All you need is love

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Barkefors, I., Le Jan, S., Jakobsson, L., Hejll, E., Carlson, G., Johansson, H., Jarvius, J., Park, J. W., Jeon, N. L., Kreuger, J., (2008). J. Biol. Chem., 283(20):13905–13912.

II Barkefors, I., Thorslund, S., Nikolajeff, F., Kreuger, J., (2009). A fluidic device to study directional angiogenesis in complex tissue and organ culture models. Lab Chip, 9(4):529-535.

III Barkefors, I., Fuchs, P. F., Bergström, T., Heldin, J., Nilsson, K. F., Kreuger, J., (2010). EXOC3l2 associates with the exocyst complex and mediates directional migration of endothelial cells. Submitted.

Reprints were made with permission from the respective publishers.

Contents

INTRODUCTION ...... 11 CELL MIGRATION ...... 11 A sense of direction ...... 13 Matrix interactions ...... 15 The role of the exocyst complex in cell polarization ...... 16 Exocyst composition and localization ...... 16 Migration and polarization ...... 18 THE VASCULAR SYSTEM...... 19 Angiogenesis...... 21 VEGF and other pro-angiogenic molecules...... 22 Vascular Endothelial Growth Factors...... 23 Fibroblast Growth Factors ...... 24 The vascular sprout and gradients in vivo...... 25 FLUIDIC CELL MIGRATION ASSAYS...... 27 Creating an in vitro environment...... 27 Controlling the flow...... 27 The matrix ...... 29 PRESENT INVESTIGATION ...... 30 PAPER I...... 30 Aim...... 30 Results...... 30 Discussion...... 31 Future perspectives...... 32 PAPER II...... 32 Aim...... 32 Results...... 33 Discussion...... 33 Future perspectives...... 34 PAPER III ...... 34 Aim...... 34 Results...... 35 Discussion...... 35 Future perspectives...... 36 CONCLUDING REMARKS ...... 37 SAMMANFATTNING PÅ SVENSKA...... 38 ACKNOWLEDGEMENTS ...... 40 REFERENCES...... 43

Abbreviations

Ang1,2 Angiopoietin 1,2 CDC42 Cell division control protein 42 homolog EB Embryoid body ECM Extracellular matrix ES cell Embryonic stem cell EXOC Exocyst complex component FGF Fibroblast growth factor HFL-1 Human fetal lung fibroblast HMVEC Human microvascular endothelial cell HS Heparan sulfate HSPG Heparan sulfate proteoglycans HUAEC Human umbilical artery endothelial cell HUVEC Human umbilical vein endothelial cell MMP Matrix metalloproteinase SNARE Soluble attachment protein receptor TIE1,2 Tyrosine kinase with immunoglobulin-like and EGF-like domains 1,2 WB Western blot VEGF Vascular endothelial growth factor VEGFR Vascular endothelial growth factor receptor

10 Introduction

To function properly every cell in the body needs a constant supply of oxygen and nutrients as well as a way of getting rid of waste products. To ensure this the entire body is riddled with a fine network of blood and lymph vessels. The development and function of the vascular tree are tightly regulated and formation of new blood vessels (angiogene- sis) rarely occurs in adult individuals. However, when the system does fail the consequences can be devastating. Tumors depend on angio- genesis both to grow and to metastasize (Reviewed by Al-Mehdi et al., 2000; Folkman, 1971; Hanahan and Weinberg, 2000), and aberrant vessel growth is also a central feature of pathological conditions such as macular degeneration, atherosclerosis, chronic ulcers, psoriasis, rheumatoid arthritis and diabetic retinopathy (Aiello, 1997; Aiello et al., 1994; Folkman, 1995; Henno et al., 2009; Isner, 1999).

Directed cell migration and infiltration are important parts of angio- genesis. The endothelial cells that are lining the inner surface of all vessels are not only incredibly sensitive micro-sensors; they possess extreme communicative skills and adapt readily to changes in their environment. The aim of this thesis is to increase the understanding of directed cell migration and angiogenesis. I will describe two in vitro models designed to enable studies of cell migration in response to growth factor gradients and finally I will discuss the possible in- volvement of ExoC3l2 in angiogenesis and vascular function.

Cell migration Cell migration is a fundamental process seen in all multicellular organisms and is traditionally viewed as a mechanical process where the primary driving force of migration is remodeling of the actin cytoskeleton (Wang, 1985). This model where actin-rich protrusions at the front push the cell forward while actomyosin filaments generate contractile forces at the sides and rear is complemented by the mem- brane flow model in which the leading edge protrudes forward

11 through blebs blown out by exocytosis (Bretscher, 1976) (Figure 1). The membrane flow type of migration has been observed in e.g. ze- brafish embryos where germ cells are guided by stromal-derived fac- tor-1 (SDF-1) and its receptor C-X-C chemokine receptor type 4 (CXCR4) (Haas and Gilmour, 2006; Insall and Jones, 2006) and in amoeboid migration of tumor cells in 3D gels (Sahai and Marshall, 2003).

Figure 1. Models of cell migration. In the cytoskeletal model polymerization of actin filaments at the leading edge pushes the cell forward while the rear is contracted through forces generated by actomyosin. This model of migration is also called treadmilling (top). In the membrane flow model the front is expanded through directed exocytosis of vesicles.

Both the cytoskeletal model and the membrane flow model assume that the cell has a concept of what is front and what is rear. A cell grown in vitro, thus not receiving any directional information from the environment will move randomly, but it will move. This suggests that if no directional information is available a direction will be chosen arbitrarily and then reevaluated with certain intervals (chemokinesis) (Tono-oka et al., 1979). Studies on the molecular level of the Rho family proteins do indeed separate directed movement from motility. Leukocytes or amoebas expressing dominant negative mutants of the

12 small GTPases Cdc42 or Rac, or neutrophils from Rac2 knockout mice have a disturbed sense of direction, but are not completely im- mobile (Allen et al., 1998). It has also been shown that neither cell movement nor the actin cytoskeleton is necessary for directional sens- ing suggesting that directionality without movement is possible (Parent et al., 1998).

Luckily, the movement of cells in the body is typically not random, but directed by extracellular guidance cues which can be presented as gradients either bound to the extracellular matrix (haptotaxis) (Carter, 1967), or as soluble growth factors (chemotaxis) (Keller et al., 1979). These cues interact with plasma membrane receptors and transmit a spatially restricted intracellular signal that influences the assembly, disassembly and arrangement of the actin cytoskeleton, the endocytotic and exocytotic trafficking pathways, and the adhesive forces through which cells interact with the environment.

A sense of direction

“Without a sense of direction, potential is often never realized”

Anna Huttenlocher

A cell is really an exquisitely sensitive microsensor, capable of rapidly interpreting and responding to even quite shallow molecular gradients (Review by Huttenlocher, 2005). The process requires several positive feedback loops and is evolutionary highly conserved.

