 Principles of Inhibition of Angiogenesis in the Eye Claus Cursiefen, M.D., Ulrich Schönherr, M.D., The process of
angiogenesis Angiogenesis means the formation of new blood vessels from preexisting vessels. Angiogenesis plays an important physiologic and pathologic role in the eye. Pathologic angiogenesis is associated with major causes of human blindness such as diabetic retinopathy and age-related maculopathy. In recent years great progress has been made in understanding the mechanisms and the regulation of angiogenesis. A complex system of angiogenic and antiangiogenic factors regulate the process of neovascularization. Recent research has identified vascular endothelial growth factor as the most important mediator of ocular angiogenesis. Increasing knowledge about angiogenesis improves the chances of a causal antiangiogenic therapy in neovascularizing eye diseases. There exist several potential ways of ocular antiangiogenic therapy: one way is the blockade of ocular angiogenic factors such as vascular endothelial growth factor, another way is to interfere with the interaction between the endothelial cells of the newly forming vessels and their extracellular matrix. In addition, local preexisting antiangiogenic factors, which usually inhibit angiogenesis, could be strengthened. Recent progress in angiogenesis research will result in new causal antiangiogenic drug therapy in ophthalmology with promising prospects for the treatment of ocular neovascularizing diseases. Because of its easy accessibility the eye is especially suitable for a topic antiangiogenic therapy. Key words Angiogenesis - inhibition of angiogenesis - eye - vascular endothelial growth factor - extracellular matrix - diabetic retinopathy - endothelial cells Angiogenesis means formation of new blood vessels from preexisting blood vessels. The term was first used by Hertig in 1935 related to neovascularization in the placenta (1). New blood vessel formation occurs under a lot of physiologic and pathologic conditions. Physiologic angiogenesis can be found in reproduction and development as well as in wound healing. Pathologic angiogenesis plays an essential role in tumor growth, in certain chronic inflammatory conditions such as rheumatoid arthritis and in ocular neovascularizing diseases. In the eye, angiogenesis is pathogenetically responsible for common vision-threatening diseases such as proliferative diabetic retinopathy, retinopathy of prematurity, rubeosis iridis and secondary glaucoma after branch and central retinal vein occlusion, age-related maculopathy and corneal neovascularization (2, 3). Although inhibition of angiogenesis in cancer therapy was already suggested by Folkman in 1971 (4), only the recent progress in understanding the process and the regulation of angiogenesis has made this therapeutic option a realistic one (2-7). Here, the general process of angiogenesis and its regulation will briefly be described. Then the possibilities for pharmacological inhibition of this process in ocular neovascularizing diseases are outlined (2). In the adult, new blood vessels arise by sprouting from preexisting vessels. This process can simplified be separated in three steps (2; fig. 1): 1 Angiogenic activation of endothelial cells and degradation of basement membrane. Angiogenic factors like vascular endothelial growth factor (VEGF) induce an angiogenic, activated state of the endothelial cells lining the blood vessel. These activated cells then secrete proteases which degrade the underlying basement membrane and the surrounding extracellular matrix (e.g. plasminogen activator factor). 2 Endothelial proliferation and migration. Angiogenic factors like VEGF are mitogens specific for endothelial cells, which in response proliferate and migrate in direction of the angiogenic stimulus out of the preexisting blood vessel. 3 New vessel formation. Outgrowing endothelial cells degrade extracellular matrix in their surrounding. On the other hand they need certain extracellular matrix (ECM) proteins to adhere at. These adhesion proteins vary depending on the angiogenic factor which stimulates the endothelial cell: VEGF induces expression of the integrin a Vb 5, which binds to the ECM-protein vitronectin, fibroblast growth factor (FGF) for example induces the expression of the integrin a Vb 3. This is of special interest because pharmacological blockade of these binding proteins can inhibit endothelial cell attachment to the ECM and induce apoptosis (programmed cell death) of the outgrowing endothelial cells (12, 13). Endothelial cells then arrange to form a new lumen and eventually fuse with another blood vessel and circulation is established. Other