Introduction

Numerous gene products participate in angiogenesis, but that participation may vary somewhat in different settings. Vascular beds differ with regard to surrounding cell types, constitutive expression of angiogenesis-related proteins, and/or their induced expression, which may alter the angiogenesis cascade and may even alter the effects of particular proteins. Some proteins promote angiogenesis in one setting and suppress it in others. Proteins depend upon other proteins for their actions and lack of expression of a binding partner in a particular locale may have a profound effect on the action of a protein.

In order to define the potential actions of a protein and its interactions with other proteins in angiogenesis, it is useful to study its effects in several well-characterized vascular beds and in different types of angiogenesis. The eye provides a valuable model system for such investigations, because it contains several vascular beds separated by avascular tissue. The vascular beds can be visualized in vivo and the presence of neovascularization can be unequivocally identified and quantified because of the surrounding avascular tissue. Also, retina-specific promoters combined with inducible promoter systems provide a useful way to control expression of proteins of interest. By observing the different effects of proteins in different ocular vascular beds, at different stages of development, and in different disease models, a more complete picture of the protein's actions and interactions with other proteins can emerge.

Vascular beds within the eye

In the eye, there are three vascular beds that have been intensively studied, the hyaloidal, retinal, and choroidal circulations. The hyaloidal vasculature develops in the embryonic eye and extends from the optic nerve through the vitreous to surround the developing lens. Near the end of development, when other circulatory systems in the eye are completed, the hyaloidal vessels regress. This provides an opportunity to study molecular signals involved in vascular regression.

The retinal circulation, unlike the hyaloidal circulation, can be studied in adult animals as well as during its development. In rodents, retinal vascular development occurs postnatally, facilitating the study of developmental or physiologic angiogenesis. On the day of birth, postnatal day 0 (P0), there are no retinal vessels and flat mounts of the retina after perfusion with fluorescein-labeled dextran show hyaloidal vessels, which have not yet regressed, but no retinal vessels (Figure 1a). The central retinal artery enters the eye through the optic nerve and retinal vessels develop from the optic nerve to the periphery of the retina. At P4, superficial retinal vessels extend from the optic nerve halfway to the peripheral edge of the retina (Figure 1b). At P7, vessels cover the entire surface of the retina (Figure 1c) and sprouts from superficial vessels begin to grow into the retina to form the intermediate and deep capillary beds. By P18, all the three vascular beds of the retina have been formed and remodeled, resulting in an adult retinal circulation, which is relatively stable thereafter (Figure 1d). Retinal vascular endothelial cells have tight junctions and modified vesicular transport, and are the sites of the inner blood–retinal barrier. This is in part due to their interactions with pericytes and retinal glia (Janzer and Raff, 1987).

Figure 1.

Retinal vascular development. Mice were perfused with fluorescein-labeled dextran on the day of birth, postnatal day (P) 0 (a, b), P4 (c), P7 (d), P10 (e), or P18 (f), and retinal flat mounts were examined by fluorescence microscopy. The large vessels seen in flat mounts from P0 mice are hyaloidal vessels; retinal vessels are just beginning to develop at the border of the optic nerve (b, arrowheads). By P4, the margin of the developing blood vessels on the surface of the retina is located halfway between the optic nerve and the peripheral edge of the retina (c, arrowheads). Large hyaloidal vessels are still seen overlying the avascular peripheral retina. At P7, superficial vessels cover about 80% of the retina (d, arrowheads). The hyaloidal vessels have started to regress. At P10, the superficial vessels have reached the peripheral edge of the retina and the deep capillary bed has begun to develop posteriorly (e). By P18, retinal vascular development and remodeling is completed (f)

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The retinal circulation supplies the inner half of the retina (Figure 2a and b). The outer nuclear layer, which is made up of the cell bodies of the photoreceptors, and the inner and outer segments of the photoreceptors, make up the outer avascular half of the retina. It receives its oxygen and nutrients from the choroidal circulation, a high-flow system supplied by multiple long and short posterior ciliary arteries, which all feed into an extensive network of fenestrated capillaries, the choriocapillaris. The choriocapillaris allows plasma to pool beneath the retinal pigmented epithelium (RPE), which has tight junctions and specialized transport systems, and it constitutes the outer blood retinal barrier.

Figure 2.