It is maybe natural to assume that receptor polymerization accounts for some of the sensitivity. However, it has been shown in e.g. neutro- phils (Servant et al., 1999) and amoebas (Xiao et al., 1997) that the receptors responsible for binding the chemoattractant are not clustered at the leading edge of chemotaxing cells. Considering the large mem- brane rearrangement going on during migration it is actually rather strange that the receptors remain so evenly distributed in the mem- brane of many polarized cells. The actin filaments do gather at the leading edge during directed migration (Iwasa and Mullins, 2007), but this polarization also occurs during random migration and thus is not the key to chemotaxis. Instead the establishment and maintenance of polarity occur at the intermediary points in the signaling pathways

13 where the response becomes localized (Reviewed by Insall and Jones, 2006; Van Haastert and Devreotes, 2004). These pathways include integrins, phosphoinositides, cytoplasmic adaptor proteins and Rho family GTPases.

The polarization mechanisms and their crosstalk differ between cell types. For example, the motility of several immune cells requires rapid changes in direction and the sensing of quite shallow gradients of at- tractants; numerous studies on chemotaxis have been performed using e.g. neutrophils and leukocytes. These cells apply an amoeboid type of migration similar to the one described by the membrane flow model. Since they move quite fast it is not necessary to produce stable gradi- ents to induce recordable polarization and migration. For example it is possible to use the micropipette assay (Futrelle et al., 1982) where the chemoattractant is positioned on one side of the study subject using a pipette. When the tip is moved the cell will “follow” the source of attractant around the culture dish. However, to study chemotaxis in tissue cells such as epithelial and endothelial cells is more complicated (Reviewed by Lamalice et al., 2007). The locomotion of these cells depends more on actin reorganization and the gradient created in the micropipette assay is far to transient to guide these slow moving cells.

There are probably many different proximal signaling pathways by which cell polarization can be regulated, but common for most of these pathways is the localized activation of Rac at the leading edge (Reviewed by Ridley et al., 2003). The polarization is also frequently accompanied by a re-organization of the microtubule-organizing cen- tre, microtubules and Golgi apparatus to the front of the nucleus (Palazzo et al., 2001). RhoA seems to be an important regulator for intracellular contractility and tension (Alblas et al., 2001).

G protein-coupled receptors (GPCRs) play an important role in regu- lating chemotaxis and they can also promote migration by transacti- vating growth factor receptors (Cotton and Claing, 2009). Stimulation of the GPCRs results in activation of heterodimeric G-proteins which in turn activate enzymes such as phospholipase C (PLC), different types of protein kinase C (PKC) and phosphoinositide 3-kinase (PI3K) (Selvatici et al., 2006). However, chemoattractant–receptor binding also induces responses that do not depend on functional G proteins. Most pathways, however, involve Cdc42, which seems to be some- thing of a chemotactic mastermind, but also the PAR proteins and

14 atypical protein kinase (aPKC) in the initial polarization (Etienne- Manneville and Hall, 2003; Itoh et al., 2002).

The production of phosphatidylinositol trisphosphate (PIP3) from PIP2 at the leading edge by PI3K is also implicated in polarization (Funamoto et al., 2002). PIP3 enhances actin polymerization and pseudopod formation and is degraded by PTEN that localizes to the rear and sides of the cell. As a result of positive feedback, internal gradients of PI3K and PTEN are generally much steeper than external gradients of chemoattractant which increases the sensitivity. Members of the WASP family are also present at the front of the migrating cell as they are important regulators of actin assembly (Myers et al., 2005).

Once polarization is initiated it is maintained by a set of positive feed- back loops involving PI3K, microtubules, Rho family GTPases, in- tegrins and vesicular transport. These events rapidly subside in the presence of persistent stimulation (Reviewed by Parent and Devreotes, 1999). This rapid inhibition allows the cell to ignore the background concentration of attractant and more accurately sense the gradient.

Matrix interactions Chemotactic factors are not the only external effectors of cell migra- tion. The extracellular matrix (ECM) interacts with the cell in many different ways including integrin signaling and modification of recep- tor-ligand complexes. The matrix can also provide directional infor- mation.

There is no doubt that growing cells in vitro in a monolayer is a con- venient way to obtain biological information; an enormous amount of important results have been achieved this way. In vivo, however, cells rarely find themselves in a uniform environment and important fea- tures such as cell-cell and cell-matrix interactions may be lost in a 2D culture. The 3D matrix does not only provide physical support, but can also induce certain differentiation pathways and contact guidance. One example of this is the well-oriented organization of tumor fibro- blast matrices that clearly direct the alignment of cells parallel to the fibers (Amatangelo et al., 2005). The random changes in direction frequently observed when a cell migrates on a 2D surface are not as pronounced in a 3D culture. This behavior correlates to increasing levels of Rac, and reducing the level of this signaling molecule in fi-

15 broblasts grown in 2D stabilizes the migration (Pankov et al., 2005). Another factor affecting cells in 3D matrices is the rigidity of the sub- strates, and most cells tend to migrate towards increasing stiffness, a phenomenon known as durotaxis (Lo et al., 2000).

When moving through a solid substrate the cells usually have to de- grade the matrix in order to penetrate it. In this context the most stud- ied and controversial molecules are members of the metalloproteinase family. MT1-MMP is the major cell-associated protease in invasive activity and deficiency of this protein causes severe abnormalities in mice (Chun et al., 2004; Holmbeck et al., 1999). Several secreted MMPs (e.g. MMP-2 and MMP-9) have also been proposed to be im- portant for infiltration (Brooks et al., 1996; Kenagy et al., 1997), but the dependence of proteases has been questioned, especially in tumor cells that seem to be able to acquire an ameboid type of migration similar to what is seen in migrating leukocytes (Even-Ram and Ya- mada, 2005).

The role of the exocyst complex in cell polarization Migration and polarization of a cell require correct localization of receptors in the plasma membrane. Membrane proteins are continuously internalized, transported in endosomes and externalized at another location in the cell membrane or transported to the lysosomes for degradation. The fusion of endosomes with the membrane is catalyzed by the SNARE protein complex (Sollner et al., 1993), but the initial contact or “tethering” is mediated by the proteins in the exocyst complex and it is therefore believed that the function of the exocyst is to ensure proper localization of the vesicle (Reviewed by He and Guo, 2009; Wu et al., 2008).

Exocyst composition and localization The exocyst, that was originally identifyed in yeast, consists of eight subunits (Novick et al., 1980; Novick and Schekman, 1979; TerBush et al., 1996) and its structure and functions are largely conserved between species (Reviewed by Munson and Novick, 2006) (Figure 2).

16

Figure 2. The exocyst complex. The eight subunits of the exocyst was first discovered and characterized in yeast (Sec3, Sec5, Sec6, Sec8, Sec10, Sec11, Exo70 and Exo84), but the composi- tion and structure is conserved in higher eukaryotes (ExoC1-8). Black ovals represent the interactions between the human exocyst proteins as described by Munson and Novick (Munson and Novick, 2006). White ovals represent the interactions between the homologues in yeast.

