Aus der Universitäts - Augenklinik der Albert - Ludwigs - Universität Freiburg im Breisgau

ROLE OF

EphrinB2 AND EphB4

IN MOUSE RETINAL ANGIOGENESIS

I N A U G U R A L - D I S S E R T A T I O N

zur Erlangung des Medizinischen Doktrogrades

der Medizinischen Fakultät der Albert-Ludwigs-University Freiburg im Breisgau

vorgelegt 2003

von Eleni G. Gogaki geboren in Nicosia / Zypern 2

Dekan: Prof. Dr. rer. nat. M. Schumacher 1. Gutachter: Prof. Dr. med. L. L. Hansen 2. Gutachter: Prof. Dr. med. vet. H. Augustin, PhD Jahr der Promotion: 2003 3

CONTENTS

1. INTRODUCTION 5 1.1. Preface 5 1.2. Blood vessel formation 6 1.3. Physiological vascularisation of the retina 10 1.4. Pathological vascularisation of the retina 11 1.5. tyrosine kinases 14 1.5.1. Characteristics 14 1.5.2. Signalling 14 1.5.3. Families of RTKs 16 1.6. Eph / family 17 1.6.1. Introduction 17 1.6.2. History 17 1.6.3. Ligands 18 1.6.4. Receptors 19 1.6.5. Numbering 20 1.6.6. Bidirectional Signalling 21 1.6.7. Functions 23 1.6.8. Role in Angiogenesis: importance of EphrinB2 and EphB4 25

2. OBJECTIVE 27

3. MATERIALS AND METHODS 28 3.1. Animals 28 3.2. Reconstruction of EphB4 and ephrinB2 28 3.3. Oxygen Induced Retinopathy (O.I.R.) Mouse Model 29 3.4. Anaesthesia 31 3.5. Intraocular injection 31 3.6. Perfusion 33 3.7. Retinal whole mounts 35 3.8. Microscopy 36 3.9. Evaluation 36 3.10. RNA isolation 38 4

3.11. RT-PCR 38 3.12. Gel electrophoresis 40 3.13. Documentation 40 3.14. Statistical analysis 40

4. RESULTS 41 4.1. Animals 41 4.2. PCR procedure 43 4.3. Retinal whole mounts: control and experimental 45 4.4. Normoxic conditions 48 4.4.1. EphB4 injection during physiological retinal vascularization 48 4.4.2. ephrinB2 injection during physiological retinal vascularization 50 4.5. Relative Hypoxic conditions 51 4.5.1. Injection of dimeric EphB4 using the OIR model 51 4.5.2. Injection of dimeric ephrinB2 using the OIR model 53

5. DISCUSSION 55 5.1. Mouse model of oxygen induced retinopathy 55 5.2. Impact of EphB4 and ephrinB2 on retinal angiogenesis in the OIR model 61 5.3. The roles of ephrinB2 and EphB4 in vascular remodelling 63 5.4. Therapeutic consequences, questions and perspectives 68

6. SYNOPSIS 69

7. ZUSAMMENFASSUNG 70

8. ABBREVIATIONS 71

9. REFERENCES 72

10. ACKNOWLEDGEMENTS 82 5

1. INTRODUCTION

1.1. PREFACE

Retinal neovascularization is a widespread damaging process, involved in a number of major eye diseases and causes of blindness. These include diabetic retinopathy, retinopathy of prematurity (ROP), central retinal vein occlusion (CRVO), and age related macular degeneration. Possible complications are vitreal bleeding, retinal detachment and/or secondary glaucoma followed by severe loss of visual function [46]. The current treatment of many forms of ocular neovascularization involves laser photocoagulation or cryotherapy. Although these have been shown to be therapeutic in the majority of cases, the treatment involves irreversible destruction of neuronal tissue. A certain number of treated eyes will also show recurrence of neovascularization. It is unclear whether additional photocoagulation is beneficial, as harmful effects begin to outweigh the benefits. This raises the issue that non destructive interventions are needed to limit the damaging effects of retinal neovascularization and to ultimately prevent the development of these new vessels. In this context emerges the need for new therapeutic approaches, that would forestall the neovascularization process early during pathogenesis.

Understanding the molecular mechanisms of neovascularization in the eye would provide an additional model for understanding the process of angiogenesis in general. Neovascularization in the eye can be easily detected through ophthalmologic examinations and it reveals principles of angiogenesis that can be applied to other organs. Novel therapeutic regimens are required to prevent neovascularization, to avoid serious complications and improve the outcome of the patients, thus effectively reducing costs of neovascular eye disease. In summary, learning how to control retinal neovascularization offers the potential to achieve control over many of the major causes of visual loss and blindness today. 6

1.2. Blood vessel formation

During embryogenesis, endothelial cells differentiate from mesodermal blood islands and proliferate rapidly to form new blood vessels [71]. The vascular system develops through the mechanisms of vasculogenesis and angiogenesis. In vasculogenesis, blood vessels develop de novo from differentiating endothelial cells in situ, whereas in angiogenesis, capillaries originate from pre-existing vessels. Vasculogenesis ceases after early development, and endothelial cell proliferation nearly ceases in adults [46].

During vasculogenesis, vascular endothelial precursor cells undergo expansion differentiation, and coalescence to form a network of primitive tubules [71]. This initial lattice, consisting purely of endothelial cells that have formed rather homogenously sized interconnected vessels, has been referred to as the primary capillary plexus. The primary plexus is then remodelled by a process referred to as angiogenesis [71], which involves the sprouting, branching, and differential growth of blood vessels to form the mature vascular patterns seen in the adult organism. This latter phase of vascular development also involves the sprouting and penetration of vessels into previously avascular regions of the embryo, and also the differential recruitment of associated supporting cells, such as smooth muscle cells and pericytes, as well as fibroblasts, to different segments of the growing vasculature [22], [49], (Figure 1).

The adult vascular network is comprised of large arteries, internally lined by endothelial cells and well ensheathed by smooth muscle cells, that progressively branch into smaller and smaller vessels, terminating in precapillary arterioles that give rise to capillaries. Capillaries are comprised almost entirely of endothelial cells and are coated by smooth muscle cell-like pericytes. Capillaries then feed into postcapillary venules that progressively associate into venous structures. Venous structures are fully enveloped by smooth muscle cells, though not to the same degree as arterial structures. 7

Fig. 1: Processes involved in the development of the embryonic vasculature (Adapted from I. Zachary, in: Angiogenesis as a Therapeutic Target, UCL, London).

The need to regulate the multitude of cellular interactions involved during vascular development suggested that there should be a number of growth factors that specifically act on the vascular endothelium. Some well known growth factors include the VEGF (vascular endothelium ) family, the family and the most recently identified, members of the ephrin family, all having unique influence on endothelial and perivascular cell function. 8

However, highly regulated angiogenesis does not occur normally in adults and is responsible only for physiologic functions, such as wound healing, ovulation, and placental maturation [23]. When unregulated, endothelial cells can cycle and divide abnormally to cause and contribute to pathologic states, such as tumor growth and eye disease [72]. In the eye this process is referred to as ocular neovascularization (Tab. 2.b).

The resulting declination from the physiological regulation of angiogenesis can be explained through 'the `angiogenic switch hypothesis' that is based on the fact that endothelial cell turnover in the healthy adult organism is very low and the maintenance of endothelial quiescence is thought to be due to the presence of endogenous negative regulators, because positive regulators are frequently detected in adult tissues in which there is apparently no angiogenesis. This has led to the notion of the angiogenic switch, in which endothelial activation status is determined by a balance between positive and negative regulators: in activated (angiogenic) endothelium, positive regulators predominate, whereas endothelial quiescence is maintained by the dominance of negative regulators [52], (Figure 2, Table 1).

The switch:

On

Off

Activators Inhibitors

Fig. 2: The changes in the balance between angiogenic activators and inhibitors can trigger or stop angiogenic phenomena. 9

Tab. 1: Activators and inhibitors of angiogenesis (modified from Folkman et al. (1995) [21] and Klagsbrun et al. (1991) [41]).

Activators Inhibitors

Vascular endothelial growth factor Angiostatin (VEGF) Platelet factor IV (PF IV) Fibrobast growth factor (FGF) Tissue inhibitor of metalloproteinases Transforming growth factor (TGFα, (TIMP 1,2,3) TGFβ) Prolactin Tumor necrosis factor (TNFα) Interferon α, γ Platelet derived growth factor (PDGF) Thrombospondine 1,2,3 Angiogenin Corticosteroids -8 (IL-8) Granulocyte colony stimulating factor (G-CSF) Cell adhesion molecule (CAM) E-Selectin

Prostaglandins (PGE1, PGE2) 10

1.3. Physiological vascularization of the retina

The retina is embryologically an extension of the diencephalon and the retinal vasculature develops in the human between the 14th and the 38th week of gestation. The retinal vasculature consists of inner and outer layers that are joined by fine capillaries. Initially, spindle-shaped cells are apparently migrating ahead of the developing inner vasculature [36].

In the retina, both angiogenesis and vasculogenesis are reported to contribute in vascularization [36], [37], [12]. The retinal vasculature is a good model system for studying the development of blood vessels in general, because its vasculature is nearly restricted to two dimensions. It simplifies the study of a vascular plexus in its entirety. In addition, development of the retinal vasculature is important in the context of retinopathies, in which abnormal vessel growth in the retina can ultimately lead to blindness [24].

In the developing human retina, the first vessels originate at the optic nerve head and spread over the inner surface of the retina, forming a dense network [36]. A network of astrocytes that also spreads from the optic nerve head precedes these vessels [83], [51]. Initially, retinal vessels seem to follow this network of retinal astrocytes

[37]. After the vascular network has spread across the entire retina, vessels start to sprout downward, into the inner plexiform layer, where they establish a second vascular network parallel to the first [84], [15]. The second vascular network is not associated with retinal astrocytes. It is widely regarded that the primary vascular development across the inner surface of the retina occurs by vasculogenesis, whereas the establishment of the secondary network in the inner plexiform layer occurs by angiogenesis. Evidence for the occurrence of vasculogenesis during the primary vascularization of the retina is based on identification of angioblasts spreading across the retina before the appearance of endothelial cells. A population of spindle-shaped cells spreading across the retina appears, before the primary vascular network and even before retinal astrocytes are detectable [12]. It is assumed that these spindle cells are angioblasts [42], [24]. 11

1.4. Pathological vascularization of the retina

Neovascularization in the developed eye almost always impairs function. Schultze has stated that "neovascularization which accompanies some ocular diseases often appears to be misguided in its purpose and may ultimately lead to blindness" [75]. These new vessels may grow within nearly all mature ocular tissue and affect the cornea, iris, retina and optic disk. Although no single factor can explain all causes of ocular neovascularization, multiple contributing factors have been implicated, such as inflammation and its molecular mediators, tumor angiogenic factors, and most important various hypoxia induced factors, like the VEGF family [46]. The new vessels that form are structurally weak, both leaking fluid and lacking structural integrity. The resultant haemorrhage, exudates and accompanying fibrosis often cause visual impairment (Figure 3). Diseases affecting the retina comprise a majority of the causes of severe loss of vision in developed countries. Diabetes mellitus for example, the main contributor to this group of diseases, is the leading cause of blindness in working-age today, and accounts for at least 12% of the new cases of blindness each year in the USA, blinding more than 8.000 people annually [46]. Neovascularization of the retina is a critical phase of the disease process not only in diabetes, but also in conditions such as retinopathy of prematurity (ROP) and retinopathy associated with occlusion of retinal vessels.

Fig. 3: Ocular neovascularization and complications. 1. Diabetic retinopathy with hard exudations and microaneurisms. 2. Neovascularization of the retina. 3. Retinal detachment. 12

Retinal neovascularization involves the growth of new capillaries from the vessels that arise from the optic disk or inner retina. Their origin is usually from the venules, but they also may arise from arterioles. At the beginning, the fragile new capillaries lie in the plane of the retina, but they may extend into the vitreous and rupture, resulting in vitreous haemorrhage and loss of vision. The greatest threats to vision are scarring, tractional detachment of the retina, and haemorrhage. New vessels may be asymptomatic until complications such as tractional detachment and haemorrhage of the retina develop [3]. (Figure 3) A list of diseases involving retinal neovascularization and ocular neovascularization in general are shown in table 2.a and 2.b respectively.