cells of the vessel wall, like pericytes or smooth muscle cells follow. Pericytes are important because they stabilize the vessel wall and seem to maintain endothelial cells (EC) in a quiescent, antiangiogenic state. At least in vitro they secrete the antiangiogenic factor TGF b when cocultured with ECs (14, 15). The homeostasis of angiogenesis, like the homeostatic system of blood coagulation principally is a very „simple" system: Either the blood vessel is quiescent or new vessel formation occurs. But the regulation of this „simple", but biologically very important decision is highly complex and is organized redundantly. Several angiogenic factors on one side are balanced out by antiangiogenic factors (tab. 1). If the local antiangiogenic system, which usually keeps ECs in a quiescent state is weakened and the concentration of angiogenic factors increases then the process of angiogenesis as outlined above starts. How, when and in which sequence the factors which regulate angiogenesis are released is still not fully understood. For one important angiogenic factor of ocular angiogenesis, the vascular endothelial growth factor, it is known that tissue hypoxia (a decrease in tissue oxygen concentration) and inflammation can stimulate its secretion (16-19). In hypoxic areas of the detached retina increased VEGF mRNA levels were found (17). Oxygen sensing haem-proteins are probably regulating this release as in the case of erytropoetin (20). In diabetic retinopathy, hypoxic nonperfused areas of retina secrete VEGF. VEGF exists in four isoforms which can bind to three different VEGF receptors. These receptors are found in very high concentrations on ocular ECs (21). In the retina the cells of the capillary wall (endothelial cells, pericytes and smooth muscle cells) can secrete VEGF. The same is true for Müller cells and retinal pigment epithelial cells (22, 23, 24). The diffusibel factor then can induces angiogenesis locally in the retina (e.g. in proliferative diabetic vitreoretinopathy) or diffuse to the anterior segment where its concentration is greatest at the pupillary margin and in the iridocorneal angle. There rubeosis iridis and rubeosis of the iridocorneal angle is then induced. The concentration of VEGF in the vitreous of patients with proliferative diabetic retinopathy was significantly increased compared to patients with non-proliferative diabetic retinopathy and was in such high concentrations which in vitro and in animal experiments can induce angiogenesis (25, 26). The VEGF-concentration decreased from the posterior to the anterior segment of the eye (25, 26). VEGF - previously known as vascular permeability factor- also increases vascular permeability and is responsible for the increased tyndall and laser flare counts in the eyes of diabetic patients (27). For several reasons VEGF at the moment seems to be the most important angiogenic factor at least in ocular neovascularizing diseases. VEGF is an endothelial cell specific mitogen, it binds to receptors on the endothelial cell, its secretion is increased by hypoxia both in vivo and in vitro, it is diffusibel and it can be found in eyes with neovascularizing diseases. Experimentally, intravitreal injection of VEGF could induce ocular angiogenesis and antibodies against VEGF could reduce the rubeosis usually occurring in monkey eyes after experimentally induced central retinal vein occlusion (2, 29). Therefore VEGF at the moment is the main target for antiangiogenic therapy by inhibition of angiogenic factors. But probably other factors like fibroblast growth factors (FGF) and others (tab. 1) are also important in ocular angiogenesis. General aspects of inhibition of angiogenesis Therapy of ocular neovascularizing diseases, e.g. proliferative diabetic retinopathy, often is unsatisfactory. Laser destruction of the non-perfused retina is yet the only established therapy to obliterate newly formed vessels. The effect of panretinal laser treatment is most likely due to reduced VEGF-production due to an increased oxygen content of the retina. An improved oxygen diffusion from the choroidal capillaries and a reduced oxygen demand of a reduced number of active retinal neurons are the most likely explanations for this (7). The rapidly increasing knowledge about the process and the regulation of angiogenesis in recent years therefore let to experimental and first clinical trials of ocular antiangiogenic therapy. From clinical trials of cancer antiangiogenic therapy a few general principles about antiangiogenic therapy of ocular neovascularizing diseases can be derived. Therapy for diabetic retinopathy, age-related maculopathy or corneal neovascularization will