ExoC1 and ExoC7 are localized in the cytosol close to the plasma membrane, where they interact with the membrane component Phos- phatidylinositol 4,5-bisphosphate (PIP2) and with members of the Rho family such as Cdc42 and Rho1 and 3 (Adamo et al., 1999; Boyd et al., 2004; Guo et al., 2001). The small GTPases are essential for cell polarization and might act as target sites for localized vesicle fusion. GTP-bound Rho1 and Cdc42 compete for binding to the N-terminal region of ExoC1 suggesting that they may control localization of ExoC1 at different cell cycle stages or growth conditions (Zhang et al., 2001).

The rest of the exocyst complex is associated with the vesicle and depends on actin fibers to reach the site of exocytosis (Boyd et al., 2004; Zajac et al., 2005; Zhang et al., 2005) (Figure 3). These exocyst components are also effectors of several small GTPases including Rab11, Arf6 and Ral A and B (Brymora et al., 2001; Prigent et al., 2003; Shipitsin and Feig, 2004; Zhang et al., 2004). The exocyst also interacts directly with the SNARE proteins and in exocyst mutants the SNARE complex fails to assemble (Grote et al., 2000). Although the

17 exocyst is often referred to as one unit, different exocyst complexes may execute diverse functions as recently described by Andersen and Yeaman. (Andersen and Yeaman, 2010).

Figure 3. The exocyst complex in directed migration. The exocytotic vesicle is associated with several exocyst complex components (ovals) and is transported along the actin filaments towards the site of exocysto- sis. EXOC1 and EXOC7 interact with small GTPases (circles) at the plasma membrane. Assembly of the exocyst complex brings the vesicle SNAREs (v- SNARE) and target SNAREs (t-SNARE) in close proximity enabling fusion of the vesicle with the plasma membrane.

Migration and polarization The exocyst has been implicated in a variety of cellular processes in- cluding cytokinesis (Chen et al., 2006), cell migration (Spiczka and Yeaman, 2008), tumor invasion (Spiczka and Yeaman, 2008), and ciliogenesis (Zuo et al., 2009). As described previously cell migration depends on localized exocytosis as well as the continuous reorganiza- tion of actin structures and the exocyst has important roles to play in both these processes (Liu et al., 2009). In migrating cells the exocyst

18 is enriched at the leading edge of the plasma membrane (Rosse et al., 2006; Zuo et al., 2006).

The role of the exocyst in receptor trafficking has been studied in sev- eral cell systems. In adipocytes, some of the exocyst complex compo- nents have been shown to participate in trafficking of Glut4 (Ewart et al., 2005; Inoue et al., 2003). Murthy et al. present evidence that ExoC2 is involved in membrane trafficking as well as in the estab- lishment of the anteroposterior axis and dorsoventral pattern in Dro- sophila (Murthy et al., 2010). The exocyst has also been shown to regulate receptor trafficking in neurons (Sans et al., 2003), and in the synapses (Gerges et al., 2006). A direct role of the exocyst in epithe- lial cell polarization has been demonstrated on several occasions, both in different in vitro models (Bryant et al., 2010; Oztan et al., 2007; Yeaman, 2003) and in Drosophila embryos in vivo (Blankenship et al., 2007).

The vascular system The vascular tree is a truly fascinating and intricate structure consist- ing of two circulatory systems which were first described in detail in 1628 by William Harvey (Harvey, 1628). The pulmonary circulation brings oxygenated blood from the lungs to the heart which is then transported by the systemic circulation to supply all tissues in the body with oxygen and nutrients. The systemic circulation is also re- sponsible for transportation of waste products to the kidneys which every day filter enormous quantities of blood. The blood leaves the heart through the large arteries via the arterioles and finally it reaches the capillary network where the exchange of oxygen, nutrients and waste products takes place. From the capillaries the blood returns to the heart through the venules and large veins. All the different vessel types have their own morphology which make them specialized to- wards a particular function (Figure 4). In a healthy individual the number and size of blood vessels change very little, however during wound healing, muscle growth, in the uterus and in some pathological conditions this pattern is broken (Girling and Rogers, 2005; Nagy et al., 2007; Prior et al., 2004; Ramakrishnan et al., 2005; Singer and Clark, 1999).

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Figure 4. The vascular system. The blood vascular system in collaboration with the heart and lungs provides oxygen, nutrients, and hormones to the cells of the tissues in the body. Blood vessels are composed of two interacting cell types, endothelial cells and smooth muscle cells. The smooth muscle cells (SMC) bind molecules released by the autonomic nervous system and respond by contracting. The contraction of the SMCs results in constriction of arterioles, which causes an increase in blood pressure and an increase in delivery of venous blood back to the heart. The capillaries are responsible for most of the exchange with the surrounding tissue and lack SMC coverage. Instead they are covered with supportive cells called pericytes. The inside of the vessels, which is in immediate contact with the blood, is lined with endothelial cells and this barrier regulates the permeability of the vessel, the adhesive properties and inflammatory responses as well as blood clotting and blood pressure.

20 Angiogenesis As opposed to vasculogenesis, which describes the formation of the vasulature in the embryo (Reviewed by Risau and Flamme, 1995), angiogenesis is the formation of new vessels from preexisting vascula- ture. Angiogenesis can occur by splitting of a mother vessel (intus- suseption), by stretching of existing vessels through mechanical forces (looping) or by sprouting (Figure 5).

Figure 5. Models of angiogenesis. Angiogenesis describes the formation of new blood vessels from existing vascu- lature. There are currently three models describing this process; intussuep- tion(1), sprouting(2) and looping(3).

Intussusception (Burri and Djonov, 2002) is a way of creating new vessels without the requirement of extensive production of new endo- thelial cells and occurs frequently during embryonic development. However, when new vessels are needed in a previously avascular area, for example during wound healing, angiogenesis occurs through loop-

21 ing or sprouting. Looping was identified as a form of angiogenesis quite recently (Kilarski et al., 2009) and our understanding of this mechanism is still relatively poor. Sprouting angiogensis on the other hand was first observed in 1939 (Clark and Clark, 1939) and has been extensively studied over the years in different model systems includ- ing the developing mouse retina (Uemura et al., 2006) and zebrafish embryos (Lawson and Weinstein, 2002). One reason for the large in- terest for sprouting angiogenesis was the assumption that this was the exclusive way of tumor vascularisation but as reviewed by Hillen and Griffioen (Hillen and Griffioen, 2007), the truth is more complex. Sprouting angiogenesis is nevertheless a major contributor to many pathological conditions and the development of improved proangiogenic or antiangiogenic therapies would be beneficial for patients suffering from diseases such as diabetes, psoriasis, macular degeneration, obesity, tumors or chronic wounds. I will continue to tell you more about vascular sprouting, but before I do so I would like to introduce some of the growth factors that are responsible for this process.