Tab. 2.a: Diseases associated with retinal neovascularization

Diabetes mellitus* Central retinal vein occlusion* Branch retinal vein occlusion* Age-related macular degeneration Retinopathy of prematurity* Sickle cell disease* Retinal detachment Systemic lupus erythematosus Eales disease Multiple sclerosis Distal large artery occlusion Coats disease Tumors

*Diseases most frequently associated with hypoxia associated retinal neovascularization. Modified from Henkind [32]. 13

Tab. 2.b: Diseases causing ocular neovascularization. Adapted from Lee (1998) [46]

Ocular neovascularization

Disease Frequent Occasional Rare Diabetes mellitus Central vein occlusion Retinopathy of Carotis- Branch vein occlusion prematurity Ischaemic cavernosus Fistula Ischaemic PHPV ophthalmopathy Sickle cell retinopathy Amotio Retinae Retinoblastoma Neoplastic Metastases Lymphoma Choroidea melanoma Behçet s. Eales d. Lupus Endophthalmitis Inflammatory / Sympathetic erythematosus Infectious Ophthalmia Uveitis Giant cell arteriitis Takayasu-aortitis Syphilis

Iatrogenic Radiation retinopathy „String“ s. Leber miliary aneurysm Idiopathic / AMD von Hippel-Lindau s. Coats s. Genetic Juvenile Retinoshisis

PHPV - persistent hypertrophic primary vitreous,s. - syndrom, d. - disease 14

1.5. RECEPTOR TYROSINE KINASES

Key signals regulating developmental cell growth and differentiation, as well as remodelling and regeneration of adult tissues, are mediated by polypeptide growth factors for and their transmembrane receptors, many of which include tyrosine kinases (RTKs) [55], [89]. Several families of receptor tyrosine kinases are characterized and some of them are endothelial cell-specific (Figure 4).

1.5.1. Characteristics Growth factor receptors with protein activity have a similar molecular topology. They consist of a large glycosylated extracellular domain that defines the receptor binding characteristics. They are anchored to the plasma membrane with their hydrophobic transmembrane region. The intracellular juxtamembrane domain carries RTK to modulate receptor functions by e.g. protein phosphorylation. The protein tyrosine kinase domain is the catalytic domain of the receptor, and indispensable for and induction of cellular responses. Lastly, they contain the carboxy-terminal tail which interacts with the substrate binding sites of the protein tyrosine kinase region, and modulates the capacity of the tyrosine kinase (TK) region to interact with exogenous substrates [88].

1.5.2. Signalling Polypeptide ligands commonly known as growth factors or cytokines activate RTKs. Signalling involves ligand binding, which induces a conformational change in the external domain of the receptor resulting in its dimerization [88]. This event results in receptor trans-phosphorylation at specific tyrosine residues and activation of the catalytic domains for the phosphorylation of cytoplasmic substrates. These phosphorylated tyrosine residues may serve to control the kinase activity of the receptor, and to create docking sites for the cytoplasmic signalling molecules, which are often substrates for the kinase. These molecules are adapters or themselves, linking RTKs to different signalling pathways. The interaction of these proteins with the activated RTKs can initiate signalling pathways leading to the nucleus or other cellular targets [31]. Following ligand binding and dimerization, 15 receptors are internalised for degradation or recycling in order to attenuate signalling [11]. RTKs typically exist as inactive monomers in the plasma membrane in the absence of ligand. Ligand binding crosslinks such monomers to form dimers or heterodimers and allows each kinase domain to phosphorylate the other, activating both kinases and possibly then proceeding with phosphorylation of other proteins. In many cases (e.g. the epidermal ), the kinases phosphorylate their partners in the activation loop thus increasing their activity. Most of the ligands, such as VEGF or the are dimers, thus allowing the receptors to dimerize. This is a very simple method allowing the ligand to generate a signal inside the cell. Tyrosine kinases autophosphorylate at a variety of sites, generally in regions surrounding the kinase domain or on inserts in the domain. The formation of these various protein complexes generally brings the into contact with its substrate (e.g. ATP). The autophosphorylated receptor can also go on to phosphorylate other proteins. Phosphorylation on tyrosine residues may affect the activity of an enzyme. It may also allow the phosphorylated protein to bind to other proteins by means of SH2 domain interaction.

Fig. 4: Three families of factors and receptors involved in blood vessel formation and maturation. (Modified form Gale and Yancopoulos (1999), [28]). 16

1.5.3. Families of Most of these growth factor receptors are single membrane spanning molecules with a tyrosine kinase in the cytoplasm. They typically have some combination of IgG, fibronectin type III or EGF repeats in the extracellular domain. There are several distinct families or receptor tyrosine kinases, each of which generally has related ligands, similar catalytic and similar extracellular domains. For example all of the related molecules (NGF, BDNF, NT3 and NT4/5) bind to the related Trk receptors (TrkA, TrkB and TrkC). Presumably each family of ligands and receptors arose from one ligand/one receptor pair which then went through cycles of gene duplication.

1. Vascular endothelial derived growth factor family (VEGF), includes platelet derived growth factor (PDGF) family, colony stimulating factor-1, (CSF-1), and others. 2. / Tie receptor family, found almost exclusively in endothelial cells and early haemopoietic cells and required for the normal development of vascular structures during embryogenesis. 3. (FGF) receptor family, bind the FGF growth factors, which are involved among many other processes, in angiogenesis and mesoderm induction in embryogenesis. 4. receptor family, including the insulin and insulin like growth factor-1. 5. (EGF), oncogenic when overexpressed, also found as transforming in tumor viruses under various names like Neu, erbB. 6. Trk receptor family including trkA, trkB and trkC. These are receptors for neurotrophins, a potent family of neuronal growth factors including NGF, BDNF, NT3, NT 4/5. 7. family (HGF). 8. family (EPH) is the largest family with more than 20 members. These are strongly expressed in the . Their ligands are the , which are membrane-anchored. EPH family receptors and their ligands are implicated in mediation of developmental events, regulating cell mixing, cell adhesion and repulsion, particularly in the nervous system, and in angiogenesis. 17

1.6. EPH/ EPHRIN FAMILY

1.6.1. Introduction

The largest subfamily of receptor protein-tyrosine kinases consists of receptors related to Eph, a receptor named for its expression in an -Producing Hepatocellular human carcinoma cell line. This subfamily has received considerable attention when receptors and their ligands, ephrins, have been copiously identified in recent years and are implicated in cell-cell interactions involved in the nervous system patterning, including guidance, and in other aspects of development. To date, fourteen distinct receptors of this subfamily and eight distinct ligands have been identified in warm-blooded vertebrates (mammals and birds), with many related proteins identified in cold-blooded vertebrates and in invertebrates.

Because of the rapid pace of discovery of receptors and ligands in various species, many different names have been used to designate them, making it difficult for the general scientific community to follow developments in this field. As a result of extensive discussions initiated by representatives of over 20 laboratories involved in research on the Eph family at the "Molecular Biology of Axon Guidance" workshop held at the EMBL, Heidelberg, September 1996, a proposal was put forth to unify and to systematize the nomenclature for these ligands and receptors, and an Eph Nomenclature Committee was elected to refine the proposal in consultation with the community at large [6].

1.6.2. History

Eph research began in late 1987 after the molecular cloning of the founding member of what is now the largest family of RTKs (Receptor Tyrosine Kinases). This receptor was detected in a screen for v-fps homologous sequences from an Erythropoietin- Producing Hepatocellular carcinoma cell line and was thus termed 'Eph' [34]. This novel receptor became a member of the RTK family when the catalytic portion of a new homologous receptor was identified the following year by using an expression 18 cloning strategy with anti-phosphotyrosine antibodies [47]. This kinase, for which a full length version was later reported [48], was termed Elk, for 'Eph like kinase'. Elk and many Eph family receptors identified thereafter have a rather specific expression patterns in developing and adult nervous system, and this feature has spurred further interest in the family. The second full receptor sequence was reported in 1990, and this receptor was termed Eck, for 'epithelial cell kinase', because of its message abundance in tissues with high proportions of epithelial cells [50]. All of these receptors share unifying features that identify them as Eph family members including a conserved intracellular catalytic domain and, more characteristically, their extracellular ligand-binding regions, which are comprised of an Ig-like motif, a single cysteine-rich region, and 2 fibronectin type III repeats (Fig. 5). In the ensuing 10 years after the cloning of Eph, at least 14 distinct receptors have joined this family mainly from various vertebrate species [27].

1.6.3. Ligands

The ligands are known as ephrins, which can be derived as an abbreviation for "Eph Family Receptor Interacting Proteins" or from the ancient Greek word "ephoros", (epsilon)(phi)(omicron)(rho)(omicron)(sigma) meaning "overseer" or "controller". The uniqueness of the ephrin ligands is that they are membrane-anchored, and this differentiates them from the rest of the ligands because the interactions are taking place at opposing cells that express the receptors and the corresponding ligands on their membranes. The ligands are divided into two structural types, being membrane- anchored either by a glycosylphosphatidylinositol (GPI) linkage or through a transmembrane domain. In addition, these two subgroups of ligands can be divided on the basis of their sequence relationships, and functionally on the basis of their preferential binding to two corresponding receptor subgroups, as described below. For this reason, the ligands are divided into the ephrin-A subclass, which are GPI- linked proteins, and the ephrin-B subclass, which are transmembrane proteins. The locus designations for human gene map positions for these two subclasses are EFNA and EFNB (previously designated EPLG), (Figure 5). 19

1.6.4. Receptors

The receptors be known as Eph receptors. The Eph family receptors can be divided into two groups based on the similarity of their extracellular domain sequences (Figure 5). This grouping also appears to correspond to the binding affinity for the ephrin-A or ephrin-B proteins. The group that includes receptors interacting preferentially with ephrin-A proteins be called EphA and the group that includes receptors interacting preferentially with ephrin-B proteins be called EphB. Human gene map locus names are EPHA and EPHB, respectively.

Fig. 5: General features of Eph receptors and ephrins. A schematic diagram, which shows an ephrin-expressing cell (top) interacting with Eph- expressing cell (bottom). Ligand-receptor interactions (green) have been mapped in detail and have ucovered dimeric and oligomeric Eph-ephrin complexes (not shown). GPI, glycosylphosphatidylinositol; SAM, sterile α-motif. Adapted from Kullander et al. (2002) [44]. 20

1.6.5. Numbering An arabic numeral designates individual receptors and ligands within each subclass, which is assigned, based on date of publication of an essentially full-length sequence. These assignments are based on sequences obtained in various warm- blooded vertebrate species, because of the observation of high sequence conservation across these species that allows unambiguous assignment of orthologs (homologs diverging by speciation rather than by gene duplication). (Figure 6)

Fig. 6: Members of the ephrin family. Ligands of A and B class and their receptors. The possible binding groups are shown. In general ligands of class A bind to receptors of class A and ligands of class B bind to receptors of class B with the exception of EphA4 that can bind members of both classes. (Adapted from Nature Reviews/Neuroscience) 21

1.6.6. Bidirectional Signalling

1. Mechanisms of Eph-receptor forward signalling: Eph-receptor activation and downstream signalling

In contrast to most other RTK receptors that are activated upon binding to ligands found in the extracellular space, Eph receptors are activated when they are bound by membrane-bound ephrin ligands. This requirement for membrane clustering of the ligand indicates that contact between cells that express Eph receptors and cells that express ephrin ligands is needed for Eph-receptor activation. Experiments have shown that membrane clustering can be mimicked by clustering tagged soluble ligands [17]. On ligand engagement, each one part of the receptor dimer autophosphorylates several tyrosine residues located in the intracellular part of the partner receptor [38]. Autophosphorylation of juxtamembrane tyrosine residues is required for full activation of the protein tyrosine kinase domain of the receptor [8], [97], [45]. The reason for this autoregulation became apparent when the X-ray crystal structure of the EphB2 cytoplasmic region was solved [93]. The structure that was used included the entire kinase domain, together with the juxtamembrane domain, but the crucial juxtamembrane tyrosine residues had been mutated to phenylalanine residues to prevent phosphorylation of these residues. The data showed that the represses catalytic activity, because interactions between the unphosphorylated juxtamembrane and kinase domains prevent proper alignment of segments in the kinase domain. But when juxtamembrane tyrosine residues are phosphorylated, the juxtamembrane domain is released from the interaction with the kinase domain, which allows the kinase domain to convert into its active state. This release also allows phosphotyrosine-binding proteins to bind to the phosphorylated juxtamembrane domain [44].

