999). PDGFRβ antibodies completely block the recruitment of pericytes to the newly formed vasculature in the retina of newborn mice which leads to retinal oedema and haemorrhages (Uemura et al. 2002). The uncovered vessels are poorly remodelled and leaky. The injection of recombinant Ang-1 almost completely rescues the phenotype mediated by the blocking PDGFRβ antibodies. This suggests that Ang-1 and PDGF-B somehow act together. However, vascular defects in Ang-1- and Tie2-deficient mice occur earlier during development than those of PDGF-B- and PDGFRβ-deficient mice which indicates a different mechanism of recruiting pericytes. Another very important player during

Introduction SMC differentiation is TGF-β which is upregulated by Ang-1 following PDGF-B stimulation. In turn, Ang-1 is downregulated by TGF-β. These data suggest that pericytes are recruited by PDGF-B which supports pericyte proliferation. Ang-1, which is upregulated by PDGF-B, promotes pericyte migration. In turn, TGF-β is responsible for SMC differentiation and to render the vasculature in its quiescent form (Hirschi et al. 1998; Oh et al. 2000; Nishishita and Lin 2004). There is not much known about Ang-2-mediated Tie2 signaling. However, recent studies have shown that Ang-2 supports RhoA and MLC activation and thereby promotes vascular leakage and endothelial cell migration (Parikh et al. 2006). Further experiments have identified Ang-2 as a pro-inflammatory cytokine. Ang-2-deficient mice cannot elicit an inflammatory response in thioglycollate-induced or Staphylococcus aureus-induced peritonitis (Fiedler et al. 2006). Ang-2 serum levels are highly increased in humans during sepsis. Normal serum levels are in the range of 1-2 ng/ml. During sepsis, Ang-2 levels may increase up to 20-fold (30 ng/ml). This elevation is also associated with mortality. More than 60% of patient with levels over 20 ng/ml die (Parikh et al. 2006; Gallagher et al. 2007; Orfanos et al. 2007; Siner et al. 2008). Furthermore, Ang-2 expression correlates with neovascularization during physiological and pathological processes, like arthritis (Fearon et al. 2003) or psoriasis (Kuroda et al. 2001). In both diseases, Ang-2 expression is not only associated with vessel remodelling but also with VEGF expression. Ang-2 and VEGF act together to induce angiogenesis and the expression of matrix metalloproteases, proteins that degrade the basal membrane (Etoh et al. 2001). However, Ang-2 induces vessel regression in the absence/inhibition of VEGF (Maisonpierre et al. 1997; Holash et al. 1999; Korff et al. 2001; Lobov et al. 2002). Ang-2 increases the expression level of the matrix metalloprotease MMP-2 (Hu et al. 2003) which is a sign for functional angiogenesis. Moreover, an anti-ang-2 therapy in the cornea of rats inhibits VEGF-induced neovascularisation (Oliner et al. 2004). Ang-2 expression is highly upregulated by angiogenesis-inducing molecules like VEGF, bfgf or TNF-α. Thrombin, an angiogenesis promoting molecule, but also hypoxia is able to increase Ang-2 expression (Mandriota and Pepper 1998; Oh et al. 1999; Kim et al. 2000e; Krikun et al. 2000; Huang et al. 2002; Yamakawa et al. 2003; Hegen et al. 2004; Lund et al. 2004; Pichiule et al. 2004). Hegen and co-workers could show that Ang-2 promoter activity is regulated by the transcription factor Ets-1 (Hegen et al. 2004). The implication of Ets-1 in neovascularization has been shown in a mouse model of proliferative retinopathy (Watanabe et al. 2004). Ets-1 dominant negative constructs injected in the eye completely block this function. Its expression is further upregulated by VEGF and shear stress which in turn increase Ang-2 expression (Goettsch et al. 2008; Milkiewicz et al. 2008). Ang-2 expression is further regulated by the transcription factor FOXO1. This family of transcription factors is involved in the upregulation of proteins during destabilization and remodelling. Ang-1