VEGF and other pro-angiogenic molecules There are tree major families of pro-angiogenic factors involved in vessel guidance. The Vascular Endothelial Growth Factor (VEGF) family is the most extensively studied, followed by the Fibroblast Growth Factors (FGFs). The third family, the angiopoietins, consists of four members that were discovered in the late nineties (Davis et al., 1996; Maisonpierre et al., 1997; Valenzuela et al., 1999). Ang-1 and Ang-2 act in a supportive manner to the VEGFA axis, but are still indispensible for angiogenesis and maintenance of vessel integrity (Reviewed by Augustin et al., 2009). The angiopoietins bind to the TIE endothelial specific tyrosine kinase receptors TIE-1 and TIE-2 (Thurston, 2003). Ang-2, but not Ang-1, is chemotactic for endothelial cells in vitro (Witzenbichler et al., 1998). The ephrin, semaphorin, slit and netrin families are also implicated in vessel guidance. These growth factors are usually referred to as axon guidance molecules but as described by several groups, axon guidance and vascular patterning have many aspects in common (Reviewed by Adams and Eichmann, 2010; Gelfand et al., 2009; le Noble et al., 2008).

22 Vascular Endothelial Growth Factors The VEGFs are secreted homodimeric glycoproteins. VEGFA was originally called vascular permeability factor (VPF) due to its effect on vascular permeability (Senger et al., 1983) but this name did not stick and recently the growth factor has received most attention as a target for antiangiogenic cancer therapy (Glade-Bender et al., 2003). The VEGF family also includes VEGFB-D and Placenta Growth Fac- tor (PlGF) and they all bind in an overlapping fashion to the VEGF receptors, VEGFR1, 2 and 3 (Reviewed by Ferrara, 2002; Olsson et al., 2006) The VEGF receptors were first identified on the surface of vascular endothelial cells but they also occur on bone marrow derived cells (Ferrara and Davis-Smyth, 1997; Tammela et al., 2005). VEGFs are secreted by a number of cell types including endothelial cells, fi- broblasts and tumor cells. VEGFC is involved in lymphangiogensis but it has also been shown to regulate blood vessel angiogenesis (Jussila and Alitalo, 2002; Lohela et al., 2008). VEGFB has generally been thought of as an angiogenic growth factor but has recently been implicated in neurogenesis (Sun et al., 2006) and in lipid up-take by endothelial cells (Hagberg et al., 2010). But I will dwell for a moment on VEGFA as it is most important for angiogenesis and plays a central role in the work I am about to present.

VEGFA can bind to both VEGFR1 and VEGFR2 although the affinity is highest for receptor 2. Data indicates that VEGFR2 is the principal signaling receptor for endothelial cells, whereas VEGFR1 is important for migration and probably functions as a decoy receptor, regulating the bioavailability of VEGFA (Rahimi et al., 2000). Although VEGFA is a potent activator of VEGFR2 the outcome of VEGFA stimulation is largely dependent of the presence of coreceptors such as neuropilin- 1 and heparan sulfate proteoglycans (HSPGs). (Jakobsson et al., 2006; Klagsbrun et al., 2002; Soker et al., 2002; Zoeller et al., 2009). The interaction between VEGFA and VEGFR2 is important in the early stages of angiogenesis, but when the vessel matures, the dependence of VEGFA seems to diminish (Lee et al., 2007). Pericyte coverage is proposed to be the key to reduced VEGFA dependence (Benjamin et al., 1999). VEGFA regulates survival (Gerber et al., 1998a; Gerber et al., 1998b) and migration in endothelial cells; it induces vascular per- meability and acts as a chemotactic factor for various cell types in- cluding endothelial cells and monocytes (Clauss et al., 1990) (Figure 6). There are several splice variants of the VEGFA protein including VEGFA121, VEGFA165 and VEGFA189 (Grutzkau et al., 1998)

23 (Figure 7). The different affinities of these isoforms for HSPGs have been proposed to regulate formation of VEGF gradients in vivo (Houck et al., 1992).

Figure 6. VEGFA/VEGFR2 signaling. VEGFA is involved in the regulation of several processes in endothelial cells. The major receptor for VEGFA is VEGFR2 but the growth factor also binds VEGFR1. Upon binding of VEGFA the intracellular part of VEGFR2 is phos- phorylated on several amino acids. The phosphorylations result in binding of signaling molecules (red ovals) and subsequent activation of signaling cascades (blue ovals).

Fibroblast Growth Factors The 22 members of the FGF family are involved in embryonic devel- opment, angiogenesis and wound healing (Reviewed by Eswarakumar et al., 2005). They bind to the FGF tyrosine kinase receptors that con- sist of two or three immunoglobulin-like domains and a heparin- binding motif. The interaction between FGF and heparin or HSPGs is crucial for FGF signaling. It stabilizes FGFs to thermal denaturation and proteolysis and may largely limit their diffusion and release (Bernfield and Hooper, 1991; Coutts and Gallagher, 1995). FGF2, or basic FGF, stimulates processes that are important for angiogenesis in

24 vivo, including endothelial cell migration, proliferation, invasion, and production of plasminogen activator (Montesano et al., 1986). The effect of FGF2 on chemotaxis of endothelial cells is somewhat un- clear.

Figure 7. VEGFA isoforms. VEGFA is produced in several isoforms with different affinities for the VEGFR2 co-receptors HSPG and neuropilin. The exons are indicated in alter- nating black and white. In VEGFA165b, exon 8 is replaced by exon 9.

The vascular sprout and gradients in vivo In sprouting angiogenesis the formation of new vessels is initiated by secretion of proangiogenic factors by e.g. hypoxic cells (Reviewed by Adams and Alitalo, 2007). Upon stimulation, the organization of the mother vessel is disrupted and the endothelial cells start to infiltrate the basement membrane, degrading the matrix e.g. by secretion of MMPs. The newborn sprout consists of two conceptual cell types; the stalk cells which make up the trunk and elongate the sprout by prolif- eration and the tip cell, the leader, sensing the direction by sending out filopodia into the matrix (Gerhardt et al., 2003). The identity of the tip cell is proposed to be maintained by lateral inhibition through the NOTCH/Dll4 pathway (Hellstrom et al., 2007), but this organization is quite plastic as recent data suggest (Jakobsson et al., 2010). The tip cell phenotype also depends on relative levels of VEGFR2 signaling and cells that are heterozygous for the VEGFR2 allele rarely make it to tip-cell position when competing with wild type cells in chimeric cultures (Jakobsson et al., 2010). The concentration gradient of proan- giogenic growth factor determines the growth direction of the sprout and probably also the tip cell phenotype. Although the concept of gra-

25 dients as directors of vascular sprouting has been accepted for quite some time, there are only a few examples demonstrating that growth factor gradients really exist in vivo (Gritli-Linde et al., 2001; Ruhrberg et al., 2002). The concentration of guidance molecules is naturally highest close to the source, the secreting cells, but if the gradient was to depend solely on free diffusion, it would probably be rather tran- sient. However, matrix proteins (e.g. HSPGs) serve to stabilize the gradients due to their affinity for growth factors such as VEGFs (Hacker et al., 2005; Mac Gabhann et al., 2006).