Although it is not yet understood how the binding of ephrin ligand releases the repressed conformation of Eph receptors, these findings implicate the juxtamembrane domain as an important autoregulator of Eph-receptor tyrosine kinase activity. This regulatory mechanism has also been proposed for other RTKs, 22 such as the platelet-derived growth factor (PDGF) receptor β [7] and the TrkB receptor [69]. Once the receptor is activated, adaptor molecules associate with it to transmit signals into the cell. Adaptor proteins are a class of proteins that contain functional protein- interaction domains, such as SRC-homologs and SH3 domains, and often lack intrinsic enzymatic function. They have a crucial role in the formation of protein complexes, and connect signalling molecules within an intracellular signalling cascade [65].

2. Mechanisms of ephrin reverse signalling

The cytoplasmic domain of ephrinB ligands contains five conserved tyrosine residues. Three of these tyrosines - residues 312, 317 and 332 - have subsequently been identified as the main in vivo sites of activated avian ephrinB1 from neural tissue [39]. These residues can be phosphorylated by several mechanisms. Stimulation of primary or endothelial cells with the soluble ectodomain of Eph receptors induces tyrosine phosphorylation of the cytoplasmic domain of endogenous ephrinB [63]. Treatment of embryonic chicken retina with fibroblast growth factor (FGF) leads to phosphorylation of endogenous ephrinB, presumably by the co-expressed FGF receptor [14]. Phosphorylation of the ephrinB cytoplasmic domain is thought to be an important event in ephrinB reverse signalling but, until recently, the identities of the kinase(s) and phosphatase(s) that regulate this process were unknown. Using primary endothelial cells and cortical neurons, it was shown that SFKs (Src-family kinases) are positive regulators of ephrinB phosphorylation and that SFK activity is required for endothelial sprouting in a three-dimensional sprout assay [63]. The existing data indicate the presence of a switch mechanism that allows a shift from phosphotyrosine-dependent signalling to PDZ-domain-dependent signalling [63]. 23

1.6.7. Functions of Eph-receptor signalling

The control of cell movement during development is essential to form and stabilize the spatial organization of tissues and cell types. During initial steps of tissue patterning, distinct regional domains or cell types arise at appropriate locations, and the movement of cells is constrained in order to maintain spatial relationships during growth. In other situations, the guidance of migrating cells or neuronal growth cones to specific destinations underlies the establishment or remodelling of a pattern. Eph receptor tyrosine kinases and their ephrin ligands are key players in controlling these cell movements in many tissues and at multiple stages of patterning.

The ephrins transduce signals on binding to an Eph receptor, such that each component can act both as 'receptor' and 'ligand' in cell-contact-dependent signalling. Eph receptors and ephrins are expressed in complex patterns throughout vertebrate development. Complementary expression of interacting Eph receptors and ephrins can lead to bidirectional activation at the interface of different cell types, whereas overlaps in their expression lead to persistent activation within the expression domain.

In contrast to most other RTK ligands, the ephrins can not induce directly mitogenic responses in target cells, suggesting that they too are involved in other types of biological processes. Ephrins also seem quite unusual in that they are obligate to act in membrane-bound form, restricting ephrin/Eph interactions to sites of direct cell-cell contact [28]. To date they have been most solidly implicated in the process of neural cell guidance. For example, Eph receptors and ligands seemingly regulate axon guidance events that establish the retinotopic map in the tectum [58]. They have also been shown to be involved in the guidance of cells as they navigate through the trunk and branchial regions of the developing embryo [43], [73], [78],

[90]. The Eph family has also been implicated more generally in patterning of the brain and somites, where they are predicted to have roles in regulating cell mixing and establishing boundaries between distinct cellular compartments [94], [95], [19],

[26]. These biological actions of the Eph family can be explained as a result from repulsive interactions between receptor and ligand-bearing cells, or instead a signal 24 that prevents two adjacent cell types from intermixing across a boundary (reciprocally expressing of Eph receptor and a cognate ligand) [27]. Recently, localization of Eph family members to suggests that they may play an important role not only in guiding neuronal processes to their connections, but in continuing to regulate these connections once they have formed [35], [87], [10].

1. Repulsion and adhesion Several components downstream of Eph receptor activation are implicated in pathways that control the local depolymerization of the actin cytoskeleton that underlies repulsion. Eph receptors can also downregulate the function of involved in cell attachment to the extracellular matrix, whereas in other contexts they can upregulate -mediated adhesion. Furthermore, ephrin-A activation upregulates integrin function.

2. Cellular responses Evidence is emerging for the role of Eph receptors and ephrins in regulating other cellular responses, such as communication through gap junctions, cell proliferation and cell death. Eph receptors and ephrins might thus couple the regulation of repulsion and adhesion to other cellular responses involved in patterning of a tissue. In addition, Eph receptors and ephrins localized at synapses might be involved in regulation of synaptic properties, such as plasticity.

3. Segmentation (formation of boundaries) One important role of reciprocal expression of Eph receptors and ephrin-B proteins is in unidirectional or bidirectional repulsion at boundaries, preventing cells or from entering inappropriate territory or "compartments". In the nervous system, this mechanism is involved in stabilizing the organization of hindbrain segments, and in the guidance of migrating neural crest cells and neuronal growth cones. 25

4. Axon guidance - (neuronal projections) A related role is in the establishment of topographic maps of neuronal projection, including the anteroposterior axis of the retinotectal map. This involves graded expression of EphA receptors in retinal neurons, which underlies a graded sensitivity of their growth cones to a gradient of ephrin-mediated repulsion in the tectum/superior colliculus. There is evidence that the degree of repulsion acts to differentially bias retinal axons in a competition for space in the tectum.

1.6.8. Role in angiogenesis: Importance of EphrinB2 and EphB4

Eph-receptors and ephrin ligands carry out essential functions during formation of the vascular system [44]. EphrinB2 is an early marker of arterial endothelial cells, whereas one of its receptors, EphB4, reciprocally labels venous endothelial cells. Mutant animals that lack ephrinB2 [2], [91] or EphB4 [29] show various cardiovascular defects. Angiogenesis in extra-embryonic tissue (yolk sac) and embryos is abolished, which results in a block at the primitive capillary plexus stage (see Fig. 7). Persistent and regulated expression of ephrins and Eph receptors in postnatal stages indicates further functions in therapeutic angiogenesis (wound healing) and pathological (tumour) angiogenesis [57], [25], [59].

The ephrinB2 gene was replaced by the lacZ gene in the study of Wang et al. (1998), thus providing an excellent marker for precisely following the expression of ephrinB2. LacZ expression analysis revealed that ephrinB2 specifically marked arterial endothelial cells at the earliest stages of vascular development [91]. Further examinations of the expression of patterns for EphB receptors, revealed that EphB4 (which only binds to ephrinB2, and has a weaker affinity for ephrinB1), specifically and reciprocally marks only the venous endothelium [91]. Embryos lacking ephrinB2 displayed severe defects in the vascular remodelling, subsequent to the stages of the vasculogenesis, in both arterial and venous parts of the vasculature [28]. The ephrinB2 cytoplasmic domain is required during development of the vasculature. Mice that carry an allele lacking the cytodomain of ephrinB2 (∆C), or that lack ephrinB2 altogether (knockout; KO) cannot undergo the remodelling step of 26 organizing the vascular bed into a vascular system that consists of large vessel and capillaries (fig. 7). As neither veins nor arteries form in ephrinB2 mutants, it has been suggested that bidirectional signalling may take place, in which ephrinB2 transduces a signal required for angiogenesis of arteries, and EphB4 transduces the signal required for the development of veins. The finding that a targeted mutation of EphB4 phenocopies the mutation in ephrinB2 provides further evidence for such bidirectional interactions taking place in vivo [29], [54].

Fig. 7: The ephrinB2 cytoplasmic domain is required during development of the vasculature. The vascular bed (centre) remodels into a hierarchically organized vascular system that consists of large vessel and capillaries (right). Mice that carry an allele lacking the cytodomain of ephrinB2 (∆C), or that lack ephrinB2 altogether (knockout; KO) cannot undergo this remodelling step. (Adapted from

Kullander et al. (2001) [45]) 27

2. OBJECTIVE

Retinal neovascularization is a widespread damaging process, involved in a number of major eye diseases and causes of blindness. The aim of many applied research projects today is to study and manipulate the course of angiogenesis in order to provide measures against disease development and progression. The efforts are divided in two directions, i.e. targeting the stimulation or the inhibition of angiogenesis. We already know that a large number of factors is regulating the inhibition or the promotion of angiogenesis. As already presented in the introduction part, there are two members of the ephrin family of receptor tyrosine kinases (RTKs) that have proved to be involved in the process of angiogenesis. These substances are ephrinB2-ligand and EphB4-receptor, which are selectively expressed on the endothelial cells of arteries or veins, respectively. What differentiates the ephrin family from the other receptor-ligand systems, is that the ligand is bound to the and possesses an intracellular domain, which can as well, transduce a signal upon binding to the receptor.

This study was designed to examine the biological effects of ephrinB2 ligand and EphB4 receptor on the development of newly forming vessels in the retina under physiological and pathological conditions in a mouse model of oxygen induced retinopathy. The purpose of the following experiments is - to verify the presence of ephrinB2 and EphB4 in the developing mouse retina, - to find out whether ephrinB2/EphB4 influence the physiological vascularization of the mouse retina, and - to determine whether ephrinB2/EphB4 influence the retinal vascularization under ischaemic conditions. 28

3. MATERIALS AND METHODS

3.1. ANIMALS

For the following experiments C57 Black/6J mice were used. All animal procedures were approved by the animal welfare committee of the University of Freiburg, according to the animal welfare laws of the Federal Republic of Germany. All young animals were kept together with their nursing mothers, until the end of the experiments. Circadian rhythmus was simulated by alternating luminance every 12 hours, at ambient room temperature. Care was taken to prevent intense noise or vibration. Mice were provided from the animal care facilities of Neurozentrum and Klinik für Tumorbiologie in Freiburg.

3.2. RECONSTRUCTION OF EphB4 AND ephrinB2

EphrinB2-Fc and EphB4-Fc are recombinant proteins fused with the Fc portion of human immunoglobulin G (IgG). To make a functionally active multimerized form of ephrinB2-Fc or EphB4-Fc, 5µg of IgG was used for 500ng of ephrinB2-Fc or EphB4- Fc.

EphB4 (Recombinant mouse EphB4/Fc chimera, cat. No: 446-B4, R&D systems)

The extracellular domain of mouse EphB4 was fused to the carboxy-terminal Fc region of human IgG. The reduced mouse EphB4-Fc monomer has a molecular weight of 85,2kDa. The recombinant EphB4-Fc dimer is a 110kDa protein. A stock solution was prepared after adding 1000µL of phosphor buffer saline solution (PBS) to the initial amount of 200µg of the lyophilized form. The stock solution was divided in 6 aliquots (each containing 167µL of stock solution) and the aliquots were kept at - 20°C. Before each use the stock solution was reconstructed to a final volume of 1000µL after adding 833µL of PBS solution. 29

Final concentration of EphB4: 200µg in 6 x1000µL 1 mol EphB4 = 110 kDa

= 33,33 µg/mL 1M EphB4 = 110 kDa/L = 0,3 µM

ephrinB2 (Recombinant mouse ephrinB2/Fc chimera, cat. No: 496-EB, R&D systems)

The extracellular domain of mouse ephrinB2 was fused to the carboxy-terminal Fc region of human IgG. The reduced mouse ephrinB2-Fc monomer has a molecular weight of 49,6kDa. The recombinant ephrinB2-Fc dimer is approximately a 65kDa protein. A stock solution was prepared after adding 1000µL of phosphor buffer saline solution (PBS) to the initial amount of 200µg of the lyophilized form. The stock solution was divided in 10 aliquots (each containing 100µL of stock solution) and the aliquots were kept at -20°C. Before each use the stock solution was reconstructed to a final volume of 1000µL after adding 900µL of PBS solution.