Introduction negatively interferes with FKHR-associated gene expression and thereby suppresses the production of Ang-2 (Daly et al. 2004). 1.6.4 Angiopoietin expression in tumors and tumor-associated angiogenesis Ang-2 is only weakly expressed in endothelial cell under physiological conditions. However, Ang-2 expression dramatically increases during vascular remodelling, e.g. during tumor growth (Vajkoczy et al. 2002). Glioblastoma for example show increased levels of Ang-2 in the endothelium (Stratmann et al. 1998). Here, Ang-2 is highly expressed in necrotic regions where it induces vessel regression (Koga et al. 2001). Vessels in these necrotic areas are not covered by smooth muscle cells. Only small vessels in glioblastomas express high amounts of Ang-2 but not the larger ones (Stratmann et al. 1998). Overexpression of Ang-2 in the rat glioma model results in aberrant vessels with low SMC coverage (Machein et al. 2004). Ang-2 is also detectable in significant concentrations in the circulation of tumor patients bearing other tumors, e.g. in oesophageal squamous cell cancer (Zhou et al. 2007), hepatocellular carcinoma (Kuboki et al. 2007), lung cancer (Park et al. 2007) and in melanomas (Helfrich et al., 2008). Furthermore, Ang-2 expression is correlated with tumor progression in melanomas. Tumor cells have been also shown to express Ang-2, e.g. in stomach (Etoh et al. 2001), colon (Ahmad et al. 2001) and bladder carcinoma (Oka et al. 2005) but also NSCLC (non small cell lung cancer) (Takanami 2004). In addition to promoting vessel regression as in glioblastomas, Ang-2 induces tumour neovascularization in combination with VEGF or bfgf. Blocking experiments with Ang-2 neutralizing antibodies or fusion proteins massively decreased tumor growth (Oliner et al. 2004). Antibodies against Ang-2 clearly inhibit Ang-2- but also VEGF-induced endothelial cell migration and proliferation during angiogenesis (Cai et al. 2003) which shows Ang-2 promoting functions during VEGF-induced angiogenesis. Moreover, Ang-2 aptamers (RNAs that bind and thereby block proteins) inhibit bfgf-induced angiogenesis in the rat corneal assay (White et al. 2003). Besides promoting vessel regression and neovascularization, Ang- 2 can also stimulate breast cancer metastasis formation in a Tie2 independent pathway directly by binding to integrin α5β1 (Imanishi et al. 2007). The role of Ang-1 in tumor-associated angiogenesis remains controversial. Ang-1 overexpression leads in many tumor models to reduced tumor growth (Hayes et al. 2000; Hawighorst et al. 2002; Stoeltzing et al. 2003). Pericyte coverage of the tumor vasculature is massively increased and thereby stabilized (Tian et al. 2002; Stoeltzing et al. 2003). Yet, Ang-1 is also able to promote tumor growth in rat gliomas (Machein et al., 2004) and in plasma cell tumors (Nakayama et al. 2004). A downregulation of Ang-1 in HeLa cells by antisense RNA inhibits tumor growth and angiogenesis (Shim et al. 2001). These findings

Introduction suggest that Angiopoietin-1 promoting or inhibiting functions are dependent on the tumor cell type, the dosage and maybe on the amount of Ang-2 in the tumors. 1.6.5 Angiopoietin functions during development and in the adult Angiopoietin-2 transgenic mice show severe vascular defects including disruption of vessel integrity (Maisonpierre et al. 1997). The endocardial lining is collapsed and detached from the underlying myocardium. Trabecular folds are completely absent. The systemic Ang-2 overexpression phenotype is highly reminiscent of the phenotype of Ang-1 and Tie2-deficient mice which supported the hypothesis that Ang-1 has agonistic functions and Ang-2 antagonistic functions on Tie2. Endothelial-specific overexpression of Ang-2 in adult mice confirms this hypothesis. These mice show a complete suppression of Ang-1-mediated Tie2 phosphorylation in addition to angiogenesis defects (Reiss et al. 2007). It was recently observed that the perinatal lethality of Ang-2-deficient mice is straindependent. Essentially all Ang-2-deficient mice in the 129/J background die postnatally within 14 days after births (Gale et al. 2002). Ang-2-deficient mice in the C56/BL6 background are viable with only 10% lethality (Fiedler et al. 2006). They show no vascular defects but develop severe chylous ascites after birth which indicates major defects in the lymphatic system.further investigation revealed that the lymphatic vessels shows that the large vessels are disorganized form a lacy network and are only poorly covered with smooth muscle cells. The small lymphatic vessels in the intestine are disorganized and irregular (Gale et al. 2002). Ang-2-deficient mice show no vascular defects. Therefore vessel formation in the retina has been investigated. The retina in wild type mice is completely avascular after birth. Only the lens shows a hyaloid vasculature. Yet, these vessels shortly regress after birth; not so in Ang-2-deficient mice where these vessels are not regressed. This is a hint for a vessel remodelling defect of these mice (Gale et al. 2002; Hackett et al. 2002). A replacement of the Ang-2 gene with the Ang-1 gene in the Ang-2 locus completely rescues the lymphatic phenotype but not the vascular remodelling defects supporting the hypothesis that Ang-2 is agonistic in lymphatic vessels and antagonistic in blood vessels. All those studies show that Ang-2 is completely dispensable during early development but is necessary for vessel remodelling during later stages. Angiopoietin-1 deficiency results in lethality at E11 to E12.5 (Suri et al. 1996). The phenotype of the knock out mice is very similar to the phenotype of Tie2-deficient mice but not as severe. These mice have growth retarded hearts with a less complex ventricular endocardium. The endocardium is collapsed and retraced from the myocardial wall. The endothelial lining in the atria is almost collapsed and the trabeculae are absent. The importance of Ang-1 during vascular remodelling has been established in Ang-1-deficient