Mice with oxygen-induced ischemic retinopathy develop severe retinal neovascularization. (a, b) Paraffin-embedded ocular sections stained with Griffonia simplicifolia lectin (GSA), which selectively stains vascular cells, from a normal postnatal day (P) 17 mouse shows normal superficial, intermediate, and deep capillaries. There are no vessels extending above the internal limiting membrane (ILM). Hematoxylin counterstain shows retinal neurons. (c, d) Sections from P17 mice with ischemic retinopathy stained with GSA show prominent neovascularization that has broken through the ILM and is on the surface of the retina (arrows). Dropout of the normal retinal blood vessels throughout the posterior retina is seen in the low magnification view in (c). This is also illustrated by the black (nonperfused) area in the retinal flat mount from a fluorescein dextran-perfused P17 mouse with ischemic retinopathy (e)

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Developmental retinal neovascularization

Retinal astrocytes enter the retina from the optic nerve (Watanabe and Raff, 1988). As they migrate away from the optic nerve into avascular retina, they become hypoxic and express increased levels of vascular endothelial growth factor A (VEGF) (Stone et al., 1995). VEGF stimulates the growth of blood vessels from the optic nerve to the periphery of the retina and as the blood vessels become functional; they alleviate hypoxia, resulting in decreased expression of VEGF. The relative hypoxia of the deeper layers of the retina results in a VEGF gradient that favors sprouting from the superficial vessels, resulting in penetrating branches that form the intermediate and deep capillary beds. VEGF also acts as a survival factor for the endothelial cells of the newly formed retinal vessels (Alon et al., 1995). Dependence on VEGF is transient and is probably eliminated by the development of cell–cell contacts, particularly association with pericytes that provide an alternative source of survival factors. Platelet-derived growth factor-B (PDGF-B) secreted by endothelial cells is critical for the recruitment of pericytes, and PDGF-deficient mice lack pericyte envelopment of retinal vessels (Lindahl et al., 1997).

Pathologic retinal neovascularization

During the period of retinal vascular development, alteration of VEGF expression in the retina causes problems. This is achieved experimentally by placing neonatal mice in high oxygen for several days, which causes downregulation of VEGF, stopping further vascular development and causing vascular regression. This results in large areas of nonperfused retina and when the mice are placed in room air, there is severe retinal ischemia resulting in high expression of VEGF and retinal neovascularization (Figure 2c–e). This constitutes a murine model of ischemic retinopathy (Smith et al., 1994), which is most analogous to retinopathy of prematurity, but also mimics important aspects of proliferative diabetic retinopathy, the most common ischemic retinopathy in patients (Klein et al., 1984).

Choroidal neovascularization

Choroidal neovascularization occurs in diseases in which there are abnormalities of Bruch's membrane and/or the RPE, the most common of which is age-related macular degeneration (The Macular Photocoagulation Study Group, 1991). Bruch's membrane is a five-layered extracellular membrane structure that separates the choriocapillaris from the RPE; it seems to provide a physical and biochemical barrier to vascular invasion of the subretinal space. Choroidal neovascularization can reliably be produced in experimental animals including monkeys and mice, by rupturing Bruch's membrane with laser photocoagulation (Miller et al., 1986; Tobe et al., 1998). The murine model of choroidal neovascularization due to laser-induced rupture of Bruch's membrane (Figure 3) has been particularly useful for exploring the effect of gene products and drugs on choroidal neovascularization (Tobe et al., 1998; Seo et al., 1999; Kwak et al., 2000; Ozaki et al., 2000).

Figure 3.

Rho/VEGF transgenic mice with expression of VEGF in photoreceptors develop subretinal neovascularization. The rhodopsin/VEGF transgene begins expression at about postnatal day (P) 7. By P10, endothelial cells are seen migrating into the outer nuclear layer (a). At P14, well-formed blood vessels are seen extending from the deep capillary bed of the retina into the subretinal space (b), and at P21 networks of vessels are seen in the subretinal space (c). At P21, retinal flat mounts of fluorescein dextran-perfused transgenics show numerous tufts of new vessels partially surrounded by retinal pigmented epithelial cells located along the outer surface of the retina. Feeder vessels are seen extending from retinal vessels that are out of focus in the background

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VEGF and ocular neovascularization