Final concentration of EphB4: 200µg in 10 x1000µL 1 mol EphB4 = 65 kDa

= 20 µg/mL 1M EphB4 = 65 kDa/L = 0,3 µM

3.3. OXYGEN INDUCED RETINOPATHY MOUSE MODEL

The mouse model of oxygen induced retinopathy (OIR) was introduced by Smith et al. (1994). It is reproducible and suitable for examining pathogenesis and therapeutic intervention for retinal neovascularisation in retinopathy of prematurity (ROP) and other ischaemic vascular diseases [79].

On postnatal day 7 (P7) newborn mice and their nursing mother were placed in an airtight incubator, ventilated by a mixture of oxygen and compressed air to a final oxygen (O2) concentration of 75±2%. The oxygen level was checked at least 3 times 30 daily using an oxygen analyser, (Oxygen Monitor 810, KONTRON INSTRUMENTS, Figure 9) On postnatal day 12 (P12), mice were returned to room air. As a consequence, the retina undergoes a relative hypoxia, which leads to angioproliferation in the immature retina over the next three to five days. On postnatal day 17 (P17), mice were perfused by injection of fluorescein-labelled dextran under general anaesthesia. Mice were then sacrificed and both eyes were enucleated and fixed in formaldehyde. Retinal whole mounts were prepared and their blood vessel patterns were quantified in a double blinded fashion using a scoring system adapted from Higgins et al. [33]. (Figure 8)

Intravitreal injection

Birth O2 Enucleation

Day 1 Day 7 Day 12 Day 17

Fig. 8: Schematic representation of the oxygen induced retinopathy model.

Oxygen

%O2 Press air

Fig. 9: Schematic representation of the oxygen incubator. 31

3.4. ANAESTHESIA Systemic anaesthesia was given by inhalation of 2,5-3,5% of isofluran (ISOFLURAN- BAXTER, BAXTER INTERNATIONAL), mixed with oxygen at a flow of 2l/min using a vaporiser (VAPOR 19.3, DRÄGER LÜBECK, GERMANY) for intraocular injections and transcardial perfusion of the mice. Mice were first anaesthetised in an airtight case for 2 min and then placed on the surgical table under the microscope where they inhaled additional anaesthetic by having their nose placed in a fitting plastic tube. The width of the tube was calculated according to the age of the animals, due to the rapidly increasing size of the nose during the intervals of the experiments. A second larger suction tube surrounding the afflux tube drew excess anaesthetic leaking out of the inhalation tube away.

3.5. INTRAOCULAR INJECTION The right eye received always the substance to be tested and the left eye was used as a control being injected only with the same volume of buffer solution.

Intravitreal injections were performed with glass-pipettes pulled until the diameter of the tip reached approximately 150µm (fig. 11). A novel injection system (Figure.10) was used for delivering 2µl of the solution into the vitreous by connecting a three-way tab with: • the plastic tube of a butterfly cannula at one site. The metallic needle was cut off and the plastic tube was fixed to the micropipette made of glass. • at the second site a 10µl Hamilton Pipette (calibrated for microliter volumes) was connected to deliver the proper volume for intraocular injection. • finally, at the third end of the three-way tab a 2ml syringe containing buffer solution was connected, serving as a reservoir.

Extra care was taken to avoid air bubbles being trapped in the system while it was filled up with the appropriate solution, as air is compressible and its presence would distort the final volume of the injected liquid. 32

1 2 3 5

4

Fig. 10: Three-way system for intraocular injections. 1. Glass micropipette. 2. Plastic tube of a batterfly canulla. 3. Three-way tab. 4. Reservoir 2ml syringe. 5. Hamilton pipette. 6. Collibri tweezers. 7. Small hook.

The lid fissure of the mice being still closed at P12 was opened with a small hook usually used for the reposition of artificial lenses in humans and the eye was proptosed by gentle pressure to the animals neck. To minimise pain caused by contact with the conjunctiva, a drop of local anaesthetic was administered (OPTOCAIN, DR. WINZER PHARMA GMBH OLCHING, GERMANY). The eye was injected with the glass-micropipette at the upper nasal limbus and a volume of 2 µl of the test solution or of the control solution was injected, in the right and left eye, respectively. Since some reflux of intraocular fluid is unavoidable when removing the pipette from the injection site, the glass-pipette was left a few seconds in the vitreous to allow diffusion of the solution before the glass-pipette was slowly withdrawn. The pressure to the neck was then released and the lids were closed over the cornea.

Fig. 11: Comparative image of a glass micro-pipette to a ruler numbering and a match. 33

Fig. 12: 1. Opening the lid fissure. 2. Administration of local anaesthetic. 3. Stabilizing the eye with the tweezers. 4. Intraocular injection with the glass micro-pipette.

3.6. PERFUSION

On postnatal day 17 (P17), mice were firmly fixed on surgical table under light microscope, the abdomen facing up-wards. The procedure was performed under deep general anaesthesia, (see above). Using a VANNAS scissors (STORZ/KLEIN, GERMANY) a small incision was made above the end of the sternum. The skin was then prepared over a large area covering thorax and abdomen and was posed from the front to the side-walls of the abdominal and thoracic cavities. An incision was made parallel to the last ribs cutting through abdominal muscles and the peritoneum. Peritoneal cavity was then opened. With light manipulations the animal's liver was brought downwards, thus making the abdominal surface of the diaphragm accessible. With the rounded blade of the surgical scissors an incision was made cutting through the diaphragm and the front-left side of the thoracic cage. The incision along the left anterior axillar line was then extended to the 4th rib. The diaphragm was dissected along the front ligation with the thoracic cage up to the right anterior axillar line. At this point a similar incision along the right anterior axillar line was made extending along to the 4th rib. 34

Fig. 13: Fluorescein-dextran perfusion after a thoracotomy.

Access in the thoracic cavity is provided, and the animal's heart is released from the peripheral tissues by incision of the ligamentii connecting the heart to the front thoracic walls. A third cut aligned with the two previous was made parasternal left extending up to the 3rd rib from the end. The motile part of the left thoracic cage is excised, bringing the heart to direct access for the next procedures.

The left ventricle was entered with an insulin syringe, which was filled with 1ml of fluorescein-labelled dextran in sodium chloride solution (0,9%) at a concentration of 50mg/ml, (FLUORESCEIN ISOTHIOCYANATE-DEXTRAN, FITC-DEXTRAN, SIGMA, 110K5301, Molecular Weight: 2,5 x106 Da). The solution was injected slowly directly into the left ventricle. As soon as yellow- green spots were observed on the liver surface, the right atrium was tipped with the tweezers so that excessive circulatory volume could escape. After the injection was delivered, the animal was sacrificed and both eyes were enucleated and embedded in 4% buffered formaldehyde, for 2-6 hours, in a dark place (light sensitive), at room temperature. 35

3.7. RETINAL WHOLE MOUNTS

Whole mounts of the retinal tissue were prepared under light microscope (OLYMPUS DE PLANAPO IX, OLYMPUS, light source: SCHOTT MAINZ KL 150 B, SCHOTT). The anterior eye segments, including the iris, were removed with a round cut along the limbus using a VANNAS scissors (STORZ/KLEIN, GERMANY). The lens was removed. The retina was gently separated from the retinal pigmented epithelium and the choroidal-scleral complex. After the connection to the optic nerve was cut, the retinal "hemisphere" tissue was transferred on a microscope slide (SUPERFROST PLUS, MENZLER-GLÄSER, BRAUNSCHWEIG) with the help of two brushes (3 DA VINCI, KOLINSKY MARDER 1510, 975). It was radially incised with 2 horizontal and 2 vertical incisions at 3, 6, 9 and 12 clock hours, starting from the edge towards the optic nerve and cutting 2/3 of the distance from the periphery to the centre, giving the retina a cross-like form. Retinas were flat mounted in glycerine (MICROSCOPIE GLYCERIN, MERCK, GERMANY) with photoreceptors facing downwards. Cover slips were placed over the tissue and sealed with Entellan (MICROSCOPY ENTELLAN, MERCK, GERMANY). For long-term storage preparations were kept in a dark place, at +4°C. Slides could be stored for up to 5 months.

Fig. 14. Representative image of a fluorescein perfused retinal whole- mount under UV light microscopy (1) and a detail of proliferative neovascularization (2). 36

3.8. MICROSCOPY Flat-mounted preparations were examined under fluorescence microscopy, at 490nm, in a dark room. (OLYMPUS BH2-RFCA, OLYMPUS).

3.9. EVALUATION Evaluation of the angioproliferative changes in the retinal preparations was performed in a blinded fashion by two independent observers on coded preparations.

Retinopathy scoring system was adapted from Higgins et al. that was developed after modification of a scoring system based on clinical observations in the neonatal intensive care unit [33]. The following features were taken into consideration: • central vasoconstriction • blood vessel tuft formation • large cluster of blood vessel tufts • tortuosity of the vessels • neovascularisation of the optic disc

Retinal haemorrhages were not taken into account as they may also result from the intraocular injection.

In detail: 1) Optic disc proliferation: Yes/No => 1/0 Point

2) Blood vessel tufts: Every clock hour of the preparation surface was divided in 4 zones. Zone 1 (optic disc zone) was evaluated in addition Zone 2 max 4 P (one for every wing) Zone 3 max 12 P (one for every clock hour) Zone 4 max 12 P (one for every clock hour) * provided no avascular areas exist => max 28 P * complete avascularization => max 12 P 37

3) Large clusters of Every large cluster of blood vessel tufts blood vessel tufts: covering more than 3 sectors counted as one point * provided no avascular areas exist => max 8 P * complete avascularization => max 4 P

4) Central vasoconstriction: >50% of zone 1 => 1 Point >50% of every wing of zone 2 & 3 =>1 Point Max: Zone 1: 1 P Zone 2: 4 P Zone 3: 4 P Zone 4: 4 P 13 P

5) Tortuosity of the vessels: <1/3 of the vessels: 1-2 P between 1/3-2/3: 3-4 P >2/3 of the vessels: 5-6 P

Points of each criterion were summed up; the higher the score, the worse the induced retinopathy.

Comments: 1. The theoretical maximum of points was never reached. For example: Zone 4 never shows avascular zones. 2. Proliferation in avascular zones was never observed. This is in principle a mutual exclusion of criteria; the existence of vascular proliferation has a higher impact on the retinopathy score than large avascular areas (see *). 3. Therefore the maximum score ever counted with this system was 38 points. 38

3.10. RNA ISOLATION

For RT-PCR analysis, total cellular RNA was prepared using the Qiagen RNeasy Mini Kit according to the manufacturer's protocol. All procedures were performed using RNase-free devices and solutions. During tissue isolation the collecting tubes were placed on ice. 21 newborn mice, treated according to the mouse model of OIR, were sacrificed and their eyes were enucleated. Retinal tissue was obtained from the eyes and was put directly into an RNA extraction solution (600µl of the RTL lysis buffer containing 6µL β-Mercaptoethanol, Qiagen RNeasy Mini Kit) immediately after isolation from the eye. Retinal tissue was homogenized using the Qiagen Shredder Kit. After centrifugation at 20°C for 3min at a maximum speed (14.000 rpm, rotor diameter: 16cm), the aqueous phase was collected, and total RNA was precipitated with an equal volume of 70% ethanol at room temperature. The sample was then applied to an RNeasy mini silica column that adsorbs RNA molecules, and centrifuged at 20°C for 15s at 14.000 rpm. Flow through was discarded and 700µL of buffer RW1 was added to the silica column and centrifuged again for 15s at 14.000 rpm. Using a new collecting tube, 500µL of buffer RPE was added to the silica column and centrifuged for 15s. The procedure using buffer RPE was repeated centrifuging for 2min. RNA was then eluted from the silica column by adding 70µL RNase-free water and centrifuging for 1min at maximum speed. The total amount of RNA extracted was measured using a UV-visible spectrophotometer (CARY 50 BIO, VARIAN) at 260nm.