The high levels of VEGFR2 and the morphology with numerous filo- podia make the tip cell ideal as a VEGFA gradient sensor, but similar to other types of chemotaxis receptors, VEGFR2 seems to be rather evenly distributed in the tip cell (Jakobsson et al., 2010). To prevent saturation and diffusion of activated receptors in the membrane, a lo- calized response depends on internalization of the receptor-ligand complex upon stimulation, and it has been shown on several occasions that internalization of VEGFR2 affects VEGFA signaling. For exam- ple, vascular endothelial cadherin inhibits cell proliferation by retain- ing VEGFR2 at the membrane (Lampugnani et al., 2006) and the VEGF-mediated arterial morphogenesis also depends on internaliza- tion of the VEGFR2/VEGFA signaling complex (Lanahan et al., 2010). In a recent study, Sawamiphak et al. demonstrate that VEGFR2 internalization depends on interaction between this receptor and the PDZ domain of ephrin-B2 (Sawamiphak et al.). Moreover, the intra- cellular localization of VEGFR2 is tightly regulated and involves in- teractions with motor proteins such as dynamin-2 (Bhattacharya et al., 2005). Relatively little is known about the recycling of VEGFR2 but the Rab proteins are clearly implicated (Jopling et al., 2009). Concern- ing the effects of VEGFA on VEGFR2 degradation contradictory re- sults have been reported. One study suggests that VEGFR2 undergoes almost complete degradation after VEGFA stimulation (Ewan et al., 2006), whereas another implies that VEGFR2 degradation is not sig- nificant (Gampel et al., 2006). One interesting observation is that VEGFR2 seems to be redistributed to the plasma membrane through a recycling pathway not observed for any other tyrosine kinase receptor (Gampel et al., 2006). The VEGFR2 trafficking is reviewed by Bruns et al. (Bruns et al., 2009).

26 Fluidic cell migration assays

Creating an in vitro environment The cell microenvironment can be described in terms of numerous biochemical and physical parameters, the most obvious being tem- perature, gas concentration, pH, salt concentration and physical sup- port. But cells in vivo also experience stimuli from other cells through various growth factors and cytokines, stimuli from the ECM both through interactions with surface receptors and through mechanic ten- sion, pressure from the interstitial fluid, shear stress, etc. When study- ing cells in vitro, it is important to remember that when you deprive the cells of any of these factors, they will no longer behave exactly like their fellows in vivo (Reviewed by Meyvantsson, 2008). As a consequence, cell culture is always a trade-off between simplicity and adequacy. Regular cell culture dishes are simple to work with and are suitable for many types of experiments. However, when a more com- plex environment is required, microfluidics has proved to be an excel- lent tool to provide additional control over cell culture conditions.

Microfluidics is the science and technology of systems that process or manipulate small amounts of fluids. It originated as a method for mo- lecular analysis but has during the past decades found its way into cell biology. Microfluidics is now regularly used to increase our under- standing of processes such as horizontal gene transfer, signaling, mo- tility, chemotaxis, viability and persistence (Reviewed by Kim et al., 2010; Weibel et al., 2007; Whitesides, 2006).

Controlling the flow Cells in vivo experience flow under different circumstances including blood flow and interstitial fluid flow. In the in vitro system flow is a convenient way to mimic the conditions in vivo or to deliver growth factors, inhibitors, oxygen, nutrients etc., in a controlled fashion. One of the first studies of cell cultures in microfluidic channels was per- formed by Tilles et al. who studied the effect of oxygenation and flow on the viability and function of rat hepatocytes (Tilles et al., 2001). The microfluidic approach has also proved an excellent tool to create molecular gradients in vitro. Gradient devices have been used primarily to study chemotaxis but also as tools to increase throughput in screening, e.g. in the search for optimal concentrations (Thompson

27 et al., 2004; Walker et al., 2007; Wang et al., 2007) and as an efficient way to locally remove cells from a monolayer (Nie et al., 2007). In the chemotaxis field Jeon et al. developed the technique of flow splitting and diffusive mixing to maintain stable gradients in solution (Jeon et al., 2002; Wang et al., 2004) and used this to study for example neu- trophil migration. Irimia et al. also studied neutrophils in a micro- fluidic device where the direction of the gradients could be rapidly switched (Irimia et al., 2006).

The assays described above rely on continuous flow in the cell culture to maintain the gradients. However, this flow inevitably results in shear stress on the cells and washes away released growth factors, both of which might be beneficial, but it may also create problems in some applications. For example, when growth factors are washed away it results in a somewhat cleaner assay, but it also deprives the cells of an important means of communication. A narrow channel connecting to big medium reservoirs or a hydrogel PDMS sandwich are both examples of microfluidic gradient devices without flow (Abhyankar et al., 2006; Shamloo et al., 2008; Wu et al., 2006), and gradients in 3D matrices have also been generated (Cheng et al., 2007; Rosoff et al., 2005).

The flow in the in vitro systems discussed in this thesis has two im- portant functions. It creates the prerequisite for stable gradients and it continuously supplies the system with oxygen and nutrients (Figure 8). The need for proper oxygen delivery is important especially when studying angiogenesis since one of the triggers for this process is hy- poxia-induced VEGFA production (Reviewed by Pugh and Ratcliffe, 2003). Glucose levels should be high enough to avoid necrosis, which could lead to the release of unwanted factors. The flow system also allows for the use of very small amounts of reagents.

Shear stress is sometimes discussed when culturing endothelial cells on the premise that these cells are constantly exposed to shear in the in vivo situation (Reviewed by Reneman et al., 2006). It is beyond the scope of this thesis to discuss the effect of this phenomenon in cell culture and I will settle by stating the potential of fluidics to create this type of stress.

28

Figure 8. The use of flow to create in vitro environments. To the left is an example of how mixing of fluids from different channels can be used to create a gradient in a 2D system. The system to the right is a quite large fluidic system, but still requires minimal amount of medium and growth fac- tors.

