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Aus der Universitäts - Augenklinik der Albert - Ludwigs - Universität Freiburg im Breisgau
EphrinB2 AND EphB4
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
1. Gutachter:
2. Gutachter:
Jahr der Promotion:
Prof. Dr. rer. nat. M. Schumacher
Prof. Dr. med. L. L. Hansen
Prof. Dr. med. vet. H. Augustin, PhD
1. INTRODUCTION5 1.1. Preface5 1.2. Blood vessel formation6 1.3. Physiological vascularisation of the retina10 1.4. Pathological vascularisation of the retina11 1.5. Receptor tyrosine kinases14 1.5.1. Characteristics 14 1.5.2. Signalling 14 1.5.3. Families of RTKs 16 1.6. Eph / Ephrin family17 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
3. MATERIALS AND METHODS 3.1. Animals 3.2. Reconstruction of EphB4 and ephrinB2 3.3. Oxygen Induced Retinopathy (O.I.R.) Mouse Model 3.4. Anaesthesia 3.5. Intraocular injection 3.6. Perfusion 3.7. Retinal whole mounts 3.8. Microscopy 3.9. Evaluation 3.10. RNA isolation
   28  28  28  29  31  31  33  35  36  36  38
3.11. RT-PCR 3.12. Gel electrophoresis 3.13. Documentation 3.14. Statistical analysis
 38  40  40  40
4. RESULTS41 4.1. Animals41 4.2. PCR procedure43 4.3. Retinal whole mounts: control and experimental45 4.4. Normoxic conditions48 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. DISCUSSION55 5.1. Mouse model of oxygen induced retinopathy55 5.2. Impact of EphB4 and ephrinB2 on retinal angiogenesis in the OIR model61 5.3. The roles of ephrinB2 and EphB4 in vascular remodelling63 5.4. Therapeutic consequences, questions and perspectives68
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.
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 developde novofrom differentiating endothelial cellsin situ, whereas in angiogenesis, capillaries originate from pre-existing vessels. Vasculogenesis ceases after early development, and endothelial cell proliferation nearly ceases in adults [46].
Duringvasculogenesis, 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 asnegosisegian [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.
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 growth factor) family, the angiopoietin family and the most recently identified, members of the ephrin family, all having unique influence on endothelial and perivascular cell function.
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 asocular 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
Fig. 2: The changes in the balance between angiogenic activators and inhibitors can trigger or stop angiogenic phenomena.
Tab. 1: Activators and inhibitors of angiogenesis (modified from Folkman et al. (1995)[21]and Klagsbrun et al. (1991)[41]).
Vascular endothelial growth factor (VEGF) Fibrobast growth factor (FGF) Transforming growth factor (TGFα, TGFβ) Tumor necrosis factor (TNFα) Platelet derived growth factor (PDGF) Angiogenin Interleukin-8 (IL-8) Granulocyte colony stimulating factor (G-CSF) Cell adhesion molecule (CAM) E-Selectin Prostaglandins (PGE1, PGE2)
Angiostatin Platelet factor IV (PF IV) Tissue inhibitor of metalloproteinases (TIMP 1,2,3) Prolactin Interferonα,γ Thrombospondine 1,2,3 Corticosteroids
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 14thand the 38thweek 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]the entire retina, vessels start to. After the vascular network has spread across 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].
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.