3.11. RT-PCR

A volume corresponding to 2µg of total RNA was reverse transcribed with a cDNA synthesis (INVITROGEN BIOTECHNOLOGIES) at 42°C for 50min in a 40µL reaction volume containing a 12-18 oligo(dT) primer (0,5µg/µL) and 1µL (200U/µL) reverse transcriptase. A 1µL aliquot of the reaction product was subjected to 35 cycles of PCR for amplification of ephrinB2 and EphB4 cDNA. PCR was performed at 35 cycles in a 20µL reaction volume containing 5µM of each primer (table 3), 200µM dNTP, 2µL PCR-buffer with 15mM MgCl2 and 0,5U Hot Star 39

Taq (QIAGEN) in a thermal cycler (MJ RESEARCH, PTC-200, PELTIER THERMAL CYCLER). The thermal cycle was for 15min at 95°C and for 35 cycles: 30s at 94°C; 30s at 55°C and 1min at 72°C. The nucleotide sequences of the PCR primers for ephrinB2 were designed according to the locus NM_010111 from the Gene Bank (for the short fragment of EphrinB2 sense and antisense primer corresponding to nucleotides 497-613 and for the long fragment of EphrinB2 sense and antisense primer corresponding to nucleotides 443-726). In the same way PCR primers for EphB4 were designed according to the locus NM_010144 from the Gene Bank (for the short fragment of EphB4 sense and antisense primer corresponding to nucleotides 2563-2682 and for the long fragment of EphB4 sense and antisense primer corresponding to nucleotides 2522-2803) (Table 3). As positive controls of the primers, total RNA was harvested from mouse liver and RT-PCR was performed in the same manner. As a positive control of the PCR primers for β-actin were used.

Tab. 3: Sequences of the primer pairs for ephrinB2 and EphB4.

Primer Sequence Genebank No. PCR product efnB2-qa cagacaagagccatgaagatcc NM_010111 497 }117bp efnB2-qb tctcccatttgtaccagcttct " 613 efnB2-1 tctacatcaaatgggtctttggag " 443 } 284bp efnB2-2 cctgcgaataaggccacttc " 726

EPHB4-qa tactgggacatgagcaacca NM_010144 2563 }120bp EPHB4-qb gtccttctgccaacagtcca " 2682

EPHB4-1 ggatcgtcatgtgggaggt " 2522 }282bp EPHB4-2 ggtccaagagtggatgtgagg " 2803 40

3.12. GEL ELECTROPHORESIS

An aliquot of the PCR product was electrophoresed in a 2% agarose gel and stained with ethidium bromide. Agarose powder (1,4g) (AGAROSE, ELECTROPHORESIS GRADE, INVITROGEN, UK) was diluted into 70ml TBE buffer solution and heated in a microwave for 4min at 600W. Ethidium bromide (6µl) (ETHIDIUM BROMIDE, SIGMA-ALDRICH CHEMIE GMBH, STEINHEIM, GERMANY) at a concentration of 0,5µg/ml was added to the solution and the gel was left to polymerize at room temperature. The agarose gel was placed into an electrophoresis box (STRATAGENE) filled with TBE buffer solution (1x). 15µl of the amplified cDNA was mixed with 5µl loading buffer solution (PERQLAB DNA LEITER-MIX) and pipetted into the agarose gel pockets. One pocket was used as size standard, filled with DNA ladder buffer (MBI FERMENTAS, GENE RULER, 100bp DNA LADDER). Constant voltage of 100V for the electrophoresis was provided by a power source (POWER PAC 300, BIO RAD) for 1 to 1,5 hours. DNA bands were visualized under UV light (TRANSLUMINATOR 2040EV, STRATAGENE) and photographs were taken with a digital camera (CANON, POWER SHOT G2).

3.13. DOCUMENTATION All images were photographed using a digital camera (HAMAMATSU C4742-95) connected to a fluorescence microscope (ZEISS AXIOPHOT).

3.14. STATISTICAL ANALYSIS Results for treated and control eyes were statistically analyzed using a paired Wilcoxon test. Wilcoxon test is recommended for comparing data that emerge from ordinal numbers. P<0,05 was considered as statistically significant. 41

4. RESULTS

4.1. ANIMALS

We established the oxygen induced retinopathy model in order to assess the degree of retinal neovascularization after intravitreal injection of soluble dimeric ephrinB2 or EphB4. During the process of the above mouse model, newborn mice were exposed to 75% oxygen from P7 to P12. In order to compare the effects of ephrinB2 and EphB4 on physiological retinal blood vessel development, we injected the above substances intravitreally and the animals were not exposed to oxygen. A total number of 216 animals were required for the following procedures. Table 4 shows the number of animals used for each experiment and the distribution of loss of material due to death of animals or because of improper material.

Tab. 4: Distribution of animals used for the experiments.

Total Death during Improper material Rest mice for Purpose due to technical number experiments results problems n % n % n % n% EphB4+O2 low dose 26 12,0 0 - 5 13,5 21 14,4 Eph B4 +O2 57 26,3 21 63,6 13 35,1 23 15,6 EphrinB2 +O2 42 19,4 4 12,1 11 29,7 27 18,5 EphB4 noO2 37 15,7 7 21,2 5 13,5 25 17,1 EphrinB2 noO2 22 10,2 1 3,0 3 8,1 18 12,3 ICH* EphB4 7 3,2 0 - 0 - 7 4,8 ICH* EphrinB2 4 1,9 0 - 0 - 4 2,7 PCR 21 9,7 0 - 0 - 21 14,4

Total 216 100 33 100 37 100 146 100

*IHC: Immunohistochemistry 42

33 animals died during the course of the experiments, some of which (9,1%) were devoured by their mothers. Other reasons of animal death were maternal death due to inability to tolerate the high oxygen concentration, or difficulty in acclimatising back to normal room air. Toxicity of the substances accounting for the animal deaths was not considered, bearing in mind the low dose together with the fact that the substance was injected in a closed cavity (intravitreal injection). Tissue material in some cases was also discarded, owing to inadequate perfusion with fluorescent dye, malformation of the retina or the whole bulbus, and because of technical problems during preparation. Table 5 shows the number of animals who died during the experiments with regard to cause of death and treatment procedure. It is obvious that more spontaneous deaths appeared in correlation with oxygen treatment, both for new-born mice as for their mothers. However, we are not able to say if the spontaneous deaths (unkown cause) were due to oxygen treatment only or due to any other reason, such an epidemic disease in the same family of animals.

Tab. 5: Cause of Death in animals during experiments in relation to treatment

Cause of death Total EphB4 +O2 EphB4 EphrinB2 EphB4 EphrinB2 low dose +O2 +O2 –O2 –O2 According to protocol 151 26 36 38 30 21

Death of mother 7 - 7 - - -

Devoured by mother 3 - - - 2 1

Unknown cause 23 - 14 4 5 - during experiments 43

4.2. PCR PROCEDURE

An amplification of the retinal RNA was carried out using RT-PCR in order to verify the presence of ephrinB2 and EphB4 RNA in the retinal tissue. Retinal RNA of mice of different age who had undergone the oxygen treatment was isolated at P12 and P17 and a reverse transcription into cDNA followed. Primers were designed to span a region of the gene which includes an intronic sequence. DNA sequence for murine ephrinB2 and EphB4 was downloaded from GeneBank (National Centre for Biotechnology Information). The positioning of the primers is shown on table 3.

Figure15 indicates the gel electrophoresis of retinal cDNA of mice at different ages. The length of the products matches to the expected product length as clarified on table 3.

Mouse retina P17 Mouse retina P17 +O2 Mouse retina P12 +O2 +O2 + stich efnB2 (1+2) efnB2 (qa+qb) EphB4 (1+2) EphB4 (qa+qb) efnB2 (1+2) efnB2 (qa+qb) EphB4 (1+2) EphB4 (qa+qb) efnB2 (1+2) efnB2 (qa+qb) EphB4 (1+2) EphB4 (qa+qb) ACT (1+2) 500bp 400bp 300bp 200bp

100bp

Fig. 15: Gel electrophoresis of the retinal cDNA of mice of different age followed by a PCR using primer pairs designed for ephrinB2 and EphB4. 44

The first four lanes are loaded with material from P17 mouse retinas that were exposed to oxygen. The first two lanes were amplified with specific primers for ephrinB2. The observed length of the long fragment is almost 300bp and of the short fragment more as 100bp (designed to be 284bp and 117bp, respectively). The same applies for the next two lanes that are loaded with material which was amplified using specific primers for EphB4. The length of the long fragment is almost 300bp and of the short fragment above 100bp (designed to be 282bp and 120bp, respectively). The next four lanes were loaded with retinal tissue material of P17 mice, exposed to oxygen and after returning to room air were injured in the eye by puncturing at the injection site using a glass micropipette, without injecting any substance, in order to see if alone the injection trauma could have a change in the response. Both substances are detected in this case, as well as in the proceeding four lanes loaded with retinal tissue material of P12 mice exposed to oxygen. The last lane has mouse retina cDNA amplified with primers for ACT, as a control for the experiment. 45

4.3. RETINAL WHOLE-MOUNTS: CONTROL AND EXPERIMENTAL

In order to have a representative image of the physiological development of mouse retina, whole mounts of postnatal day 17 (P17) mice were prepared after perfusion with fluorescein-dextran (FITC-dextran). The animals were neither exposed to oxygen nor injected intravitreally. We observed that during the physiological development of the retina the vasculature comprises of a uniform plexus of capillaries and precapillary arterioles or postcapillary venules that spreads from the centre of the retina to the periphery (figure 16). On whole mount preparations of mouse retinas we observed that the superficial layer of the retina is covered by a dense network of capillaries and in the deep layer of the retina numerous larger vessels arise radially from the optic nerve (figure 17). No apparent vasoproliferative features such as neovascularization blood vessel tufts, extraretinal neovascularization clusters, or avascular areas or tortuosity of the central vessels were observed, as seen in oxygen induced retinopathy (compare to fig. 18-20).

Fig. 16: Whole-mount of a P17 mouse retina. A uniform plexus of capillaries and precapillary arterioles or postcapillary venules that spreads from the centre of the retina to the periphery is observed. 46

Fig. 17: Detail of the optic nerve vasculature. Both pictures were taken from the same preparation and focused at two levels. Left: Superficial layer of the retina. Here, a dense network of capillaries is seen. Right: Deep layer of the retina, showing numerous larger vessels arising radially from the optic nerve.

Fig. 18: Mouse retinal whole-mount after oxygen treatment (P17). Central vascular occlusion is displayed (white arrows), together with multiple blood vessel tufts (blue arrows). 47

Fig. 19: Retinal whole-mount which indicates tortuosity of the central retinal vessels (white arrows). Blood vessel tufts and large clusters of blood vessel tufts are marked with blue arrows.

Fig. 20: This image represents a variation of neovascularization features on mouse retinal whole mount (P17) with numerous blood vessel tufts. 48

4.4. NORMOXIC CONDITIONS

The physiological development of retinal vasculature in mice takes place in the first postnatal days and not during embryonic development as happens in humans. In order to investigate the effect of EphB4 and ephrinB2 on physiological vessel development of the retina, newborn mice were injected intravitreally on postnatal day 6 or 7 (P6-P7) with ephrinB2 (0,3µM) or EphB4 (0,3µM). The animals remained in room air for the next 7 days and their eyes were enucleated after dextran-fluorescein perfusion on P13 and P14 respectively. The proliferation score was determined and statistically analysed using the paired Wilcoxon test.

4.4.1. EphB4 INJECTION DURING PHYSIOLOGICAL RETINAL VASCULARIZATION

Intravitreal injection of soluble dimeric EphB4-Fc (0,3µM) in 25 mice which remained in room air throughout the experiment, had no significant difference on the proliferation score of whole-mount retinas, in comparison to the contralateral eye that was injected with the same volume (2µl) of PBS buffer.

Tab. 6: Statistical analysis of the proliferation scores of eyes injected with EphB4 without oxygen exposure. EphB4 (0,3µM) without Oxygen exposure

Paired Wilcoxon test (n=25) p-Value

Proliferation score 0,2829

Figure 21 shows the effect on the physiological blood vessel development after intravitreal injection of dimeric soluble EphB4 0,3µM. 49

10 EphB4 no O2

8

6

4 Untreated Eye

Wilcoxon test: p>0,05 2 n = 2

0 0 2 4 6 8 10 Treated Eye

Fig. 21: Proliferation score in mice injected with EphB4 (0,3µM) without oxygen exposure.