The matrix As I pointed out earlier in the text the 3D environment is vital for many types of cells and cell systems, especially when studying migra- tion. There exists a multitude of artificial ECMs ranging in complexity from matrigel, which consists of many different components and growth factors, to single component gels containing collagen, laminin or fibrin. The 3D milieu is however not only determined by the mo- lecular composition of the matrix but also by the concentration, orien- tation and the degree of cross-linking. Collagens and laminins have an intrinsic capacity to polymerize and form a 3D gel spontaneously, whereas fibrin gels are a result of enzymatic cleavage of fibrinogen (Even-Ram and Yamada, 2005). The collagen gels are likely to be more easily penetrated than basement membranes in vivo due to their organization and relatively low concentration, while fibrin gels pre- sent a denser fibrous network. The gels also have their own way of communicating with the migrating cells. Collagen modulates growth differentiation in osteoblast-like cells (Green et al., 1995) and fibrin fragments released upon matrix degradation are proposed to have chemotactic effect on numerous cell types including endothelial cells (Bootle-Wilbraham et al., 2001; Kodama et al., 2002). Several studies also investigate the importance of matrix stiffness on endothelial cell behavior (Reviewd by Califano and Reinhart-King, 2010; Yamamura et al., 2007).

29 Present investigation

Paper I

Aim Growth factor gradients are important regulators of angiogenesis in vivo but recreating these gradients in vitro has proved to be a chal- lenge. The aim of this study was to develop an uncomplicated and reliable assay where the effect of molecular gradients on adhesive cells could be studied at high resolution, and to evaluate VEGFA and FGF2 as chemotactic agents.

Results In this report we describe the development of an endothelial cell chemotaxis assay based on a migration chamber designed by Noo Li Jeon (Chung et al., 2007). In the chamber the cells migrate on a gela- tin coated surface in response to diffusion based gradients maintained by a laminar flow through the system (Figure 9). Gradient profiles are assessed by measurement of fluorescent dextran and the experimental data corresponds well to computer simulations of the gradient profile. Using these simulations the concentration profile of any molecule can be predicted given that its diffusion coefficient is known. Using the chemotaxis assay we show that a gradient of FGF2 is able to attract venular but not arterial endothelial cells when the cells are grown sparsely on a gelatin coated surface. The attraction to FGF2 is how- ever much weaker than that to VEGFA165. The results also demon- strate that constant levels of VEGFA165, but not of FGF2, are able to reduce chemokinesis. The truncated version of VEGF (VEGF121) lacks the binding site for HS and has reduced, but does not lack, at- tractive properties. Systematic exploration of different gradient shapes and ranges led to the identification of a minimal gradient steepness required for efficient cell guidance and the conclusion that chemotaxis of endothelial cells is more dependent on gradient shape than on the absolute concentrations.

30

Figure 9. The 2D migration chamber. The system is placed directly on top of the cell culture and is held in place by a web of vacuum channels. The growth factor to be studied is placed in one of the three vacuum reservoirs which are connected to the main channel. The gradi- ent is formed by diffusion in the main channel and it is maintained by a con- stant flow through the system. To the right in the figure is a gradient visualized by FITC-Dextran which has been added to the middle reservoir producing a hill shaped concentration profile (blue line).

Discussion Historically, the most common way to assess chemotactic responses has been to use the Boyden chamber, or modifications of this assay (Boyden, 1962). In the Boyden chamber the cells migrate from an upper well to a lower well through a porous membrane. The factor to be studied is placed in the lower well and the gradient is thus based on diffusion and will change over time until the concentration difference between the wells is abolished. As shown in our study the chemotactic response to a specific growth factor is largely dependent on cell type and gradient shape, but also on the absolute concentration of the chemotactic molecule. The classical chemotaxis assay might thus be too blunt a tool to properly evaluate chemotaxis. In addition, it has been proved difficult to separate chemotaxis from chemokinesis and other phenotypes such as proliferation, survival and adhesion. Hence, if the cells fail to penetrate the membrane in the Boyden chamber this could in fact be the result of defects in any of these processes. For example, our data indicate that FGF2 is a rather poor chemotactic fac- tor, which is at odds with the reputation of FGF2 as a potent inducer of endothelial cell chemotaxis (Kanda et al., 2000). This might in part explain the lack of an obvious vascular phenotype observed in FGF2 knockout mice (Miller et al., 2000). It is also interesting to note that

31 VEGFA121 is not unable to attract endothelial cells. This suggests that the importance of HS on endothelial cell chemotaxis is not mainly at the signaling level but that HS rather plays a role in the creation and maintenance of molecular gradients in the tissue.

Another obvious advantage of the microfluidic assay over the Boyden chamber is that the cells can be monitored in real time during the course of the experiment. One argument raised against microfluidic devices generally has been that they are hard to use. The chamber used in this study is however quite simple to handle thanks to the vacuum assembly enabling standard cell culture in regular Petri dishes.

Future perspectives Since this study was published in 2008 the 2D chemotaxis assay de- scribed in the paper has already been used at several occasions to in- vestigate different phenomena in endothelial cells, but also in other cell types. It has proved very useful to evaluate the effect of siRNA mediated gene silencing on chemotaxis (Barkefors et al., under revi- sion) and we have also used the assay to study the effect of micro- RNA on migration (Larsson et al., 2009). But although the system has been functional to study attraction, we have not yet been able to vali- date the chemotaxis assay with a repulsive guidance cue. The sub- strate aspect is also very interesting, as observations indicate that the adhesive properties of the cells are crucial for a good migratory re- sponse.

Paper II

Aim The information you can extract from a cell culture in 2D is valuable but limited; for example it is not possible to study the behavior and dynamics of tip and stalk cells or migration in 3D. Several in vitro models exist where it is possible to study the development of vascular structures with close resemblance to angiogenic sprouts (Go and Owen, 2003; Goodwin, 2007; Jakobsson et al., 2007; Staton et al., 2009) and the aim of paper II was thus to create a system where stable gradients could be combined with 3D cell systems.

32 Results In this paper we describe a new assay enabling studies of complex cell structures in presence of controlled molecular gradients. The system can be used to produce stable diffusion based gradients in 3D matri- ces, and the gradients that are formed within hours can be maintained for several days. The sample is cast into the matrix directly in the chamber in its open configuration. By closing the system and connect- ing it to a pump it is possible to apply the gradient at any time during the experiment (Figure 10, top panel). The shape of the gradient de- pends on the affinity between the growth factor and the matrix, but is roughly linear in the central part of the chamber (Figure 10, bottom panel). We use this assay to study directed angiogenesis in mouse em- bryonic kidneys and embryoid bodies (EBs) that are placed in a colla- gen I matrix and stimulated with VEGFA gradients. In both models directed sprouting is detected towards increasing levels of growth fac- tor.

Figure 10. The 3D migration chamber. (Top) Schematic outline of how the system is assembled and used. The sample (1.) and the matrix (2.) are placed in the open chamber and incubated for several days if necessary. The system is then closed and the gradient is applied by the introduction of flow. The samples can be monitored with an inverted microscope and is easily recov- ered when the experiment is finished. (Bottom) Example of gradient.