Introduction mice (Suri et al. 1996). The mice show a much simpler and immature primary capillary plexus. The distinction between the larger and smaller vessels is much less pronounced. Periendothelial cells are scarce in Ang-1-deficient embryos and not associated with endothelial cells but separated from rounded endothelial cells. Overexpression of Ang-1 in cardiac myocytes under the control of the doxycycline-inducible promoter shed further light in the importance of Ang-1 during heart development. Most of those mice (90%) die between E12.5 and E15.5 as a result of cardiac haemorrhages. The myocardial walls of both atria and the ventricles are thinned and trabeculae density is dramatically decreased. Those mice show haemorrhages around the heart and the atria are enlarged. The outflow tract is collapsed and mice lack an intact endocardium and coronary arteries. Ten percent of the mice survive with cardiac hypertrophy and a dilation of the right atrium (Ward et al. 2004). The studies show that Ang-1 overexpression dramatically affects early development of the mice. Yet, overexpression in the adult has little effect on vessel structure and heart development. Transgenic mice overexpressing Ang-1 under the control of the keratin 14 (K14) promoter are viable and generally healthy (Suri et al. 1998). Newborn mice show larger vessels in the skin. Additionally, the skin of older mice is more reddish than those of normal mice. Transmission electron microscopy analysis confirmed that these mice have normal cell-cell contacts between endothelial cells and between endothelial and perivascular cells. The interendothelial cell distance is slightly increased but the mice show no plasma leakage or edema. The experiments demonstrated that the vasculature is largely intact and functional. 1.7 Endothelial barrier function and vascular permeability Only one in every 10,000 endothelial cells (0,01%) is in the cell division cycle in a normal healthy adult blood vessel (Engerman et al. 1967; Hobson and Denekamp 1984). Cells are tightly connected to each other. They maintain stable interactions with the underlying extracellular matrix and proliferation is contact-inhibited (Lampugnani et al. 1997; Vinals and Pouyssegur 1999). Additionally, cells are less sensitive to growth factors (Grazia Lampugnani et al. 2003), they are protected form apoptosis and permeability is reduced. This status is called endothelial cell quiescence. Endothelial cell quiescence is mediated by cell-cell junctions and cell-matrix protein interactions. Cell-cell junctions are classified in tight junctions (TJs) and adherens junctions (AJs). In the epithelium, TJs are located at the apical side whereas endothelial cells have intermingled TJs and AJs. TJs and AJs have similar features but are composed of different molecules. Tight junctions are formed by claudins and junctional adhesion molecules (JAMs) whereas AJs mainly contain VE-cadherin (Dejana 2004). Following activation, VE-cadherin is