The correlation of increased expression of VEGF and retinal neovascularization suggested a role for VEGF as a stimulatory factor. Subsequent studies have confirmed that VEGF plays a central role, because retinal neovascularization is suppressed by agents that bind VEGF (Aiello et al., 1995; Adamis et al., 1996; Saishin et al., 2003) or block VEGF receptors (Seo et al., 1999; Ozaki et al., 2000). In fact, VEGF receptor kinase inhibitors, which are very effective at decreasing VEGF signaling, completely block retinal neovascularization, indicating that VEGF is a necessary stimulator (Seo et al., 1999; Ozaki et al., 2000). To determine if increased expression of VEGF is sufficient to cause neovascularization, the rhodopsin promoter was coupled to a full-length cDNA for human VEGF₁₆₅ and transgenic mice (rho/VEGF mice) were generated that express VEGF in photoreceptors (Okamoto et al., 1997). Rho/VEGF mice develop neovascularization that originates from the deep capillary bed of the retina (Figure 4a, b), but not from the superficial retinal capillaries or choroidal vessels. The deep capillary bed is the last vascular bed to develop starting around P7, the same time that expression of the VEGF transgene begins, raising the possibility that expression of a developmentally regulated permissive factor is responsible for the differential sensitivity of the deep capillary bed. This hypothesis was tested by using the rhodopsin promoter in combination with the reverse tetracycline-inducible promoter system (Gossen et al., 1995); double transgenic mice with doxycycline-inducible expression of VEGF in photoreceptors were generated (Ohno-Matsui et al., 2002). Administration of doxycycline to adult double transgenic mice resulted in sprouting of neovascularization from the deep capillary bed, but not the superficial retinal capillaries or the choroidal vessels just as was the case in rho/VEGF mice. This suggests that even in adult retina, there is likely to be a gene product that is expressed in close proximity to the deep capillary bed that permits VEGF to induce neovascularization. Therefore, VEGF is necessary, but not sufficient for retinal neovascularization, which originates from the superficial capillary bed. This is supported by the demonstration in primates that repeated intraocular injections of VEGF (Tolentino et al., 1996) or sustained intravitreous release of VEGF (Ozaki et al., 1997) results in several changes to retinal vessels including dilation, leakage, and microaneurysms, but not retinal neovascularization.

Figure 4.

Choroidal neovascularization at Bruch's membrane rupture sites is almost completely prevented by oral administration of PKC412. (a) At 2 weeks after rupture of Bruch's membrane with laser photocoagulation, there is prominent choroidal neovascularization stained red with GSA and HistoMark Red. (b) Oral administration of PKC412 during the 2-week period after rupture of Bruch's membrane, markedly suppresses the development of choroidal neovascularization

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It is not known if hypoxia plays any role in choroidal neovascularization due to age-related macular degeneration (AMD), but it is very unlikely to play a role in choroidal neovascularization that occurs in young patients with high myopia or angioid streaks in which the major problem is rupture of Bruch's membrane. As noted above, this clinical situation is modeled by laser-induced rupture of Bruch's membrane in mice, which results in choroidal neovascularization (Tobe et al., 1998). Despite the absence of hypoxia in this model, VEGF is a necessary stimulator, because VEGF receptor kinase inhibitors or other agents that specifically and efficiently bind VEGF, essentially eliminate the choroidal neovascularization (Seo et al., 1999; Kwak et al., 2000; Saishin et al., 2003). While VEGF is necessary, it is not sufficient, because increased expression of VEGF in photoreceptors or RPE cells does not cause choroidal neovascularization (Okamoto et al., 1997; Ohno-Matsui et al., 2002).

Clinical trials using VEGF inhibitors have been initiated in patients with subfoveal choroidal neovascularization due to AMD. In an uncontrolled phase IA trial, in which 15 patients received intraocular injection of an aptamer that binds VEGF, 80% of patients had stabilization or improvement in vision after 3 months (The EyeTech Study Group, 2002). Visual acuity was improved by three lines or more in 27% of patients. A humanized rhuMAb VEGF Fab antibody is also being tested. Intravitreous injections of ¹²⁵I-labeled VEGF Fab antibody showed penetration throughout the retina and localization in the RPE for up to 7 days (Mordenti et al., 1999). The half-life in the vitreous was 3.2 days. Preliminary results in a phase I trial testing intravitreous injection of the VEGF Fab antibody in patients with subfoveal choroidal neovascularization due to AMD, showed a substantial number of patients with visual improvement during the treatment phase (Schwartz et al., 2001). Although judgment of efficacy for each of these VEGF antagonists must be reserved until results of ongoing randomized, controlled trials are obtained, these preliminary results are encouraging because spontaneous improvement in vision is very unusual in patients with subfoveal choroidal neovascularization due to AMD.

VEGF plays a major role as a stimulator of angiogenesis in some tumors such as renal cell carcinoma and glioblastoma, and it contributes to angiogenesis in almost all tumors (Plate et al., 1992; Shweiki et al., 1992; Brown et al., 1993; Takahashi et al., 1994; Takano et al., 1996; Nicol et al., 1997; Edgren et al., 1999). Outcomes in early stage oncology trials can be difficult to assess, because patients with advanced stages of multiple different tumor types that may respond differently are included. However, occasional partial or complete responses have been noted in patients with advanced disease treated with inhibitors of VEGF or VEGF receptors. This and the encouraging preliminary results in patients with choroidal neovascularization, support the strategy of targeting tumor angiogenesis with VEGF antagonists.