Discussion The effect of gradients on more complex vascular structures is intrigu- ing; it is fascinating that the vasculature of an embryonic kidney can be reprogrammed by only an external VEGF gradient. The results pre-

33 sented in this paper are to be considered as proof of concept, but still provide some useful information about the model systems. The EB is a convenient in vitro model of angiogenesis, but in this assay it proved to be unreliable as the onset of sprouting was hard to predict. The kid- ney on the other hand was more robust. The 3D gradient system is a good model to study initiation of sprouting and maybe also vessel remodeling. It is probably most useful as a way of detecting specific defects in vascular patterning in knock-out or knock-in animal models.

Future perspectives When we had finished this study and the proof of concept phase of the 3D migration chamber, it was obvious that some optimization needed to be performed, especially regarding “user-friendliness” and the bub- bles that tend to form in the channels over time. Several of these prob- lems have already been solved by the development of a polystyrene capsule (Gradientech), which reduces the bubble formation to a mini- mum and also makes the system easier to handle. However, some challenges still remain concerning e.g. matrix degradation and inter- pretation and quantification of the data produced in the chamber. It would also be beneficial to have systems where several chambers could be studied simultaneously as well as chambers where different gradient shapes could be produced. For example it has been shown using the 2D migration system that an exponential gradient is more potent than a linear in directing endothelial cells. When these prob- lems have been sorted out the system should be very useful to study both vascular and axon guidance.

Paper III

Aim A lot is known about the mechanisms of angiogenesis, but the field is continuously growing as is the list of molecules implicated in the process. The original aim of this project was to identify new players involved in angiogenic sprouting but over time the project has come to involve characterization of the protein EXOC3l2 in endothelial cells.

34 Results Using the microarray technique on aggregates of differentiating mouse embryonic stem cells we are able to identify the gene exoc3l2 as po- tentially involved in angiogenesis and endothelial cell migration. The gene fingerprint of the invasive cells in a VEGFA stimulated EB cul- ture is compared to the stationary cells as well as to the invasive cells in the un-stimulated control. Genes already implicated in angiogenesis such as Tie1, PECAM, Cxcr4, VWF, CD43, NOTCH4&1, Dll4, VEGFR1 and VEGFR2 are all upregulated in the invasive cell fraction of the VEGFA stimulated EBs, which lends credibility to the array as a method to predict genes involved in vessel formation. The previ- ously rather unknown gene exoc3l2 is also upregulated several times, which is why we decided to continue exploring the expression pattern and function of this gene. In this study we use co-immunoprecipitation of a Myc-tagged EXOC3l2 fusion protein to show that EXOC3l2 is associated with the exocyst complex, an important regulator of e.g. cell polarity. The expression of exoc3l2 is upregulated in the endothe- lial cell population of both VEGFA stimulated EBs and different mouse organs. However, in endothelial cells, exoc3l2 mRNA levels are comparable to the levels in for example fibroblasts. Growing the endothelial cells on collagen gel in presence of VEGFA drastically increased exoc3l2 expression, a response not seen in the fibroblasts. We also show that downregulation of the exoc3l2 mRNA results in impaired VEGFR2 phosphorylation as well as loss of directionality in response to a VEGFA gradient.

Discussion Exploring a new gene is as frustrating as it is exciting and lack of veri- fied antibodies and other reagents is a constant concern. The exocyst has not previously been described in the context of angiogenesis, but the connection is not unexpected. The role of the exocyst as a hub in the vesicle transport machinery makes it important for polarity, tran- scytosis and receptor recycling (He and Guo, 2009), all important as- pects of angiogenesis and vascular function. The recent finding that differently composed exocyst complexes may execute diverse func- tions (Andersen and Yeaman, 2010) opens up for the idea of special- ized exocyst complex components regulating e.g. receptor trafficking. If different exocyst complex components are responsible for transport of different tyrosine kinase receptors, this might partly explain the specialized recycling pathways observed for VEGFR2 (Gampel et al.,

35 2006). The finding that EXOC3l2 is expressed continuously in the endothelial cell layer of mature vessels might suggest a function in apical basal polarity or permeability, but this has to be considered as wild speculations at this stage. Although there seems to be a connec- tion between exoc3l2 and VEGFA/VEGFR2 interaction, it is not unlikely that EXOC3l2 also exerts functions completely unrelated to the vasculature.

An unexpected challenge that we encounterd during this project was to determine the protein size. Although both antibodies recognized the protein when it was introduced in the cells by transfection of the open reading frame (ORF), they did not detect a protein of that same size in the endogenous proteome. Instead, the antibodies detected what seemed to be two distinct proteins, and a good guess at this point seemed to be that these represented unspecific interactions. However, the concentration of both proteins was reduced upon siRNA silencing of exoc3l2 leaving degradation products or splice variants as possible explanations. The observation that the smaller variant of EXOC3l2 was only detected in endothelial cells makes a vascular specific splice variant an intriguing possibility. In any circumstances the EXOC3l2 protein is a lot smaller than the other proteins of the exocyst complex including the close relative EXOC3. In fact EXOC3l2 seems to com- pletely lack the N-terminal equivalent of EXOC3 which might give a clue to the functionality of this protein (Sivaram et al., 2006). For ex- ample, is has been shown that several protein-protein interactions are abolished if the N-terminus is removed from the EXOC3 protein.

Future perspectives For future studies of EXOC3l2 it is important that the protein be char- acterized in terms of structure and size e.g. by mass spectrometry analysis of the proteins recognized by the antibodies. If EXOC3l2 exists in different splice forms, it would be interesting to investigate the expression of these variants in different cell types and conditions. If the endogenous composition(s) of the protein(s) were known, it would also be possible to create a fluorescently labeled protein for time-lapse imaging of vesicle transport. The idea of different exocyst complexes with specific functions is exciting, and a first step towards elucidating the specificity of EXOC3l2 would be to investigate the effect of exoc3l2 knockdown on the phosphorylation of other surface receptors.

36 Concluding remarks

In the introduction I tried to summarize some of the theory describing angiogenesis and cell migration. There are a billion more things to be said about this, but I hope that I have at least been able to put my work in a context. When reviewing vascular sprouting, I have put some ex- tra effort into the description of receptor trafficking due to my work on EXOC3l2, the “new” member in the exocyst complex. I can’t re- frain from reflecting on the fact that two years ago I didn’t even know there was something called the exocyst. It is fascinating how chance influences the course of things, and I am really curious to see what the future will bring in terms of scientific curiosities.