be a long-term treatment, lasting for month or years. Because of its easy accessibility the eye and especially the cornea are suitable for a local antiangiogenic therapy e.g. in corneal diseases with neovascularization (2, 6). In cancer patients sometimes initial disease progression occurred, but this should not be a reason for discontinuation of therapy, which also should not be interrupted once begun (6). Endothelial cells obviously do not develop resistance against antiangiogenic drugs, whose effect often had a quite prolonged action (6). Regarding possible side-effects of a local or systemic antiangiogenic therapy one has to consider, that physiologically angiogenesis in the adult only occurs during wound healing, in the female reproductive system and in embryonic and fetal development. Antiangiogenic drugs can be contraceptive or can have disastrous effects in pregnancy, such as the antiangiogenic drug thalidomide (30). Recently a basal VEGF-secretion even in normal adult tissues such as the kidney, lung and the eye (ciliary body, corneal epithelium) was discovered, which might have an important „housekeeping effect" in normal tissues, e.g. to maintain endothelial fenestration in kidney tubules and the ciliary body (7-10). What effects a systemic antiangiogenic therapy will have on these putative VEGF housekeeping functions is yet unknown. Effects of antiangiogenic therapy on preexisting blood vessels in others organs have not been noted. Therefore in summary great care has to be kept in testing antiangiogenic drugs. Nevertheless, antiangiogenic therapy in the non-pregnant adult should to our present knowledge be quite safe. This of course is especially true for a local application of antiangiogenic agents which could be used in ophthalmology. Application of antiangiogenic drugs in ocular neovascularizing diseases could either be locally in form of eye drops - especially in corneal neovascularizing diseases. A local injection or local intraocular application e.g. during vitrectomy in proliferative diabetic retinopathy might also be feasible. In addition or alone a systemic application has already been successful tested in animal experiments, either by subcutaneous injection or orally. The subcutaneous injection of a cyclic peptide antagonist against vitronectin binding integrins e.g. reduced new vessel formation in a mouse model of ischaemia-induced neovascularizing retinopathy (13). What are the potential starting-points for antiangiogenic therapy? The easiest and best antiangiogenic therapy would be the avoidance of the causal stimuli for the liberation of angiogenic factors. But e.g. proper regulation of blood glucose levels in diabetic patients and prevention of diabetic angiopathy and tissue hypoxia are often difficult to achieve. Therefore the next and first pharmacological way to influence the process of angiogenesis is the blockade of angiogenic factors. One general problem of this therapeutic approach is the above mentioned abundance of angiogenic factors and their redundant organization. Blockade of a single angiogenic factor probably will not be sufficient (2, 6, 7). The mosaic of angiogenic factors differs between different diseases; the antiangiogenic factor interferon alpha-2a e.g. accelerated the regression of eyelid hemangiomas, but failed to interfere with subretinal neovascularization in age-related maculopathy (6). Therefore the specific underlying pathology for every ocular neovascularizing disease has to be established together with the locally active „cocktail" of angiogenic factors before in a „multiple warhead" strategy a specific antiangiogenic cocktail for each disease entity can be applied (6). A second approach of pharmacological antiangiogenic treatment is the inhibition of the outgrowth of proliferating endothelial cells from their parental blood vessel by interfering with the interaction between endothelial cells and their surrounding extracellular matrix (fig. 2). This can be achieved by blocking endothelial cell adhesion proteins (integrins) or by blocking their counterparts in the extracellular matrix. As mentioned above, blockade of integrins on angiogenically active ECs by cyclic peptides induced endothelial cell apoptosis and reduced new vessel formation in a mouse model of ischaemia-induced neovascularizing retinopathy (12, 13). Clinical trials with Vitaxin, an inhibitor of integrin a Vb 3 are in progress (12, 13, 31). Factors which interfere with the ECM-degrading proteases, which normally clear the way for the outgrowing ECs, could act here as well: Batimastat, an inhibitor of matrix metalloproteases, or TIMPs 1-3 (tissue inhibitors of matrix metalloproteases) are such