Introduction phosphorylated and internalized. This process supports loosening of the AJs leading to an increase in transendothelial and consequently increased permeability (Esser et al. 1998; Andriopoulou et al. 1999; Angelini et al. 2006; Gavard and Gutkind 2006; Wallez et al. 2007). TJs and AJs bind to the cytoskeleton or other signaling molecules relaying signals into the cell. In addition to the classical junctional molecules, endothelial cells express molecules like PECAM-1 or S-endo-1 which also link the cells via homophilic trans interactions (Dejana 2004). Other molecules including occludin might not be essential for TJ formation, but they support intercellular adhesion mediated by VE-cadherin and claudin-5 (Furuse et al. 1993). Occludin is upregulated by Ang-1 (Hori et al. 2004) which performs vessel sealing and maturation promoting activities (Suri et al. 1996; Thurston et al. 2000). Additionally, Ang-1 can decrease VE-cadherin phosphorylation thereby supporting the quiescent phenotype of the vessel (Gamble et al. 2000). VE-cadherin signaling is not only involved in connecting cells to each other but it has also important functions in inducing survival signals via the activation of Akt through phosphatidylinositol 3-kinase (PI3K) (Gerber et al. 1998; Pece et al. 1999). Mutant mice lacking VE-cadherin die at E9.5 and have an increased number of apoptotic cells (Carmeliet et al. 1999). The association of VEGF-R2 with VE-cadherin has been linked to a decrease in VEGF-induced proliferation and to increased endothelial cell survival (Carmeliet et al. 1999). This could be due to the activity of the Density Enhanced Phosphatase (DEP-1), which interacts in confluent cells with the VE-cadherin/VEGFR-2 complex thereby decreasing VEGF-R2 phosphorylation (Grazia Lampugnani et al. 2003). In addition to cell-cell contacts, cell-matrix contacts also mediating EC quiescence. It is thought that endothelial focal adhesions play a key role in maintaining barrier function (Wu 2005). Focal adhesions are primarily composed of integrins that connect the extracellular matrix via linker proteins to the cytoskeleton. These adhesive complexes not only provide an anchorage for the cells to the matrix but also transmit forces and biochemical signals between the cell and the matrix (Lehoux et al. 2006). Recent studies have shown that inhibition of integrins binding to vitronectin (like αvβ3) and fibronectin (like α5β1) with RGD peptides leads to an increase in venular permeability (Wu et al. 2001) and therefore disrupt barrier function. Focal adhesion kinase (FAK) plays a central role in the dynamic control of focal complex assembly and distribution (Schaller 2001; Parsons 2003; Schlaepfer et al. 2004; Mitra et al. 2005). Signal transduction through focal adhesions has been related to many physiological but also pathophysiological processes, inlcuding angiogenesis, wound healing, and vascular remodelling in response to physical or chemical stress (Petit and Thiery 2000; Ingber 2002; Wozniak et al. 2004; Chien et al. 2005). Despite these advances, the specific contribution of focal adhesions to the regulatory mechanism of barrier function

Introduction remains a mystery. The same controversial discussion exists about how focal adhesions regulate permeability. 1.8 Cell adhesion Cell adhesion is mediated by cell surface receptors which can be primarily classified in four groups: the cadherins, the immuno-globulin-superfamiliy, the selectins, and the integrins. Direct cell-cell contacts are primarily mediated by cadherin superfamiliy members whereas the interaction with the extracellular matrix (ECM) is mediated by integrins. Cadherins play an important role in cell-cell recognition and cell sorting during early development (Takeichi 1991; Takeichi et al. 2000) and are also involved in pathological processes like the switch from the quiescent to the angiogenic phenotype of the endothelium (Wallez et al. 2006). Cell matrix interactions are primarily established by integrins. They are not only responsible for the stability of the tissue but also for migration of cells and activation of intracellular signaling pathways. They bind to extracellular matrix proteins like collagen, fibronectin, laminin or proteoglycans (Gumbiner 1996). The adhesive structure of the integrins is named focal adhesion which connects the extracellular matrix and the cytoskeleton of the cell. They are restricted to certain areas of the cell and are highly dynamic. 1.8.1 Integrins and extracellular matrix The integrins play an important role in physiological processes like embryogenesis (Darribere et al. 2000), angiogenesis (Schwartz et al. 1995; Stupack and Cheresh 2004), cell adhesion and cell migration (Cox and Huttenlocher 1998; Schwartz and Horwitz 2006) and in the control of the cell cycle (Schwartz and Assoian 2001). They are also involved in pathological processes such metastasis during tumor growth (Ramsay et al. 2007). Integrins are essential for the establishment and stabilization of endothelial monolayers, because interruption of integrin-matrix binding causes cell detachment from the substratum (Dejana et al. 1990; Cheng et al. 1991). Integrins are type I transmembrane glycoproteins comprising an α subunit (120-180 kda) and a β subunit (90 110 kda). Both subunits form heterodimeric complexes that are linked via disulfide bonds (Hynes 1992). Each subunit crosses the membrane once with most of each polypeptide in the extracellular space and two short cytoplasmic domains. They are receptors for extracellular matrix proteins and membrane bound polypeptides on other cells. Over 16 α and 8 β subunits can combine to form a diverse array of over 20 different integrins (Hynes 1992; Eliceiri and Cheresh 1999) (Figure 6). Vascular endothelial cells express multiple integrins with distinct combinations of α/β subunits, including α3β1, α6β1 and α6β4 which all bind to laminin; they also interact with the peptide binding motive arginine-glycine-aspartic acid (RGD) from fibronectin (FN), like α5β1 or vitronectin (VN), like αvβ3 and αvβ5 and with native collagens, like α1β1 and α1β2 (Figure