VEGF isoforms

There are three isoforms of VEGF-A due to alternative splicing of pre-mRNA (Park et al., 1993). In mice, the shortest isoform is VEGF₁₂₀. It lacks exons 6 and 7, which promote binding to extracellular matrix, and therefore is the most soluble isoform. The longest isoform is VEGF₁₈₈, which is poorly soluble because it binds avidly to extracellular matrix and cell surfaces. An intermediate isoform, VEGF₁₆₄, lacks exon 6, and is more soluble than VEGF₁₈₈, but does some binding to extracellular matrix unlike VEGF₁₂₀. Mice engineered to selectively express VEGF₁₆₄ had normal retinal vascular development and were healthy, while mice that expressed only VEGF₁₂₀ had severely impaired outgrowth and patterning of developing retinal vessels and died early due to impaired myocardial angiogenesis resulting in heart failure (Carmeliet et al., 1999; Stalmans et al., 2002). Mice that selectively expressed VEGF₁₈₈, showed normal outgrowth of developing retinal vessels, but im-paired development of arterioles. Therefore, VEGF₁₆₄ appears to be a critical isoform for developmental retinal angiogenesis.

Other members of the VEGF family

Other members of the VEGF family include VEGF-B (Olofsson et al., 1995), VEGF-C (Joukov et al., 1996; Lee et al., 1996), VEGF-D (Achen et al., 1998), VEGF-E (Meyer et al., 1999), and placental growth factor (PlGF) (Maglione et al., 1991). VEGF-B does not play an important role in retinal neovascularization, because mice deficient in VEGF-B have normal retinal vascular development and no difference in hypoxia-induced retinal neovascularization compared to wild type mice (Reichelt et al., 2003). VEGF-C and -D have their primary action on lymphatic vessel development and maintenance through VEGF receptor-3 (Kukk et al., 1996; Achen et al., 1998). VEGF-E is a viral homolog of VEGF-A that has similar angiogenic activity despite activation of VEGF receptor-2, but not VEGF receptor-1 (Meyer et al., 1999). It is not known if VEGF-C or -D play any role in the eye, but PlGF acts synergistically with VEGF to promote retinal neovascularization, because PlGF-deficient mice have significantly less ischemia-induced retinal neovascularization than wildtype mice (Carmeliet et al., 2001). Since PlGF binds VEGFR-1, and not VEGFR-2, this implies that VEGFR-1 plays an important role in the development of retinal neovascularization, and this has been confirmed by the demonstration that anti-VEGFR-1 antibodies suppress ischemia-induced retinal neovascu-larization (Luttun et al., 2002).

Potential participation of other peptide growth factors in ocular neovascularization

Ever since the demonstration that hypophosectomy has an ameliorative effect on proliferative diabetic retinopathy, it has been suspected that growth hormone (GH) and/or insulin-like growth factor-1 (IGF1) contribute to retinal neovascularization (Wright et al., 1969). In support of this hypothesis, antagonists of GH or IGF-1 decrease retinal neovascularization by 30–50% (Smith et al., 1997). Intraocular injection of gutless adenoviral vectors expressing IGF-1 does not cause retinal or anterior segment neovascularization (unpublished data). Also, coinjection with vectors expressing VEGF and IGF-1 does not cause retinal neovascularization. Therefore, IGF-1 may contribute to retinal neovascularization, but increased expression of IGF-1 even when combined with increased expression of VEGF is not sufficient to initiate retinal neovascularization.

Basic fibroblast growth factor (FGF2) was one of the first angiogenesis factors to be identified and has been suspected to participate in ocular neovascularization. However, FGF2-deficient mice exhibit normal retinal vascular development and compared to wildtype mice, show comparable amounts of choroidal neovascularization at Bruch's membrane rupture sites (Ozaki et al., 1998; Tobe et al., 1998). Transgenic mice that express high levels of FGF2 in the retina do not develop neovascularization, nor do they show increased amounts of ischemia-induced retinal neovascularization (Ozaki et al., 1998). However, if photoreceptors expressing high levels of FGF2 are damaged providing the FGF2 access to the extracellular compartment, then increased choroidal neovascularization occurs compared to wild type mice (Yamada et al., 2000). Thus, increased expression of FGF2 in the retina does not stimulate angiogenesis, but rather confers a latent angiogenic potential that is not manifested unless there is cell injury.