In the graph above, the relation of the proliferation score of treated eye versus untreated eye of each mouse is represented. Each dot indicates the proliferation score of both eyes, with values for the treated eye on the horizontal axis and values for the untreated eye on the vertical axis. The diagonal on the graph is the midline dissecting the axes at +45°. It represents no regression line but it is designed in order to give an overview to the reader. The dots found under the diagonal midline indicate that the proliferation score is greater for the treated eye. Conversely, the dots lying above the diagonal midline indicate that the proliferation score is greater for the untreated eye. The dots that are lying on the diagonal midline show that the proliferation was the same for both eyes. In addition, it should be made clear that the proliferation scoring system uses an ordinal scale and therefore, no standard deviation or regression coefficient can be estimated. As already mentioned, the appropriate test for statistical analysis of the data emerging from an ordinal scale is the Wilcoxon test. 50

4.4.2. EphrinB2 INJECTION DURING PHYSIOLOGICAL RETINAL VASCULARIZATION

Intravitreal injection of soluble dimeric ephrinB2-Fc (0,3µM) in 18 mice which remained in room air throughout the experiment, had no significant difference on the proliferation score of whole-mount retinas, in comparison to the contralateral eye that was injected with the same volume (2µl) of PBS buffer. Figure 22 shows the effect on the physiological blood vessel development after intravitreal injection of dimeric soluble EphB4 0,3µM.

10 EphrinB2 no O2

8

6

Wilcoxon test: 4 Untreated Eye p>0,05 n = 2 n = 3 2

0 0 2 4 6 8 10 Treated Eye

Fig. 22: Proliferation score in mice injected with ephrinB2 (0,3µM) without oxygen exposure.

Tab. 6: Statistical analysis of the proliferation scores of eyes injected with ephrinB2 without oxygen exposure. ephrinB2 (0,3µM) without Oxygen exposure

Paired Wilcoxon test (n=18) p-Value

Proliferation score 0,1090 51

4.5. RELATIVE HYPOXIC CONDITIONS

To investigate the effects of EphB4 and ephrinB2 on ischaemic retinal angiogenesis in vivo, a highly reproducible murine model of retinal ischaemia (OIR) was used. Intraocular injections of soluble dimeric EphB4 and ephrinB2 at a concentration of 0,3µM at P12 enhanced morphologically evident retinal neovascularization at P17, compared with an equivalent injection of control buffer in the contralateral eye. No retinal detachment or other damage related to the needle puncture was observed.

4.5.1. INJECTION OF DIMERIC EphB4 USING THE OIR MODEL

Intravitreal injection of soluble dimeric EphB4-Fc (0,3µM) in 23 mice which were exposed to oxygen as described in materials and methods chapter (see 3.3), showed significant difference on the proliferation score of whole-mount retinas, in comparison to the contralateral eye that was injected with the same volume (2µl) of PBS buffer. EphB4 was able to enhance the prolifefation score of the whole-mount retinal tissue, in comparison to the control eye. Table 8 shows the statistical analysis using the paired Wilcoxon test of the proliferation score between treated and untreated eye in mice who were injected with EphB4 0,3µM.

Tab. 8: Statistical analysis of the proliferation scores of eyes injected with EphB4 in the OIR model.

EphB4 (0,3µM) after Oxygen exposure

Paired Wilcoxon test (n=23) p-Value

Proliferation score 0,0430 52

Figure 23 shows the effect on the physiological blood vessel development after intravitreal injection of dimeric soluble EphB4 0,3µM.

40

EphB4 + O2

30

20 Untreated Eye

10 Wilcoxon test: p<0,05

0 0 10 20 30 40 Treated Eye

Fig. 23: Proliferation score in mice injected with EphB4 (0,3µM) after exposure to oxygen. 53

4.5.2. INJECTION OF DIMERIC EphrinB2 USING THE OIR MODEL

Intravitreal injection of soluble dimeric ephrinB2-Fc (0,3µM) in 27 mice which were exposed to 75% oxygen, showed significant difference on the proliferation score of whole-mount retinas, in comparison to the contralateral eye that was injected with the same volume (2µl) of PBS buffer. EphB4 was able to enhance the prolifefation score of the whole-mount retinal tissue, in comparison to the control eye.

Figure 24 shows the effect on the physiological blood vessel development after intravitreal injection of dimeric soluble EphB4 0,3µM.

30 EphrinB2 + O2

25

20

15 Untreated Eye

10

Wilcoxon test: 5 p<0,05 n = 2

0 0 5 10 15 20 25 30 Treated Eye

Fig. 25: Proliferation score in mice injected with ephrinB2 (0,3µM) after exposure to oxygen. 54

Table 9 shows the statistical analysis using the paired Wilcoxon test of the proliferation score between treated and untreated eye in mice who were injected with ephrinB2 0,3µM.

Tab. 9: Statistical analysis of the proliferation score of eyes injected with ephrinB2 after oxygen exposure. ephrinB2 (0,3µM) after Oxygen exposure

Paired Wilcoxon test (n=27) p-Value

Proliferation score 0,0140

As observed in the graphs shown above, there is a noticeable difference in the proliferation score concerning the experiments with and without oxygen exposure. Mice that were not exposed to oxygen displayed a reduced proliferation score. Either injection of ephrinB2 and EphB4 in the normoxic mice has no measurable effect on angioproliferation or their effect can not be detected using our model. 55

5. DISCUSSION

With our advanced mouse model of oxygen induced retinopathy we were able to show that ephrinB2 and EphB4 are expressed in mouse retina at different stages of postnatal development and that intravitreal injection of the above substances using the OIR model showed increased neovascularization of the retina in comparison to the control eye. On the other hand, intravitreal injection of the above substances under normoxic conditions had no visible effect on the physiological blood vessel development of the retina. Prior to the discussion of these results, our methods with its advantages and associated problems will be addressed for a proper rating of the results.

5.1. Mouse model of oxygen induced retinopathy

Development of the mouse model. In 1994, Smith et al. have described a reproducible and quantifiable mouse model of oxygen-induced retinal neovascularization that proved useful for the study of pathogenesis of retinal neovascularization as well as for the study of medical intervention for ROP and other retinal angiopathies [79]. There have been numerous attempts in the past to establish a reproducible animal model for the induction and quantification of the proliferative retinopathy. After the early trials using cats and dogs, [20], [68] it emerged that mice are the most suitable animals, because the blood vessel formation in the mouse retina reflects the situation in humans very well and because of the increased rate in population growth which can offer a larger number of animals to be used in the experiments in a little time. The variation in the degree of proliferative retinopathy of the first trials was explained through the exposure of the animals to multiple factors. The point of time by which the animals are exposed to oxygen as well as the oxygen concentration are decisive for the development of the oxygen induced retinopathy. The hyaloideal vascular system is completely developed at the time of birth of the mice and it regresses progressively during the maturation of the animals [9]. When the animals are exposed to oxygen too early (before P7) then an angiopathy of the hyaloideal artery is observed but no reproducible proliferation of the retinal vessels. In addition, when 56 the animals are exposed to a higher oxygen concentration, a higher mortality of the mother and the young animals is observed [79]. Comparing the various exposure schemes, the model proposed by Smith et al. turned out to be the most suitable for a reproducible and quantifiable oxygen induced retinopathy. This was confirmed by other experimental groups [30], [62], [82]. The mouse model of oxygen induced retinopathy today is a well established model and it exhibits an extended compatibility with the pathophysiology concerning retinal neovascularization in humans. Not only the pathogenic mechanisms involved, but also the clinical outcome and the appearance of the fluorescein-perfused whole mounted mouse retina under the microscope are very similar to the fundoscopic results in the human eye using fluorescein angiography.

Evaluation of the retinal changes. There are different methods to estimate the extend of ischaemic retinopathy in the OIR-model. The histological evaluation of retinal endothelial cells lying interiorly to the internal limiting membrane in PAS- stained sections is often used to estimate the retinal angioproliferation. This is a laborious and time-consuming procedure, which in our eyes is not well reflecting the extend of OIR. Endothelial cell counting technique takes into consideration only one parameter of vasoproliferative changes, that is the extraretinal growth of vessels, while the retinal vessel tufts, central vasoconstriction, and tortuosity of the blood vessels are not accounted for. We therefore used retinal flat mounts after fluorescein-dextrane perfusion. This allows for evaluation of simple and multiple criteria to estimate the degree of proliferative retinopathy. Compared to the section-based estimation of preretinal endothelial nuclei as measure of OIR, the microscopic examination of the mouse retinal whole mounts allows for a three-dimensional observation of the tissue and reveals potential changes and detailed information concerning additional features such as the tortuosity of the vessels, the avascular zones, the existing vasoconstriction and leaking capillaries. All the above information deploys the advantages of the OIR-model with the similarity of its retinal vascular changes to the clinical appearance in human eye. On the other hand, the quantification of the proliferative state of the retinal whole mounts requires the experience and thorough training of the investigator. 57

The perfusion with fluorescein ligated to high molecular dextran (molecular weight: 2,5x106 Dalton) is an established method, described already in 1993 [16]. A perfusion with low molecular weight fluorescein-dextran (40.000-500.000 Dalton) is not reasonable, since it would leak out of the loose, newly-formed endothelial cells due to the small size of its molecule. Fluorescein leaking out of the endothelial vessels, increases background fluorescence, which in turn makes the whole-mount evaluation difficult. Fluorescein-dextran angiography, as used in our experiments, delineated the entire vascular pattern, including neovascular tufts in flat-mounted retinas. Hyperoxia-induced neovascularization occurs at the junction between the vascularized and avascular retina in the mid-periphery. With this method retinal neovascularization was detected in all animals between postnatal day 17 and postnatal day 21.

The difficulty of our score system for retinal ischaemia and proliferation is the lack of clearcut criteria or numbers to characterize the parameters. Already in 1977 a point-system was developed for the estimation of the severity of ROP in cats [67]. Because there were differences observed on the impact of oxygen exposure for the different animal species, a score system for mice was developed [33], whose criteria closely refer to changes in human ROP [5]. Due to the fact that the retina of mice cannot be observed with accuracy through indirect ophthalmoscopy, the retina has to be prepared as whole mount. In this study the evaluation scheme was adapted from Higgins, because the quantification of multiple criteria was more relevant than other scoring systems, as for instance the one from Ricci et al. which took only three criteria into account [70]. On the other hand, the quantification of the proliferative state of the retinal whole mounts requires the experience and thorough training of the investigator. We overcame this problem at least partly by a blind evaluation by two observers.

It was proposed that the evaluation of the retinal whole mounts could be supported by a computerised system. However, it was technically difficult for the comparison of different samples since the eyes of the animals did not all have the same size at the end of these experiments leading in a diameter difference of the whole mounts. The diameter difference was also subject to compression after sealing with the cover slip 58 on the microscope slide. In order to overcome this problem for future experiments it is proposed to use glass plates with appropriate excavations where the tissue would be placed into and as a result no pressure is exerted on the tissue by the cover slip (EMS, ELECTRON MICROSCOPY). These glass plates are on the other hand more expensive and one should decide if the benefits outweigh the costs. For manual evaluation the absolute diameter of the whole mount is not of the importance it has for the computerised evaluation in order to asses the extend of the vasoproliferative damage.

There are individual differences in the degree of retinal neovascularization between animals. In a control group of mice with hyperoxia induced retinopathy the retinal score for proliferative changes - using the whole mount preparation for evaluation - could vary up to 20 points between animals. No significant difference was found when both eyes of an animal were compared to each other. This was reported as well from Mc Leod et al. for a canine model of OIR [53]. This emphasises the necessity for intraindividual comparison (treated eye versus untreated eye) to avoid camouflage of therapeutic effects by interindividual variability, instead of the substance being systematically administered and the effect being compared among different animals.