37 Sammanfattning på svenska

För att fungera normalt måste alla kroppens celler försörjas kontinuer- ligt med syre och näring samtidigt som de behöver ett sätt att göra sig av med avfall och föroreningar. För att tillgodose dessa behov är hela kroppen täckt av ett finmaskigt nät av blod- och lymfkärl som alla är specialicerade för ett visst ändamål. Blodkärl är uppbyggda av ett inre cellager, endotelcellerna, som är i direkt kontakt med blodet. Endotel- cellerna är täckta av olika typer av stödceller som stabiliserar kärlen och hjälper till att reglera blodtrycket. Kärlens funktion och tillväxt är noga reglerad, och normalt växer kärlen mycket långsamt. Nybildning av kärl (angiogenes) sker väldigt sällan i vuxna individer, men ibland förlorar kroppen kontrollen vilket kan få förödande konsekvenser. Tumörer, till exempel, behöver blodkärl för att kunna leva och växa och använder kärlsystemet för att sprida sina celler till andra delar av kroppen, så kallad metastasering. En del av problematiken i sjukdoms- tillsånd som psoriasis, reumatism och diabetes beror på att kroppen förlorat kontrollen över kärltillväxten. Patologisk angiogenes kan ock- så orsaka makuladegeneration vilket innebär att gula fläcken förstörs och således medför synbortfall. Men vad är det då som orsakar dessa systemkrascher och hur kan man normalisera nybildningen av kärl? Vi vet redan ganska mycket om vad som får kärltillväxt att löpa amok och anti-angiogenesbehandlingar är redan en etablerad del av exem- pelvis cancerterapin. Ändå har vi långt kvar tills vi förstår alla sam- band och varje liten pusselbit tar oss ett litet steg närmare bättre och effektivare behandlingsmetoder.

En viktig del i regleringen av angiogenes är tillväxtfaktorn VEGF (va- scular endothelial growth factor). När celler upplever syrebrist använ- der de VEGF för att signalera detta och kärlsystemet svarar med att producera nya kärl. Endotelcellerna i de nybildade kärlen känner av i vilken riktning som koncentrationen av VEGF ökar mest och kan på så sätt växa i riktning mot den syrefattiga vävnaden. För att känna av dessa VEGF-gradienter har endotelcellerna utvecklat ett komplicerat

38 signalsystem som gör att de kan detektera även mycket små koncent- rationsskillnader.

I arbete I har vi undersökt hur olika typer av VEGF-gradienter påver- kar migrationen hos enstaka endotelceller som isolerats från mänskli- ga blodkärl. Endotelceller migrerar väldigt långsamt, så för att kunna studera deras beteende måste vi producera en VEGF-gradient som är stabil under flera timmar. I en statisk kultur är detta nästan omöjligt, men genom att använda en migrationskammare där vi kontinuerligt pumpar vätska genom kulturen kan vi upprätthålla gradienten tillräck- ligt länge. Med den här metoden visar vi bland annat att en exponenti- ell gradient är bättre än en linjär när det gäller att dirigera endotelcel- ler till rätt ställe. Vi kan också se att VEGF är mycket mer potent än FGF, en annan molekyl inblandad i kärltillväxt.

Trots att metoden med enstaka endotelceller är väldigt informativ så finns det saker som den inte klarar av. Eftersom cellerna migrerar på en tvådimensionell yta kan man till exempel inte studera hur ett helt blodkärl beter sej i en gradient. För att kunna studera även dessa fe- nomen har vi utvecklat en tredimensionell migrationskammare som vi beskriver i arbete II. Vi tittar bland annat på hur en njure från ett mus- embryo utveckals om man introducerar en extern gradient av VEGF.

Vår kunskap om angiogenes växer hela tiden. Genom att studera gen- uttrycket i nybildade blodkärl i en kultur av stamceller har vi försökt identifiera nya gener som skulle kunna vara inblandade i kärltillväxt. En av generna som visade upp en inressant uttrycksprofil var den tidi- gare relativt okända genen exoc3l2 (exocyst complex component 3 like 2) och vi bestämde oss därför att studera denna gen noggrannare. Vi fann bland annat att exoc3l2 verkligen har en roll i att reglera hur endotelceller migrerar i en VEGF-gradient, vilket vi beskriver i papper III.

39 Acknowledgements

This work was carried out at the department of medical biochemistry and microbiology at BMC, and partly at the department of genetics and pathology at the Rudbeck laboratory. Many people have sup- ported me in different ways during this work and I would like to ex- press my warmest gratitude to you all. Especially, I would like to thank the following people.

My supervisor Johan Kreuger. I am really grateful for the opportunity to work with research. You have given me an interesting perspective and been extraordinarily patient over the years.

My co-supervisor Lena Kjellén. You have such a wonderful attitude both scientifically and socially and it was a pleasure to organize CPID with you. Thank you for all the clever advice.

My co-supervisor Pär Gerwins. It has been a couple of beers over the years and I really appreciate your creativity and enthusiasm.

My examinator Staffan Johansson. You know so much and you al- ways seem to be truly interested.

My opponent Ferdinand Le Noble. I am really glad that you have taken the time to scrutinize my work and to travel all the way to Swe- den.

The Kreuger group, past and present. Ed, Sébastien, Zsolt, Katarina and Johan Heldin. Special thanks to Katarina for taking the time to introduce me to zebrafish and for struggling with the EXOC3l2 MOs.

The Gerwins group, past and present. Witek, you are demented but I love you for it, Peder, thanks for all the times you have rescued my experiments and for having solutions to most problems, and Ludde, our personal dictionary.

40 Lena Cleasson Welsh. I could never reach your level of knowledge and efficiency, but I am really happy to have worked in your lab, if only for a while. Science is lucky to have you.

Colleges at Rudbeck, past and present. Lars, Pacho, Dan, Sofie, Anna, Lothar and Sonia. A special thanks to Jakobsson et al. for making me look extremely stupid on your dissertation party and to Pacho for good trekking-advice and for a pleasant midsummer in the archipelago.

Noo Li Jeon and Jean Wong Park. Though it is now a long time since your visit in the lab, your influence on my work is still substantial.

Sara Thorslund and Fredirk Nikolajef. Without you I would still be lost in the clean-room.

My student Anja. I really admire your dedication and how you take everything seriously.

The D9:4 crowd past and present. What would science be without a good laugh from time to time? I’ll never forget our lunch conversa- tions where no topic was too weird. Anna and Elina, you are just lovely and never boring, Christoffer, sorry…. Dr. Taaaaaaamm, thanks for mutual exchange of reagents and Ahn-Tri for skeonsk veis- dom.

All other IMBIMers past and present. A special thanks to Kathrin, you are a very kind person and an excellent source of secondary antibod- ies.

Åke Engström, you are a hero.

The admin people, Erika, Marianne and Rehné. Thanks also to Kerstin for putting up with my PCs.

Karin Holmström. You were there when things got tricky and I won’t forget it.

Annea, my beloved sister and friend. I hope I can be a support for you too during your PhD studies.

My dear college/friend;______. Thank you for everything!

41 Min familj. Ni finns alltid där för mig och jag skulle inte ha klarat mig utan er kärlek och ert stöd.

Morfar, mormor och farmor. Ni är bäst!

Familjen Orméus-Carlson och framför allt Gustav, min underbare äls- kade, medförfattare och bäste vän. Du är mitt allt!

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