Tie receptors

Tie1 and Tie2 receptors are selectively expressed on vascular endothelial cells and are required for embryonic vascular development (Dumont et al., 1994; Sato et al., 1995). Mice with targeted disruption of Tie1, die between E14.5 and birth with severe edema and hemorrhage, suggesting that Tie1 signaling promotes vascular integrity (Puri et al., 1995; Sato et al., 1995). Mice with targeted disruption of Tie2 or mice expressing a dominant-negative Tie2 mutant die between E9.5 and E10.5, showing lack of development of the endothelial lining of the heart, lack of remodeling of the primary-capillary plexus to form more complex higher order branching vessels, and failure of vascular invasion of neuroectoderm (Dumont et al., 1994). The ligand for Tie1 has not been identified. The first binding partner identified for Tie2, angiopoietin-1 (Ang1), binds with high affinity and initiates Tie2 phosphorylation and downstream signaling (Davis et al., 1996). Ang1 is a critical Tie2 agonist, because mice deficient in Ang1 die around E12.5 and show vascular defects similar to, but less severe than those seen in Tie2-deficient mice (Suri et al., 1996). The second Tie2-binding partner identified, Ang2, binds with high affinity, but does not stimulate phosphorylation of Tie2 in cultured endothelial cells (Maisonpierre et al., 1997). In vitro, Ang2 acts as a competitive inhibitor of Ang1, because it decreases Ang1 binding to Tie2 and Ang1-induced phosphorylation. With regard to embryonic vascular development, Ang2 also acts as an Ang1 antagonist, because transgenic mice overexpressing Ang2 have a phenotype similar to Ang1-deficient mice (Maisonpierre et al., 1997).

The functions of Ang1 and Ang2 and the manner in which Ang/Tie2 signaling modulates VEGF signaling after embryonic development are less clear. In the female reproductive system, coexpression of Ang2 and VEGF occurs in developing follicles in association with neovascularization, while high expression of Ang2 alone occurs in atretic follicles in association with vascular regression (Maisonpierre et al., 1997). This observation led to the hypothesis that Ang2 blockade of Tie2 signaling disrupts stabilizing input to endothelial cells from the extracellular matrix and surrounding cells, making endothelial cells more responsive to VEGF and thereby stimulating NV, but Ang2 also disrupts survival signals from the surrounding environment, so that in the absence of VEGF. endothelial cells undergo apoptosis, resulting in vessel regression (Maisonpierre et al., 1997). Subsequent studies in the female reproductive system, tumor vasculature, and brain vasculature have been supportive of this hypothesis (Beck et al., 2000; Hazzard et al., 2000; Pichiule and LaManna, 2002), but it does not apply to all vascular beds. In adult heart, retina, or choroid, high expression of Ang2 when there is low expression of VEGF, does not cause vascular regression (Visconti et al., 2002; Oshima et al., manuscript submitted). In the retina and choroid, new vessels are sensitive to Ang2, but established blood vessels are not. When Ang2 expression is increased in the setting of ischemic retinopathy, there is a striking increase in retinal neovascularization, but in several other situations, even with moderately increased expression of VEGF, high expression of Ang2 causes regression of neovascularization. These data suggest that increasing Ang2 levels, particularly when combined with inhibition of VEGF, may be a useful way to achieve regression of pathologic neovascularization.

It has been postulated that Ang1 counters the effects of Ang2 and acts a vascular stabilizing factor. Testing of this hypothesis has been difficult because, Ang1 forms multimers complicating its isolation and use for in vitro studies (Procopio et al., 1999). A genetically engineered chimeric protein, Ang1*, that does not form large multimers, but still activates Tie2 receptors, has allowed investigation of the effects of activating Tie2 in various in vitro systems (Koblizek et al., 1998). Ang1^* promotes survival of cultured endothelial cells and stabilizes tubular networks (Kwak et al., 1999; Papapetropoulos et al., 1999; Kim et al., 2000b). Since Ang1^* does not behave exactly like Ang1, it cannot be concluded that Ang1 has the same functions, but such studies at least provide some support for a potential Ang1 vascular maintenance function. But there is also evidence suggesting that Ang1 may stimulate chemotaxis, tubule formation, and vascular sprouting and hence act as a proangiogenic agent (Koblizek et al., 1998; Witzenbichler et al., 1998; Hayes et al., 1999; Kim et al., 2000a). This is supported by increased expression of Ang1 in skin under control of the keratin-14 promoter in transgenic mice, which caused a moderate increase in number and a large increase in diameter of the dermal vessels (Suri et al., 1998). Transgenic mice with increased expression of VEGF in skin showed a large increase in leaky dermal vessels, and double transgenic mice with coexpression of Ang1 and VEGF had an additive effect on angiogenesis, but the vessels did not leak spontaneously and were resistant to inflammation-induced leakage (Thurston et al., 1999). Increased expression of Ang1 in adult mice by intravascular injection of adenoviral vectors expressing Ang1 did not cause increased vascularity, but conferred leakage resistance to existing vessels (Thurston et al., 2000). Overexpression of Ang1 in the retina of transgenic mice causes suppress of retinal or choroidal neovascularization, and suppresses VEGF-induced vascular permeability (unpublished data). Therefore, increasing levels of Ang1, even without inhibition of VEGF, may be a good strategy for treatment of neovascularization.