Technical modifications and problems of the OIR-model. Most of the experimental groups use 32 GA gauge needles with a diameter of about 200µm for intravitreal injections, [4], [60], [76]. In order to minimize the retinal damage caused by the intravitreal injection, in this study self-pulled glass pipettes are used. Our glass pipettes were pulled at the tip to final diameter of about 150 µm. In this way, it is possible to reduce the trauma at the injection site to a minimum. It is already evidenced that the perforation of the bulbus alone, in this case the intravitreal injection, is able to decrease the degree of angioproliferative retinopathy [76], [66], (Nagy D, Gogaki E, EVER03). However, Drixler et al. could not confirm this observation [18]. Because in this study both of the eyes were perforated for the intravitreal injection, the possible antiangiogenic effect can be neglected as a systematic error. A further advantage of the use of glass pipettes with a reduced diameter is that the above supposed error is kept as low as possible. The use of the glass pipettes with a 59 conical end provides a good seal at the injection site without visible leakage during the injection. The glass pipette is left a few seconds in the vitreous to allow diffusion of the solution before the glass-pipette is slowly withdrawn. Nevertheless a minimal reflux of intraocular fluid is unavoidable when removing the glass pipette from the injection site, and must be taken into consideration.

The three-way system used for intraocular injections has the advantage of being relative inexpensive and can therefore be dispensed after each use (except the Hamilton microliter pipettes). This prevents contamination of the injection system in multiple injections. In addition, the three-way system is connected to a reservoir which provides an easy and economical way to substitute the injected volume in the Hamilton pipette without having to refill large volumes of the solution to be tested. This saves a lot of the tested substance during the injection process.

The size of the lens is not to be disregarded during the experiment since it occupies a high percentage of the total volume of the eye. For a 12 days old mouse the total volume of the eye is about 25µl. Consequently, the danger of injuring the lens during the injections is always existing. In the case of lens damage, this could change the outcome of the experiment, since an avalanche of other metabolic reactions could be triggered. In order to avoid this, the angle of incidence of the glass pipette through the sclera during the injection was kept low, so that the glass pipette encountered the lens surface tangentially, if at all.

Comparing oral vs intravitreal administration of the substance to be tested, it was shown previously that oral treatment with a tyrosine kinase inhibitor can reduce retinal neovascularization in the OIR-model. Seo and colleagues [77] have demonstrated complete inhibition of retinal neovascularization via oral administration of the staurosporine derivative CGP 41251, a partially selective kinase inhibitor that blocks phosphorylation by VEGF and platelet-derived growth factor (PDGF) receptors as well as several isoforms of C (PKC). In another study by the same group [62] the orally administered RTK inhibitor PTK 787 that blocks phosphorylation by VEGF and PDGF receptors, also led to complete inhibition of retinal neovascularization in the mouse model of oxygen-induced retinopathy. On the 60 other hand, drugs that selectively block PDGF receptor kinase activity had no significant effect on retinal neovascularization. Intravitreal administration of the substance to be tested directly in the eye has several advantages. In the case of substances with a short half-life the OIR-model is ideal. The fact that the intravitreal cavity is a closed cavity, does not allow much of the injected substance to diffuse through it. In this way, the concentration of the substance remains high within the bulbus. The low diffusion of the substance provides no or only low systemic side-effects. Notable differences can also be accounted in the total amount of the substance required, comparing intravitreal injection to systemic administration, since multiple amount of the substance needs to be administrated systematically several times in order to achieve the desired concentration in the eye tissues. Problems of intravitreal therapy, however, are the risk of intraocular infection, which was not seen in any of our 151 cases (Tab. 5). As far as the dwell time of the drug in the eye is concerned, slow drug release devices such as intravitreal polymer pellets in future might offer the possibility for a more continuous treatment in humans.

Reconstruction of the substances: In this study EphB4 was injected according to the OIR protocol at a concentration of 1,0 ng/ml (≈ 0,01µM) but no significant results were observed (data not shown). The reason why the concentration of 1,0 ng/ml has no significant effect after the performance of the experiments is that the injected substance is diluted once more in the intravitreal cavity. For a mouse of 12 days the estimated intravitreal volume is 25µl. This means that in order to have the same end- concentration at the target cells, the substance should have a higher concentration during the injection. We estimated that when using an initial (injection) concentration of 0,3µM it should be adequate for the end-concentration of 1,0ng/ml. For ephrinB2 the concentration is multiplied by a factor of 20 (1µg/ml=0,017µM) and for EphB4 the concentration is multiplied by a factor of 33 (1µg/ml=0,009µM). Both factors lie in the region of the dilution factor in the intravitreal cavity (25µl).

Finally, the death rate of the animals, as well as of their nursing mothers was observed to be higher after oxygen treatment, but it was not able to ascertain whether the spontaneous deaths were due to oxygen treatment only or due to any other additional reason. 61

5.2. Impact of EphB4 and ephrinB2 on retinal angiogenesis in the OIR model

The role of ephrinB2 and EphB4 on the development and maturation of the retinal vasculature was examined throughout this study. It was found that a single intravitreal injection of dimeric EphB4 at a concentration of 0,3µM enhances the neovascularization features in the OIR-model. The proliferation score of the injected eye was significantly higher (p<0,05) compared to the score of the contralateral eye which received the same volume of PBS buffer solution. The development of the retinal vasculature in mouse occurs during the first postnatal days, and not during the embryonic life as happens in humans. So a possible impact of EphB4 on this postpartal angiogenesis under normoxic conditions was examined as well. As expected an effect of EphB4 on the physiological process of mouse retinal blood vessel formation could not be found. The proliferative score of the retinal whole-mounts showed no statistically significant difference between treated and control eye.

For the study of the role of ephrinB2 on the development of the retinal vasculature the same experiments as for EphB4 were performed. It was found that a single intravitreal injection of dimeric ephrinB2 at a concentration of 0,3µM is able to enhance the neovascularization features on the mouse retina in the OIR-model. The proliferation score of the injected eye was significantly higher (p<0,05) compared to the score of the contralateral eye which received the same volume of PBS buffer solution. Furthermore, the effect of ephrinB2 on the physiological process of mouse retinal blood vessel formation was studied. For this procedure 18 animals received an intravitreal injection of the same volume and concentration of ephrinB2 on P6 or P7 and remained however at room air throughout the following seven days. The proliferative score of the retinal whole-mounts showed no statistically significant difference between treated and control eye.

Comparing the effects of the two substances to one another it is clear that after performing the same experiment and using the same amount of the substances after oxygen exposure, the proliferation score p-value of the Wilcoxon test is higher by a factor of about 3 for EphB4 to ephrinB2. This may be interpreted as increased effect 62 on the retinal neovascularization of ephrinB2 compared to that of EphB4. The comparison of the analysis of the proliferation scores is shown in table 11.

Tab. 10: Comparison of the statistical analysis of the proliferation scores for ephrinB2 and EphB4.

Paired Wilcoxon test ephrinB2 EphB4 p-value p-value

Proliferation score 0,014 0,043

The stronger impact of ephrinB2 on the proliferation score is consistent with the known importance of the action of ephrinB2 for the development and maturation of the primitive vascular network as it was shown on ephrinB2 wild and knock out animals [2], [91]. The potency of ephrinB2 ligand to bind to and activate more than one receptors (EphB1, EphB2, EphB3, with the greater affinity for EphB4) could explain the more potent effect seen after ephrinB2 injection [2], [28].

There is a noticeable difference in the proliferation score concerning the experiments with and without oxygen exposure. Mice that were not exposed to oxygen displayed a reduced proliferation score. This difference could be explained as either injection of ephrinB2 and EphB4 in the normoxic mice has no measurable effect on angioproliferation or their effect can not be detected using our model. 63

5.3. The roles of ephrinB2 and EphB4 in vascular remodelling

Despite the reciprocal expression of ephrinB2 and its EphB4 receptor in developing arterial and venous endothelium, it remains unclear as to precisely where and how the critical interactions occur between cells expressing these molecules [91], [96]. Because both partners are membrane bound, it is presumed that signalling must be occurring at sites of cell-cell contact.

Embryos lacking ephrinB2 displayed severe defects in the vascular remodelling, in both arterial and venous domains. This led to the assumption that the observed vascular defects arose from defects in bidirectional signalling, normally mediated by the reciprocally expressed ephrinB2 and EphB4, that occurs between arterial and venous vascular beds during embryonic angiogenesis [91]. Interdigitation of arterial and venous vessels in the primary vascular plexus was absent in mice lacking ephrinB2, and remodelling of the primary yolk sac plexus into large and small branches also did not occur, consistent with a critical role for ephrinB2 in these processes [28]. Arterial vessels appeared as though they failed to delaminate from endodermal layers, and in some cases vessels were dilated and proved to lack the normal complement of supporting cells [91].

The functions of the cytoplasmic tail of ephrinB ligands have been genetically examined in mice by replacing wild-type alleles that encode full-length ephrinB protein with modified alleles that encode ephrinB protein lacking their cytoplasmic tail. Deletion of the cytoplasmic tail of ephrinB2 caused a phenotype with defects in angiogenic remodelling and vasculogenesis, which strongly resembles the phenotype of mice that lack the entire protein [1] (fig. 7). Although the exact cellular function of ephrinB2 in endothelial cells is not understood, these results indicate that ephrinB2 reverse signalling might be required for remodelling of the embryonic vasculature. It is possible that, in order for cells to generate new vascular sprouts, cell-cell and cell-matrix adhesions have to be suppressed and that this is mediated by the repulsive actions of Eph receptors and ephrins [44]. The results of this study could be explained according to the above evidence. The increased retinal neovascularization after intravitreal injection of dimeric ephrinB2 or 64

EphB4 could be due to the propulsive or repulsive action of the receptor of ligand respectively. Since both of the substances are specifically expressed on arterial and venous endothelial cells the sprouting of the vessels resulting in retinal neovascularization is triggered after the binding of the injected soluble proteins. Likewise, mural cells (smooth muscle cells and pericytes) and other surrounding cells such as astrocytes in the retina have been proposed to act as guidance cells during sprouting angiogenesis [56] which may similarly involve propulsive and repulsive Eph-ephrin signalling.

Many questions are raised about the distribution or functions of these proteins as vascular development proceeds, in the adult vasculature. EphrinB2 continues to play an important role during the development of arteries and it continues to specifically mark arterial as opposed to the venous vessels in the adult. However, unlike the situation described in early embryos, ephrinB2 expression is not limited to the endothelial lining of the large arteries. In the adult it is prominently observed in the smooth-muscle cells that cover these large arteries. In a large number of adult organs in mice, ephrinB2 expression extends into the microvasculature, marking capillaries to approximately the midway point between arterial and venous vessels, suggesting that capillaries exhibit arterial/venous characteristics. Within this microvasculature, both endothelial cells and pericytes can express ephrinB2 [25]. Experiments on adult tissue at sites of normal and pathological angiogenesis showed high levels of ephrinB2. These new vessels arise form previously existing ephrinB2- expressing arterioles within the connective tissue [25]. Similarly, EphB4 continues to mark vein endothelium in the adult vasculature, to the extend as the above given for ephrinB2. In this study the animals required for testing the above substances were newborn mice. Although their development does not belong to the embryonal type, the retinal vasculature of the mice develops during the first days of life [79] and it provides a comparable model to the embryonal type of development. According to the above information, it is expected that retinal endothelial cells of newborn mice express ephrinB2 and EphB4 at high levels. In this study this was shown through the experiments of the RT-PCR amplification of RNA from retinal tissue. 65

The ephrins are unique among RTK ligands because they do not function as typical soluble ligands for the activation of their receptors but, have to be membrane attached to activate their receptors [17], [27]. Membrane attachment seems to promote clustering or multimerization of the ligands, and it is this clustering that seems to be necessary to activate receptors on adjacent cells [17]. Consistent with this notion, whereas monomeric soluble ligands seem to act as antagonists, artificial clustering of soluble versions of these ligands can allow them to activate their receptors [17], [92]. In this study dimeric form of the proteins was used. EphrinB2-Fc and EphB4-Fc are recombinant proteins fused with the Fc portion of human immunoglobulin G, that is able to bind two molecules of ephrinB2 or EphB4. When the dimeric complex of EphB4 for example is injected intravitreally, then it produces dimerization of the membrane bound ephrinB2 when it is bound on the cell surface of endothelial cells. In this way, the intracellular cascade beginning with the phosphorylation of the ligand, that requires its previous dimerization, is triggered [88]. And this is responsible for the enhanced retinal neovascularization features seen after injection of EphB4. Activation of ephrinB2 ligand allows for the signal transduction required for the development of arteries [54]. Similarly, the same dimerization of EphB4 receptor on endothelial cells, occurs when soluble dimeric ephrinB2 is injected. The receptor dimerization allows for the signal transduction required for the development of veins [54]. Studies have shown that ephB class receptors are capable of discriminating between the density or the extent of ligand oligomerization, and mediate tubule assembly in response to tetrameric versions of ligand, but not dimeric forms [81], [28]. It is not yet certain whether the dimeric form of the injected substance acts only as an inducer at the signal cascade or as an antagonist for the opposing ligand or receptor as well. Moreover, there are questions as to whether the ephrin ligands or Eph receptors are met on the luminal surface of the endothelial cells, or on the abluminal, or at the junctional points between adjacent endothelial cells (Augustin HW, personal communication). 66