factors (6, 32). A third approach of antiangiogenic therapy is the reactivation of the locally preexisting antiangiogenic factors, which e.g. in proliferative diabetic retinopathy are down regulated. These factors, once there distribution is exactly known, could also be applied therapeutically. In addition a great number of already existing drugs have shown to posses antiangiogenic properties and partly are already tested in clinical trials. Thalidomide is tested as an oral agent against age-related maculopathy (23, 24, 30), interferon alpha-2a is helpful in vision threatening lid hemangiomas but failed in the clinical therapy of age related maculopathy (6), tetracyclines and non steroidal antiinflammatory drugs are tested as well (33, 34). Also spironolactone, captopril, furosemide, paclitaxel, bumetanide, garlic and soy exhibit antiangiogenic properties, which still need further elucidation (6). Because VEGF seems to be the most important angiogenic factor in diabetic retinopathy and most experiments on antiangiogenic therapies have focused on VEGF, the four potential starting-points for antiangiogenic inhibition of VEGF are outlined below. Inhibition of the angiogenic factor VEGF There are four potential targets for inhibition of the angiogenic factor VEGF (2, 5, 6, 7). Inhibition of VEGF will not only result in inhibition of the angiogenic property of VEGF but will also interfere with the permeability increasing effect of VEGF. This could be especially interesting in inflammatory diseases with breakdown of the blood retina barrier associated with angiogenesis.
Regarding antiangiogenic therapy by inhibition of angiogenic factors some general principles for this special approach shall be discussed. First, that probably not a single angiogenic factors alone is responsible for pathologic angiogenesis in a definite disease (5). This implies that a cocktail of antiangiogenic factors will probably be necessary to achieve sufficient inhibition of angiogenesis. Second, that the „orchestra" of angiogenic factors probably varies between different organs and different pathologic entities. This means that the antiangiogenic cocktail will vary for different ocular neovascularizing diseases, e.g. a different set of antiangiogenic factors will be used for proliferative diabetic retinopathy than for corneal neovascularization after herpetic keratitis. Third, that the effect of angiogenic factors is contextual, which means that the effect of a certain angiogenic factors varies depending on its own concentration, the concentration of its naturally occurring antagonists and other angiogenic factors, which influence the action of the first factor, and depending on proteins of the ECM present at the specific disease location. Therefore the fundamental mechanisms underlying each ocular neovascularizing disorder need to be studied in detail before a specific and effective antiangiogenic therapy can be established. In summary, pharmacological antiangiogenic therapy offers very interesting and promising prospects for the treatment of ocular neovascularizing diseases such as proliferative diabetic retinopathy in the near future. Figures Figure 1 General process of angiogenesis (with friendly permission of F. Enke Verlag, Stuttgart from Cursiefen C, Schönherr U. Angiogenese und Angiogenesehemmung im Auge. Klin Monatsbl Augenheilkd 1997; 210: 341-351, [ 2] ; A - basement membrane, B - endothelial cell, C - extracellular matrix): 1. Normal capillary; 2. Degradation of basement membrane and extracellular matrix by the proteases which are released from the angiogenically stimulated endothelial cells; 3. Proliferation and migration of angiogenically stimulated endothelial cells; 4. A new vessel lumen and a new extracellular matrix around the vessel are formed and the new vessel anastomoses with another blood vessel. Figure 2 Potential targets for antiangiogenic therapy by influencing the interaction between outgrowing endothelial cells and their surrounding extracellular matrix (with friendly permission of F. Enke Verlag, Stuttgart from Cursiefen C, Schönherr U. Angiogenese und Angiogenesehemmung im Auge. Klin Monatsbl Augenheilkd 1997; 210: 341-351, [ 2] ; A - basement membrane, B - endothelial cell, C - extracellular matrix): 1. Blockade of endothelial adhesion receptors (E, integrins) and 2. blockade or degradation of extracellular matrix receptors (D). Tables Table 1 Angiogenic and antiangiogenic factors (with friendly permission of F. Enke Verlag, Stuttgart from Cursiefen C, Schönherr U. Angiogenese und Angiogenesehemmung im Auge. Klin Monatsbl Augenheilkd 1997; 210: 341-351, [ 2] )
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