Prostaglandins and cyclooxygenase inhibitors

Patients who regularly take nonsteroidal anti-inflammatory drugs (NSAIDs) have a 40–50% reduction in mortality from colorectal cancer (Smalley and DuBois, 1997). This and the demonstration that cyclooxygenase-2 is upregulated in colorectal and other cancers has suggested that prostaglandins (PGs) may act as tumor promoters and that inhibition of cyclooxygenases may be chemoprotective (Kawamori et al., 1998; Williams et al., 1999, 2000). At least part of the tumor-promoting effect of PGs appears to be through stimulation of angiogenesis and a proangiogenic PG effect has been noted in other systems (Form and Auerbach, 1982; Ziche et al., 1982; Diaz-Flores et al., 1994). Conversely, cyclooxygenase inhibitors suppress some types of angiogenesis (Deutsch and Hughes, 1979; Davel et al., 1985; Tsujii et al., 1998; Jones et al., 1999; Sawaoka et al., 1999; Yamada et al., 1999; Masferrer et al., 2000).

Nepafenac, a potent cyclooxygenase-1 (COX-1) and COX-2 inhibitor, readily penetrates the cornea when applied topically, and suppresses retinal and choroidal neovascularization (Takahashi et al., 2003b). Nepafenac also blunted the ischemia-induced increase in VEGF mRNA in the retina, suggesting that this is how it achieves its antiangiogenic effects. Therefore, COX inhibitors are modest suppressors of tumor and ocular angiogenesis, and are likely to provide benefits with regard to prophylaxis, but may be too weak to benefit established disease.

Tubulin-binding agents

Tubulin-binding agents, such as vincristine, vinblastine, and colchicine, cause tumor necrosis from damage to tumor blood vessels, but at doses that are too toxic for patients to tolerate (Seed et al., 1940; Hill et al., 1993). Combretastatin A-4 is a naturally occurring structural analog of colchicine that binds tubulin at the same site as colchicine, but with different characteristics (Pettit et al., 1989; Woods et al., 1995) that impart selective toxicity to tumor vasculature (Dark et al., 1997). Combretastatin A-4-phosphate (CA-4-P) is a more soluble, inactive prodrug that is converted to CA-4 by endogenous nonspecific phosphatases (Pettit et al., 1995). The selectivity of CA-4 for tumor vasculature allows for antitumor effects at doses of CA-4-P that are well tolerated (Chaplin et al., 1999). A single dose of 100 mg/kg is well tolerated in adult mice and has beneficial effects on several types of tumors (Dark et al., 1997; Horsman et al., 1998; Grosios et al., 1999; Tozer et al., 1999). This led to a Phase I study in patients with advanced cancer, which showed a reasonable safety profile for 10 min intravascular infusions of doses 60 mg/m² given every 3 weeks, and some preliminary evidence of possible efficacy in that a patient with anaplastic thyroid cancer had a complete response (Dowlati et al., 2002). Daily intraperitoneal injections of CA-4-P significantly suppress neovascularization in transgenic mice with ectopic expression of VEGF in photoreceptors and mice with laser-induced rupture of Bruch's membrane (Nambu et al., 2003). Administration of CA-4-P to mice with established choroidal neovascularization results in significant regression of the neovascularization. Therefore, CA-4-P shows promise as a novel treatment for tumors and for ocular neovascularization.