A study of Adams et al. (1999) [2] showed that other B-class receptors and ligands are expressed in and around the developing vasculature. In particular they reported that ephrinB1 is coexpressed with ephrinB2 on arterial endothelium, though its presence there is obviously not sufficient to compensate for the knockout of ephrinB2 and that venous endothelial cells produce both EphB3 and EphB4 receptors as well as an ephrinB ligand. Arterial endothelium expresses both ephrinB1 and ephrinB2 as well as EphB3 in some limited sites [2]. The widespread expression patterns of other B-class family members also play an important role in vascular development, which may in some ways overlap with that of ephrinB2 and EphB4. Repulsive interactions as the ones observed in the formation of somite boundaries, may likewise occur in the vasculature, because vessels that develop within or around somites express EphB3 and EphB4 and are observed to grow in close association with, but not within, domains of expression of ephrinB2 in the dermomyotome in wild type mice [2], [43], [90]. All of these interactions are further complicated by the possibility of bidirectional signalling in all cases, making it difficult to understand which cell is sending the signal and which is receiving it. Deletion of the EphB4 receptor alone results in a dramatic phenotype [13]. Eph receptor knockout animals have not exhibited overt phenotypes when deleted singly, presumably due to compensation by other family members [32],

[61], [64], [28]. EphB4 may be a special case due to its high specificity for ephrinB2. Alternatively it may be due to unique signalling capabilities of EphB4 that cannot be compensated by the overlapping expression of EphB3 in veins [28].

Similarities between many of the vascular remodelling defects resulting from disruption of either Ephrin or Angiopoietin signalling have been observed. It seems possible that the Angiopoietin system may act upstream of ephrinB2, but the reverse is also plausible. However, since Angiopoietins and Ties (their receptors) have not been reported to display asymmetric distributions, it seems unlikely that Angiopoietins directly establish ephrinB2/EphB4 expression patterns, or vice versa. It also remains possible that the Ephrin and Angiopoietin systems act in independent fashions, disruption of either of which leads to a similar phenotype. For example, since it has been proposed that the Angiopoietins themselves may not be instructive but rather permissive, in that they allow for proper interactions between endothelial 67 cells and supporting cells resulting in a system that can then properly respond to other cues, it is possible that ephrinB2 may be just such an instructive cue that requires prior permissive actions of the Angiopoietins [28], [74], [85].

Relative conclusions arising from this study, is that the ephrins/Ephs alone are not angiogenic. They may be, however, required as permissive factors during angiogenesis. This is evidenced by the results of this study that show increased angioproliferation features after oxygen exposure but no enhancement in the angiogenesis process in the normal course of vascular development (without oxygen exposure). If this is the case, then the main factor initiating the proliferative angiogenesis response should be another factor or angiogenic substance of the RTK family or else. The main candidate for this action is VEGF, known for its potent effect on vascular development [28], [80]. 68

5.4. Therapeutic consequences, questions and perspectives:

As it was already mentioned the angiogenesis process appears in multiple occasions most of which are pathological. Physiological angiogenesis develops only during embryonic development and growth, during reproduction and pregnancy and during wound repair. On the other hand there is a large spectrum of diseases where pathological angiogenesis is triggered, such as ocular neovascularizing disease, tumor growth, cardiovascular disease and other autoimmune diseases as rheumatoid arthritis and psoriasis.

Antiangiogenesis therapy for ocular neovascularization: Ocular angiogenesis remains 'dormant' until a switch to an angiogenic phenotype occurs. The angiogenic switch (The angiogenic switch hypothesis [52]) reflects a balance between (endogenous) positive and negative regulators of angiogenesis. Many of the properties identified across neovascularizing tissues are a result of the features of the endothelium. Endothelial cells that undergo hypoxia are abnormal, multilayered and more permeable. Bridging and splitting of vessels is more common than in normal vasculature. These endothelial cells may as well express specific epitopes which can be targeted for 'homing' anti-angiogenic therapeutic molecules.

The ephrin/Eph system remains a favourable candidate for being used in antiangiogenesis therapy for retinal neovascularization with its two major angiomodulatory members ephrinB2 and EphB4. This study has shown that ephrinB2 and EphB4 are directly implicated in the hypoxia induced retinal neovascularization in the mouse. The enhanced neovascularizing features across the retinal tissue after the intravitreal injection of ephrinB2 and EphB4 dimers proposes that the forward or backward signalling involving these substances is activated in endothelial cells under relative hypoxic conditions. For a successful intervention with therapeutic or preventive results against retinal neovascularization, manipulation of the ephrinB2/EphB4 system is under consideration. The interactions taking place at the opposing faces of endothelial cells could be modulated by blocking the forward or backward signalling of the receptor and/or ligand respectively. It is not yet certain whether the dimeric form of the injected substance acts as an inducer at the signal cascade or as an antagonist for the opposing ligand or receptor. 69

6. SYNOPSIS

Eph receptors are transmembrane RTKs that are activated by clustering that occurs on binding to membrane-tethered ephrin ligands. Two members or the ephrin family, ligand ephrinB2 and receptor EphB4 are known for their ability to distinguish arterial versus venous endothelium upon their expression on the cell surface. Both substances are playing an important role for the formation of the primary vascular network during embryonal development, as well as for the distinction and boundary formation between arteries and veins. In order to verify the presence of both substances in the retina RNA was isolated from retinal tissue of new-born mice. A reverse transcription into cDNA, was performed using the polymerase chain reaction (RT-PCR). cDNA was amplified in a PCR cycler using ephrinB2 and EphB4 specific primers. To investigate the effect on pathological retinal blood vessel formation, dimeric ephrinB2 or EphB4 was injected in the vitreous of mice using the murine model of oxygen induced retinopathy (OIR). Retinal whole mounts were prepared after fluorescein-dextran perfusion and neovascularization features were quantified in a blinded fashion. The criteria taken into account were blood vessel tuft formation, extraretinal neovascularization, non-perfusion areas, tortuosity of the vessels and neovascularization of the optic disc. In order to visualise the effect of the substances on physiological retinal blood vessel development, the substances were injected intravitreally in P6 or P7 mice and after seven days in room air, retinal whole mounts were prepared and evaluated. Concerning pathological angiogenesis, a single intravitreal injection of dimeric ephrinB2 or EphB4 increased the retinal angioproliferative changes significantly compared to the control eyes. No evident effects on physiological angiogenesis were observed. The PCR amplification experiments showed that ephrinB2 and EphB4 are detectable in the retinal tissue of mice at different stages of postnatal development.

These findings provide evidence that ephrinB2 ligand and EphB4 receptor are involved in pathologic retinal angiogenesis. It is possible to modify the angiogenesis response by manipulating their signalling in the retina. Blocking their action might open new possibilities in the treatment of angioproliferative retinopathy. 70

7. ZUSAMMENFASSUNG

Eph-Rezeptoren sind mebrandurchspannende Rezeptortyrosinkinasen, die nach Bindung der entsprechenden ebenfalls membranständigen Liganden durch Multimerisierung in der Zellmembran aktiviert werden. Zwei Mitglieder der Ephrinfamilie, EphrinB2 aus der Gruppe der Liganden und EphB4 aus der Gruppe der Rezeptoren, führen je nach Expressionsmuster auf der Zellmembran zu einer Differenzierung des Endothels zu arteriellen oder venösen Gefäßen. Während der Embryonalentwicklung spielen beide Substanzen eine wichtige Rolle für die Entstehung des primären vaskulären Netzwerkes, sowie für die Differenzierung zu Arterien und Venen. Um ihre Rolle für die physiologische und pathologische retinale Gefäßentwicklung herauszufinden, wurde dimeres EphrinB2 oder EphB4 in den Glaskörper von Mäusen unter normalen Bedingungen bzw. im Rahmen eines Mausmodells für ischämieinduzierte Retinopathie injiziert. Nach einer Perfusion mit Fluoreszein- Dextran wurden retinale Flachpräparate angefertigt und verschiedene Merkmale der vorhandenen Retinopathie quantifiziert. Die hierbei berücksichtigten Kriterien waren die Entwicklung von Gefäßknäueln, extraretinale Neovaskularisation, avaskuläre Areale, Gefäßschlängelung und Neovaskularisation der Papille. Um die Existenz beider Substanzen in der Netzhaut der Mäuse zu verifizieren, wurde RNA aus retinalem Gewebe neugeborener Mäuse isoliert und damit eine RT-PCR mit spezifischen Primern durchgeführt. Für die pathologische Gefäßentwicklung ließen sich die angioproliferativen Veränderungen der Netzhaut durch eine einzelne intravitreale Injektion von dimerem EphrinB2 oder EphB4 im Vergleich zu Kontrollaugen, denen eine Pufferlösung injiziert wurde, statistisch signifikant erhöhen. Es konnte kein Effekt auf die physiologische retinale Gefäßentwicklung festgestellt werden. Mit Hilfe der RT-PCR ließen sich EphrinB2 und EphB4 in retinalem Gewebe der Mäuse in unterschiedlichen Phasen der postnatalen Entwicklung nachweisen. Die beschriebenen Befunde deuten darauf hin, dass EphrinB2 und EphB4 bei der pathologischen retinalen Gefäßentwicklung eine Rolle spielen könnten. Es scheint möglich durch eine Differenzierung der Ephrin-Eph-Signalwege die Angiogenese zu beeinflußen. Eine Blockierung solcher Signalkaskaden könnte neue Möglichkeiten für die Behandlung angioproliferativer Erkrankungen schaffen. 71

8. ABBREVIATIONS

EGF : Epidermal Growth Factor Eph: Ephrin receptor FGF: Fibroblast Growth Factor GPI: Glycosylphosphatidylinosytol Ig: Immunoglobulin Min: minutes NV: Neovascularisation O.I.R.: oxygen induced retinopathy P: Point PBS : Phosphat Buffered Saline PCR: Polymerase Chain Reaction PDGF: Platelet-Derived Growth Factor PDZ domain: PSD-95, Dlg and ZO-1/2. A protein-protein interaction domain of around 90 amino acids that binds particularly to carboxy-terminal polypeptides. PFA: Paraformaldehyde Px: x postnatal day ROP : Retinopathy of Prematurity RPE : Retinal Pigmented Epithelium Rpm : Rounds per minute RTK: Receptor Tyrosine Kinases SFKs: Src-family kinases SH2 domain: Src-homology-2 domain. A protein motif that recognises and binds tyrosine-phosphorylation sequences, and thereby has a key role in relaying cascade of signal transduction. SH3 domain: Src-homology-3 domain. A protein sequence of around 50 amino acids that recognises and binds sequences rich in proline. TBE : Tris-Borate-EDTA (buffer) VEGF : Vascular Endothelial Growth Factor 72

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10. ACKNOWLEDGEMENTS

A very special thank for Prof. Hansen for allowing me to work on this subject and for his constant supervision and support throughout this study. I would like to thank Dr. Agostini and Dr. Martin for their support and assistance during the experiments and for providing the appropriate know-how for the technical part of this study and having always the right answers to most of the obstacles I had to come across. A very special appreciation to Eugenia Blinowa, Dr. Philip Maier and Dr. Anke Unsoeld for their assistance throughout the experiments, and for the perfect working conditions in the laboratory. I would like to thank the medical assistants of our laboratory, Mrs. Buchen, Mrs. Flügel, Mrs. Korth and Mrs. Mattes for their kind help and their pleasant companionship during long days of working at the laboratory. In particular, I would like to thank Prof. Augustin for his welcome comments on this study. Finally, I would like to thank Dr. Theodoros Argyriou and my family for their support and their encouragement.