Introduction 6). Yet, integrins, like αvβ3, can also bind to denaturated or degraded collagen (Davis 1992). They have no enzymatic activity but can be activated by talin binding (Hynes 2003; Tadokoro et al. 2003) and/or by binding of extracellular ligands which induces the formation of clusters and a conformational change of the integrins (Figure 6). Integrins are bi-directional transmembrane molecules that not only transduce signals from the extracellular matrix to the cytoplasm ( outside-in-signaling ) but they also transport signals from the cell to the extracellular matrix ( inside-out-signaling ). For example activated α5β1 is necessary for the formation of fibrilles from FN and to establish the ECM (Hynes 1992). The functions of integrins are very specific as demonstrated by different phenotypes of the corresponding knock out mice. The defects range from a complete block in preimplantation development (β1) to major developmental defects (α4, α5, αv, β8) to perinatal lethality (α3, α6, α8, αv, β4, β8) and defects in leukocyte function (αl; αm, αe, β2, β7), inflammation (β6), haemostasis (αiib, α2, β3), bone remodelling (β3) and angiogenesis (α1, β3, β5) (Hynes 1996; De Arcangelis and Georges-Labouesse 2000; Sheppard 2000; Bouvard et al. 2001). Beta3- and beta5-deficient mice show enhanced angiogenesis but these mice are perfectly viable which could indicate that these integrins can be substituted by other integrins (Reynolds et al. 2002). Taken together, integrins play a diverse and very important role in multiple biological processes.

Introduction Figure 6: Overview of integrin receptor subunits. Integrins are α/β hetereodimers whereby each subunit crosses the membrane once. 8 β subunits can assort with 18 α subunits to form over 20 different integrin heterodimers. These are divided into different subfamilies. β2 and β7 are specific for white blood cells. Subunits with grey hatching have inserted I/A domains. These units are restricted to chordates (α4 and α9 in green and the subunits β2- β8. α subunits that are specific for laminin are indicated in purple or for the RGD sequence in blue. They are much wider distributed and can also be found in the metazoa. Red circles around α- and β subunits indicate integrin subunits expressed on the vascular endothelium. Asterisks denote alternatively spliced cytoplasmic domains (Hynes 2002).

Introduction 1.8.2 Integrin structure Integrins consist of transmembrane α/β-heterodimers (Figure 7A). The subunits have a large extracellular domain each (up to 1114 amino acids for the α subunit and up to 678 aa for the β subunit). Each subunit crosses the membrane once. Integrins have a short transmembrane domain and a small cytoplasmic domain with no catalytic activity (from 15-77 aa for the α subunit and from 46-60 aa for the β subunit) (Humphries et al. 2003). The extracellular region of α-integrin-subunit consists of three to four domains with 12 to 15 aa in length each which bind divalent cations (Ca 2+, Mg 2+ or Mn 2+ ) (Figure 7). These cations are directly involved in mediating conformational changes of the integrins and are thereby responsible for their affinity and the specificity. The N-terminal region of the α subunit consists of 7 elements that show only a weak homology and folded as a seven leaved β propeller domain (Takagi et al. 2002). Domains four to seven consist of binding sites for three to four divalent cations. This domain is called the EF-hand-motif (Tuckwell et al. 1992). Some α subunits have an I (Inserted) domain between domain three and four that is about 200 aa long (Leitinger and Hogg 2000) (Figure 7). For example, the I-domain can also be found in proteins of the ECM-like collagen IV. In general, this domain is known to be involved in protein-protein interactions. Some integrins bind their ligands depending on the I-domain (Mould et al. 2000). Other α subunits do not have an I-domain. Maybe the I-domain is enzymatically cleaved in these α subunits. The cytoplasmic domain of the α subunit are only weakly homolog, but they have a highly conserved membrane proximal domain (KXGFFFKR-sequence). The integrin is constitutively active when this domain is deleted (O'Toole et al. 1994). Different splice variants of the α subunit enhance the heterogeneity of the cytoplasmic domain (van der Flier and Sonnenberg 2001).