Inhibitors of nitric oxide synthetase

Nitric oxide (NO) has been shown to have proangiogenic or antiangiogenic effects depending upon the setting. Mice with targeted deletion of one of the three isoforms of nitric oxide synthase (NOS) were selected to investigate the effects of NO in ocular neovascularization (Ando et al., 2001,2002). In transgenic mice with increased expression of VEGF in photoreceptors, deficiency of any of the three isoforms caused a significant decrease in subretinal neovascularization, but no alteration of VEGF expression. In mice with laser-induced rupture of Bruch's membrane, deficiency of iNOS or nNOS, but not eNOS, caused a significant decrease in choroidal neovascularization. In mice with oxygen-induced ischemic retinopathy, deficiency of eNOS, but not iNOS or nNOS, caused a significant decrease in retinal neovascularization and decreased expression of VEGF. These data suggest that NO contributes to both retinal and choroidal neovascularization, but that different isoforms of NOS are involved. Oral administration of N^G-monomethyl-L-arginine (L-NMMA), a broad-spectrum NOS inhibitor, caused significant inhibition of choroidal neovascularization in mice with laser-induced rupture of Bruch's membrane, and significantly inhibited subretinal neovascularization in transgenic mice with expression of VEGF in photoreceptors (rho/VEGF mice), but did not inhibit retinal neovascularization in mice with ischemic retinopathy. Triple homozygous mutant mice deficient in all three NOS isoforms had marked suppression of choroidal neovascularization at sites of rupture of Bruch's membrane and near-complete suppression of subretinal neovascularization in rho/VEGF mice, but showed no difference in ischemia-induced retinal neovascularization compared to wild type mice. These data indicate that NO is an important stimulator of choroidal neovascularization and that reduction of NO by pharmacologic means is a good treatment strategy. However, the situation is more complex for ischemia-induced retinal neovascularization, for which NO produced in endothelial cells by eNOS is stimulatory, but NO produced in other retinal cells by iNOS and/or nNOS is inhibitory. Selective inhibitors of eNOS may be needed for treatment of retinal neovascularization.

Integrin antagonists

Integrins v3 and v5 are not detectable on normal endothelial cells in skin, but are highly expressed on endothelial cells participating in angiogenesis (Brooks et al., 1994a). Treatment of tumors or chick chorioallantoic membrane with LM609, a monoclonal antibody that binds v3, causes endothelial cell apoptosis, vascular regression, and suppression of tumor growth (Brooks et al., 1994b). A humanized version of LM609, Vitaxin, is being tested in clinical trials for its effects on tumors (Gutheil et al., 2000; Posey et al., 2001).

With regard to v3, there is good correlation between neovascularization in tumors and retinal neovascularization. There is upregulation of v3 on endothelial cells participating in ischemia-induced retinal neovascularization, and agents that bind v3 and/or v5 partially suppress retinal neovascularization (Friedlander et al., 1996; Hammes et al., 1996; Luna et al., 1996). The effect is modest (about 50% inhibition) compared to some other agents, and agents that bind v3 and v5 have no significant effect in models of choroidal neovascularization (unpublished data); therefore, there has been hesitancy to move into clinical trials for ocular neovascularization with these agents. Also, their mechanism of action is uncertain. While it was initially thought that the antibodies and cyclic peptides that bind v3 act as antagonists that promote apoptosis by perturbing endothelial cell adhesion, there is evidence suggesting that they may act by stimulating intracellular signaling through v3, resulting in activation of antiangiogenic pathways (Hynes, 2002). A better understanding of the mechanism of action of antibodies and cyclic peptides that bind v3 and/or v5 may provide insights as to how to improve efficacy.

Proteinase inhibitors

Since neovascularization involves degradation of the extracellular matrix by invading endothelial cells, it has been postulated that proteinase inhibitors will inhibit neovascularization. This hypothesis has been supported by studies demonstrating that systemic administration of a nonspecific matrix metalloproteinase (MMP) inhibitor suppresses retinal neovascularization (Das et al., 1999) and mice deficient in MMP-9 develop less choroidal neovascularization at Bruch's membrane rupture sites than wild type mice (Lambert et al., 2002). However, genetic manipulation of plasminogen activator inhibitor-1 or tissue inhibitor of metalloproteinase-1 does not suggest that these endogenous proteinase inhibitors have antiangiogenic activity (Lambert et al., 2001; Yamada et al., 2001). Since several endogenous inhibitors of angiogenesis are generated by proteolytic cleavage of proteins, inhibition of proteinase activity can have proangiogenic effects that compromise antiangiogenic activity and thereby complicate the use of proteinase inhibitors to treat tumor or ocular neovascularization.

