Choroidal Neovascularization

growth factor- (TGF- ) and related family members inhibit endothelial cell migration and proliferation in vitro, but have been suggested to be proangiogenic or antiangiogenic in vivo, depending on the context (8–10). Several purported endogenous inhibitors of angiogenesis have been described including angiostatin (11), endostatin (12), antithrombin III (13), platelet factor 4 (14), thrombospondin (15), and pigment epithelium-derived factor (PEDF) (16).

Along with soluble proangiogenic and antiangiogenic factors, extracellular matrix (ECM) molecules also participate in several ways in the regulation of neovascularization. They may bind and sequester soluble factors, preventing them from activating receptors on endothelial cells until they are released from the ECM by proteolysis (17–-19). Acting through integrins on the surface of endothelial cells, ECM molecules may directly stimulate or inhibit endothelial cell processes involved in angiogenesis (20). Soluble angiogenic factors exert some of their effects through ECM molecules by altering expression of integrins on endothelial cells (21). Endothelial cells of dermal vessels have increased expression of v 3 integrin when participating in angiogenesis and v 3 antagonists block angiogenesis (22).

Angiogenesis in all tissues is likely to involve certain processes in endothelial cells, including proteolytic activity, migration, proliferation, and tube formation (23,24), but the molecular signals that mediate or modulate these processes might vary from tissue to tissue. For instance, two proteolytic systems have been implicated in the breakdown of ECM during angiogenesis, one involving the urokinase type of plasminogen activator (uPA) (25) and one involving matrix metalloproteinases (MMPs) (26,27) and the relative importance of these systems could vary in different types of angiogenesis. Tissue inhibitor of metalloproteinases-1 (TIMP-1) has been touted as an inhibitor of neovascularization (28), but it stimulates VEGF-induced neovascularization in the retina (29). Interferon 2a causes dramatic involution of hemangiomas (30) and inhibits iris neovascularization in a model of ischemic retinopathy (31), which led to the prediction that it would inhibit CNV. However, a multicenter, randomized, placebo-controlled trial demonstrated that patients with CNV who received interferon 2a did not have any involution of CNV and ended up with worse vision than those treated with placebo (32). Therefore, testing in relevant animal models is necessary to predict the effect of proteins or drugs on ocular neovascularization.

III.INFERENCES FROM RETINAL NEOVASCULARIZATION

It would be nice if information regarding retinal neovascularization could be applied to CNV, because more is known about the pathogenesis of retinal neovascularization. The clinical observation that retinal neovascularization almost always occurs in association with retinal capillary nonperfusion led to the hypothesis that retinal ischemia is the driving force (33–35). This hypothesis is supported by experimental models in which damage to retinal vessels leads to retinal neovascularization (31,36–39). Advances in the understanding of hypoxia-mediated gene regulation have suggested potential molecular signals such as hypoxia-inducible factor-1, involvement of which has been confirmed by experimental studies (40). As a result, many of the molecular signals involved in retinal neovascularization have been defined (for review, see Ref. 41).

Hypoxia has not been definitely implicated in the occurrence of CNV. While there is evidence that choroidal blood flow is decreased in patients with AMD, it is not clear whether the decrease is sufficient to cause hypoxia of photoreceptors and RPE (42, 43).

Furthermore, hypoxia cannot be invoked in patients with ocular histoplasmosis, myopic degeneration, angioid streaks, or many other diseases in which young people get CNV. Another difference between CNV and retinal neovascularization is the contribution of the RPE to CNV. Although the contribution of the RPE to CNV on a molecular level has not yet been clearly defined, it is clear that the RPE is intimately involved. Therefore, it is hazardous to use our knowledge of retinal neovascularization to draw inferences regarding CNV, unless they are confirmed experimentally.

IV. THE PATHOGENESIS OF CNV

One thing that patients with CNV share is that they all have abnormalities of Bruch’s membrane and the RPE. In patients with AMD, pathological studies have demonstrated that diffuse thickening of Bruch’s membrane is highly associated with the occurrence of CNV (44). Large soft drusen and pigmentary abnormalities are clinical risk factors for CNV (45); soft drusen indicate the presence of diffuse sub-RPE deposits and pigmentary changes suggest compromise of the RPE. Therefore, there is disordered metabolism of ECM in patients with AMD that may compromise RPE cells leading to cell dropout and proliferation, and CNV. Breaks in Bruch’s membrane and/or other abnormalities of the ECM of RPE cell occur in other diseases in which CNV occurs. Patients with Sorsby’s fundus dystrophy have a mutation in the tissue inhibitor of metalloproteinase-3 (TIMP-3) gene that results in abnormal processing of the protein so that it is deposited along Bruch’s membrane (46). This collection of an ectopic protein along Bruch’s membrane is associated with RPE and photoreceptor degeneration and a high incidence of CNV (47,48).