Introduction Figure 7: Integrin architecture. A) Organization of domains within the primary structure of integrin. Depending on the α subunit, it may contain an I-domain insertion as denoted by the dotted line. Asterisks show Mg 2+ (blue) and Ca 2+ (red) binding sites. Lines below the stick diagrams show disulfide bonds. B) Arrangement of domains within the three-dimensional crystal structure. Each domain is color coded as in (A) (Takagi et al. 2002). The β integrin subunit is less variable than the α subunit. Each β subunit has in the N-terminus, the extracellular part, a large loop, a highly conserved domain of about 250 aa. This structure is very similar to the I-domain of the α subunit. This region is therefore named the I-like domain. The I-like domain contains similar to the I-domain the MIDAS motive (Loftus et al. 1994; Lee et al. 1995). Next to the transmembrane region the β subunit contains four cysteine-rich domains (about 40 aa long) which are homolog to the EGFmodule (Mould 1996). The cytoplasmic region of the β subunit consists of 15-65 aa and are very homolog in contrast to the α subunits (Sastry and Horwitz 1993). All β integrin subunits have a highly conserved motive, the HDRR sequence, in the membrane proximal part of the protein (Williams et al. 1994; O'Toole 1997). This sequence interacts with the highly conserved GFFKR-motive of the α subunit (Briesewitz et al. 1995; van der Flier and Sonnenberg 2001). 1.8.3 Activation of integrins Many integrins are not constitutively active. They are present in an OFF state on the cell surface. This is an significiant phenomenon especially on circulating blood cells. A model for integrin activation is shown in Figure 8. Integrin activation is mediated here by a conformational change, normally due to ligand binding or cell activation. The OFF state is switched and the ON state is extended. Integrins can signal and bind to their ligands in the stretched state.

Introduction Figure 8: Model for integrin activation by global conformational change. The model of the resting integrin resembles the structure of αvβ3 observed in the crystal structure. The active integrin is shown in its extended conformation as observed in electron micrographs from αiibβ3 (Takagi and Springer 2002). The major platelet integrin, αiibβ3, is highly present on the platelet surface where it is inactive. It would directly bind to its major ligand, fibrinogen, in its active form which would result in aggregation followed by the development of thrombosis. Upon platelet activation, the integrin is immediately activated and binds its ligands (fibrinogen, von Willebrand factor (vwf) and fibronectin). This results in the strong adhesion to the vessel wall and to other platelets (Hynes 2002). Integrin activation is also highly regulated on leukocytes. They express predominantly β2 integrins that are inactive in resting white blood cells. Following activation, they are rapidly adhesive for their receptors, in this case, Ig superfamily molecules like ICAMs. These are expressed by endothelial cells and allow leukocytes to adhere to the vessel wall and support phagocytosis and cytotoxic killing. Therefore, it is important that integrins on leukocytes are present in the inactive form to avoid inflammatory responses (Hynes 2002). 1.8.4 Composition and function of integrin-mediated cell matrix adhesions It has been hypothesized that cells sense and respond to their ECM environment via integrins (Miranti and Brugge 2002). When cells anchor to the ECM, integrin engagement leads to the formation of membrane-associated protein complexes at sites called cell-matrix adhesions (Zamir and Geiger 2001). Cell-matrix adhesions are able to provide dynamic bidirectional linkages between the ECM and the intracellular cytoskeleton. These membranespanning links play critical roles in the regulation of ECM assembly, rearrangements of cytoskeleton, transduction of signaling as well as in the control of cell morphogenesis, migration, proliferation, differentiation and survival (Hynes 2004).