Endogenous inhibitors of angiogenesis

Several endogenous inhibitors of angiogenesis have been described, including angiostatin (O'Reilly et al., 1994), endostatin (O'Reilly et al., 1997), antithrombin III (O'Reilly et al., 1999), platelet factor 4 (Maione et al., 1990), thrombospondin (Good et al., 1990), pigment epithelium-derived factor (PEDF) (Dawson et al., 1999), and several others. Adenoviral vectors encoding endostatin were injected into the tail veins of mice in a model of choroidal neovascularization (Mori et al., 2001a). Constructs utilizing a CMV promoter resulted in about 10-fold higher endostatin serum levels on average than constructs utilizing a RSV promoter. There was a strong positive correlation between endostatin serum levels and inhibition of choroidal neovascularization, indicating that endostatin is a good candidate for antiangiogenic ocular gene therapy. Subsequent studies have demonstrated that intraocular injection of gutless adenoviral vectors or bovine immune deficiency viral vectors encoding endostatin inhibit VEGF-induced ocular neovascularization and vascular leakage (Takahashi et al., 2003a). As the name implies, PEDF is produced by pigmented epithelial cells in the eye (Tombran-Tink et al., 1991; Steele et al., 1993), making it a particularly good candidate for a negative regulator of ocular neovascularization. Intraocular injection of adenoviral vectors encoding PEDF resulted in high levels of PEDF in the retina and significant suppression of retinal or choroidal neovascularization compared to null vector-injected eyes (Mori et al., 2001b). Intraocular gene transfer of PEDF in eyes with established choroidal neovascularization, resulted in regression of neovascularization (Mori et al., 2001c). Adeno-associated viral (AAV) vectors provide the advantages of minimal immune response and long-term expression. At 4 or 6 weeks after intraocular injection of AAV vectors encoding PEDF, there was significant suppression of choroidal neovascularization (Mori et al., 2002). These results are encouraging, because they suggest that intraocular PEDF gene transfer may be useful for both prevention of neovascularization and treatment of established neovascularization, and have led to a phase I clinical trial investigating intraocular injection of adenoviral vectors encoding PEDF in patients with age-related macular degeneration and subfoveal choroidal neovascularization (Rasmussen et al., 2001).

Other agents that have been demonstrated to suppress ocular neovascularization after intraocular gene transfer include soluble VEGF receptor-1 (Flt-1) (Honda et al., 2000; Lai et al., 2001b; Gehlbach et al., 2003b), angio-statin (Lai et al., 2001a; Raisler et al., 2002), tissue inhibitor of metalloproteinases-3 (Takahashi et al., 2000), and soluble Tie2 (Hangai et al., 2001). While intraocular injection of vectors maximizes delivery of gene product to the retina, there is still some concern regarding the possibility of vector-related toxicity or toxicity from ectopic expression of proteins in specialized cells such as photoreceptors. Periocular injection of adenoviral vectors encoding Flt-1 (Gehlbach et al., 2003b) or PEDF (Gehlbach et al., 2003a) results in significant inhibition of choroidal neovascularization and provides an alternative, potentially safer, means of delivery.

Summary and conclusions

There are compelling data suggesting that treatment with antiangiogenic agents can have beneficial effects on tumors, but complexities in animal models and particularly in tumor patients make outcomes difficult to assess. The eye provides a model system with several vascular beds surrounded by avascular tissue, which facilitates identification and quantitation of neovascularization. Inducible, retina-specific expression of transgenes provides a powerful tool to explore the effects of angiogenic and antiangiogenic proteins. Data obtained from animal models and ocular clinical trials provide important correlative information that can help put results in tumor models and clinical trials in context. One concept that has emerged from studies in the eye is that VEGF is an extremely good target for treatment of neovascular diseases. Despite contributions of other angiogenic factors, signaling through VEGF receptors is necessary for ocular neovascularization and blockade of VEGF receptors is a very efficient approach. Another concept that is emerging is that Ang1 and Ang2 modulate the effects of VEGF, but differentially on newly developed vessels, with no identifiable effect on mature retinal and choroidal vessels. This is good news for treatment development, because a combination of Ang2 and VEGF antagonists can promote regression of established neovascularization without adverse effects on normal blood vessels. Results in eye models have supported results in tumor models, suggesting that the vascular targeting agent Combretastain A-4-P, is a promising small molecule that can also promote regression of established neovascularization. And finally, effects in tumor models of purported endogenous inhibitors of neovascularization such as endostatin and angiostatin have been confusing and controversial. Ocular gene transfer of endostatin, angiostatin, and PEDF, have shown unequivocally that these proteins are antiangiogenic and provide promising therapeutic agents. These results suggest perseverance in studies investigating their potential application to treatment of tumors.

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Acknowledgements

Supported by EY05951, EY12609, and core grant P30EY1765 from the NEI; Research to Prevent Blindness, Inc (a Lew R Wasserman Merit Award (PAC)); Dr and Mrs William Lake. PAC is the George S and Dolores Dore Eccles Professor of Ophthalmology.