Why would abnormal ECM along the basal surface of RPE cells result in cell compromise and CNV? Like most epithelial cells, the phenotype and behavior of RPE cells is regulated in part by interaction with its ECM. Cultured RPE cells display alterations in morphology and gene expression when grown on different ECMs (49). Presentation of some ECM molecules such as vitronectin or thrombospondin to the apical or basal surface of RPE cells results in small increases in fibroblast growth factor-2 (FGF-2) and large increases in VEGF in the media of the cells (50). Therefore, alterations in the ECM of RPE cells can cause them to increase production of proteins with angiogenic activity.

Is increased production of angiogenic proteins in the retina sufficient to cause CNV? To address this question, bovine rhodopsin promoter was coupled to a full-length cDNA coding for VEGF165 and transgenic mice (rho/VEGF mice) were generated (51). Three founder mice were obtained and crossed with C57BL/6 mice to generate transgenic lines. One of the lines (V6) had sustained increased expression of VEGF in photoreceptors starting on postnatal day (P) 7 and developed neovascularization that originated from the deep capillary bed of the retina and grew into the subretinal space. In contrast, transgenic mice with increased expression of FGF-2 in photoreceptors (rho/FGF2 mice) do not develop any neovascularization (52).

There are several possible explanations for why mice from the V6 line of rho/VEGF trangenics develop neovascularization that develops from deep retinal vessels, but not from choroidal vessels. One possibility is that the outer blood-retinal barrier constituted by the RPE prevents VEGF produced by photoreceptors access to choroidal vessels. Another possibility is that choroidal vessels cannot respond to VEGF. A third possibility is that Bruch’s membrane provides a biochemical as well as a mechanical barrier to the growth of CNV.

The first possibility was addressed by Schwesinger et al. (53), who coupled the promoter for RPE-65 to a cDNA for VEGF 165 and generated transgenic mice with expression of VEGF in RPE cells. These mice failed to show any CNV, although they did show increased numbers of choroidal blood vessels indicating that the choroidal vessels had some response to the excess VEGF. In wild-type mice, laser-induced rupture of Bruch’s membrane results in CNV (54). In rho/VEGF or rho/FGF2 transgenic mice, rupture of Bruch’s membrane resulted in very large areas of CNV, much larger than those in wild-type mice (55). Low-intensity laser, which ruptured photoreceptor cells but did not rupture Bruch’s membrane, resulted in CNV in rho/FGF2 mice, but not rho/VEGF or wild-type mice. These experiments demonstrate that choroidal vessels are capable of responding to excess VEGF or extracellular FGF2 when there is a concomitant rupture of Bruch’s membrane. This suggests that Bruch’s membrane constitutes a mechanical and biochemical barrier to CNV. Increased expression of VEGF or FGF2 is unable to cause a breech in the barrier. In the case of FGF2, sequestration is likely to be an important control mechanism, because lowintensity laser that ruptures photoreceptor cells and releases FGF2, but does not rupture Bruch’s membrane, results in CNV. This is not the case for VEGF, which stimulates CNV only when the Bruch’s membrane barrier has been disrupted by another means.

The importance of the Bruch’s membrane barrier for prevention of CNV may help to explain difficulties in modeling CNV. Laser-induced rupture of Bruch’s membrane, first established in primates and later adapted to rodents, has been widely used (54,56,57). All other models of CNV, whether they involve implantation of sustained-release polymers or gene transfer, have a component of surgical damage to Bruch’s membrane (58,59). Therefore, as noted in genetic experiments mentioned above, some sort of compromise of Bruch’s membrane must accompany increased levels of angiogenic factors to generate CNV.

Laser-induced rupture of CNV in mice (54) has provided a particularly valuable tool, because it can be used in genetically engineered mice to explore the role of individual gene products. Using this strategy, Ozaki et al. (52) demonstrated that mice with targeted deletion of FGF2 develop CNV similar to that in wild-type mice indicating that FGF2 is not necessary for the development of CNV after rupture of Bruch’s membrane. This approach was also used to demonstrate that nitric oxide (NO) is proangiogenic in both the retina and the choroid, but different isoforms of nitric oxide synthetase play a role (60). For retinal neovascularization, eNOS plays an important role, while for CNV, nNOS is important. This suggests that NOS inhibitors may be useful in patients at risk for CNV.

V.PROSPECTS FOR PHARMACOLOGICAL TREATMENTS FOR CNV

Since hypoxia has not been definitely implicated in the development of CNV, unlike the situation for retinal neovascularization, there is no strong rationale for suspecting that VEGF, as opposed to the many other angiogenic factors that have been identified, plays a central role in CNV. Therefore, we were somewhat surprised to find that oral administration of drugs that inhibit VEGF receptor kinases dramatically inhibit CNV as well as retinal neovascularization (61,62). Antagonizing VEGF by other means could also be beneficial. Intravitreous injection of a fragment of an anti-VEGF antibody inhibits CNV after laserinduced rupture of Bruch’s membrane in primates (63). Intravitreous injection of the same anti-VEGF antibody fragment (64) or an aptamer that binds VEGF (65) have been tested in phase 1 trials in patients with subfoveal CNV and are currently in phase 2 trials.

Another approach for treatment is to use an endogenous inhibitor of angiogenesis. Endostatin is a cleavage product of collagen XVIII that inhibits tumor angiogenesis resulting in dramatic tumor regression (12). However, proteins can be difficult to work with and some studies using the protein have suggested against a strong antiangiogenic effect. Gene transfer provides a strategy to achieve sustained release of endostatin and can circumvent difficulties arising from handling the protein. We performed intravascular injections of adnenoviral vectors containing a transgene consisting of murine Ig -chain leader sequence coupled to sequence coding for murine endostatin (66). Mice injected with a construct in which endostatin expression was driven by the Rous sarcoma virus promoter had moderately high serum levels of endostatin and significantly smaller CNV lesions at sites of laser-induced rupture of Bruch’s membrane than mice injected with null virus. Mice injected with a construct in which endostatin expression was driven by the cytomegalovirus promoter had roughly 10-fold higher endostatin serum levels and had significantly less CNV with nearly complete inhibition. There was a strong inverse correlation between endostatin serum level and area of CNV. This study provides proof of the principle that gene therapy to increase levels of endostatin can inhibit the development of CNV.

A potential advantage of gene therapy is that intraocular injection of a vector containing an expression construct provides a potential means of sustained local delivery. We investigated the effect of adenoviral-mediated intraocular transfer of the PEDF gene. Intravitreous injection of an adenoviral vector encoding PEDF resulted in expression of PEDF mRNA in the eye measured by RT-PCR and increased immunohistochemical staining for PEDF protein throughout the retina. In mice with laser-induced rupture of Bruch’s membrane, choroidal neovascularization was significantly reduced after intravitreous injection of PEDF vector compared to injection of null vector or no injection. Subretinal injection of the PEDF vector resulted in prominent staining for PEDF in retinal pigmented epithelial cells and strong inhibition of choroidal neovascularization. In two models of retinal neovascularization [transgenic mice with increased expression of vascular endothelial growth factor (VEGF) in photoreceptors and mice with oxygen-induced ischemic retinopathy], intravitreous injection of null vector resulted in decreased neovascularization compared to no injection, but intravitreous injection of PEDF vector resulted in further inhibition of neovascularization that was statistically significant. Several studies have suggested that PEDF has neuroprotective activity (67–72) and it might contribute to the trophic support of photoreceptors provided by RPE cells, because in an in vitro model of photoreceptor degeneration in which the RPE is removed from Xenopus eyecups, PEDF protected photoreceptors from degeneration and loss of opsin immunoreactivity (73). Therefore, intraocular PEDF gene transfer may provide a good approach in patients with AMD, because it could possibly benefit both neovascular and nonneovascular AMD.

Recently, it has been demonstrated that intraocular injection of an adenoassociated viral vector containing a cDNA for angiostatin inhibits laser-induced CNV. Therefore, three different proteins have been found to inhibit CNV (74).

VI. CONCLUSIONS

Current treatments for neovascular AMD do not address the underlying stimuli for abnormal blood vessel growth and are basically palliative treatments. As our understanding of the molecular signals that lead to AMD improves, opportunities for more effective

pharmacological treatments will increase. Several agents, including VEGF receptor kinase inhibitors, anti-VEGF antibodies, PEDF, and angiostatin, that effectively prevent CNV in animal models have been identified. Over the next several years many clinical trials will be performed and it is highly likely that one or more beneficial drugs and/or transgenes will be identified.

ACKNOWLEDGMENTS

This work was supported by grants EY05951, EY12609, and P30EY1765 from the National Eye Institute, the Foundation Fighting Blindness, Lew R. Wasserman Merit Awards (SV and PAC), and unrestricted funds from Research to Prevent Blindness. PAC is the George S. and Dolores Dore Eccles Professor of Ophthalmology and Neuroscience.

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