Targeting Angiogenesis in Neovascular AMD

DELIA N. SANG, MD, FACS · MARK S. HUGHES, MD, FACS

Neovascular age-related macular degeneration (AMD) is a complex condition with a multifactorial pathogenesis that has yet to be completely characterized. The development of choroidal neovascularization (CNV) involves many interacting factors, including increased vascular permeability, proangiogenic signals, anomalous matrix alteration, remodeling, and inflammation.^1,2 The introduction of anti–vascular endothelial growth factor (VEGF) therapies, such as ranibizumab (Lucentis, Genentech), was a revolutionary advance and has been shown to significantly improve visual acuity (VA) in many patients with CNV due to AMD.^3,4 But the benefits of ranibizumab therapy are not necessarily uniform nor universal.^3-5 Variability in outcomes among patients is probably related to the involvement of factors other than VEGF,^6-9 as well as the effects of inflammation and stage of CNV. Combining ranibizumab with treatments that target different steps in angiogenesis might have the potential to enhance treatment outcomes.

In this article, we consider new agents directed against VEGF, as well as some of the steps within the angiogenic process that represent additional potential targets for new treatments for CNV due to AMD.

ANGIOGENESIS

Angiogenesis is the growth of new vessels (neovascularization) from pre-existing vessels, and it proceeds through several stages, beginning with the degeneration of basement membranes and ending with the development of differentiated vessels.¹⁰ Of the factors involved in angiogenesis, most attention has been given to VEGF, which stimulates endothelial cell mitogenesis, proliferation, vascular permeability, extracellular matrix degradation,^11-15 and the formation of new vessels from existing choroid vasculature.^16-18 The role of VEGF in angiogenesis was illustrated by the beneficial effects of ranibizumab, which binds to and inhibits all isoforms of VEGF. Also, many other modalities are under investigation for their potential to inhibit VEGF, including VEGF-Trap-Eye and small interfering RNA (siRNA) therapies.¹⁹ Other investigational strategies have focused on blocking the downstream effects of VEGF and targeting other factors involved in angiogenesis.

NEW AGENTS DIRECTED AT VEGF

VEGF Trap-Eye

VEGF Trap-Eye is a recombinant protein consisting of the binding regions of VEGF receptors 1 and 2 and the constant region of human immunoglobulin G1.²⁰ The binding of VEGF Trap-Eye to VEGF forms an inert stable complex, thereby blocking proangiogenic effects.²⁰ In a phase 1 study of 25 patients with neovascular AMD, excess retinal thickness at day 71 was reduced by 63% with VEGF Trap-Eye at a dose of 1 mg/kg (the maximum tolerated dose) and 5.6% with placebo.²¹ Also, a dose-escalation study showed that single intravitreal injections of VEGF Trap-Eye at doses of 0.05 mg to 4 mg resulted in a mean increase of 4.8 letters of VA and a reduction in central retinal thickness from 298 μm to 208 μm.²² VEGF Trap-Eye has a long duration of action and maintains anti-VEGF activity for 10 to 12 weeks after a single injection.²³ It may have the potential for less frequent dosing than with other anti-VEGF agents. Phase 3 trials of VEGF Trap-Eye are ongoing.

Small Interfering RNA (siRNA)

RNA interference has a catalytic mechanism that enables one siRNA molecule to facilitate the cleavage of thousands of mRNA molecules.²⁴ Two siRNAs are under investigation as potential treatments for neovascular AMD, one targeting VEGF-A (bevasiranib; OPKO Health, Miami)²⁴ and the other targeting VEGF receptor 1 (AGN211745 or SIRNA-027; Allergan, Irvine, CA).²⁵

VEGF Tyrosine Kinase Inhibitors

Vascular endothelial growth factor binds to 2 receptor tyrosine kinases, VEGF R1 and VEGF R2. Several tyrosine kinase inhibitors may provide a means to target these receptors, including vatalanib (previously known as PTK787). Vatalanib blocks the phosphorylation of VEGF and platelet-derived growth factor (PDGF) receptors and has been shown to prevent retinal neovascularization induced by ischemia in mice.²⁶ Vatalanib is currently under investigation in clinical trials for patients with CNV due to AMD.

Multikinase inhibitors, such as TG100572 (TargeGen, San Diego), are also under investigation for neovascular AMD. Delivery of TG100572 to the retina and choroid seems to be enhanced by administration in the form of a prodrug, TG100801.²⁷ Topical use of TG100801 suppresses laser-induced CNV in mice and reduces fluorescein leakage and retinal thickening in a rat model of retinal vein occlusion.²⁷ Other multikinase inhibitors under investigation include pazopanib (GW786034; GlaxoSmithKline, Philadelphia), which inhibits VEGF receptors 1, 2, and 3, PEDF receptors α and β, and c-kit.²⁸

NEW AGENTS DIRECTED AT NON-VEGF TARGETS

Complement Cascade

Increasing evidence has implicated the complement system in the development of drusen²⁹ and in the progression from non-neovascular to neovascular AMD. The development of CNV due to AMD has been associated with the presence of basal laminar and linear deposits consisting of extracellular matrix components and vitronectin and occurring alongside activation of the complement cascade and VEGF.³⁰ Complement components C3a and C5a have been shown to upregulate expression of VEGF³¹ and have been seen in specimens from patients with AMD and in animal models of laser-induced CNV.^32,33 Complement and associated immune complexes may both be implicated in damage to the retinal pigment epithelium (RPE).³⁴

The complement factor H (CFH) gene has been implicated in AMD development.³⁵ It is estimated that 43% to 50% of cases of AMD are associated with the expression of a single nucleotide substitution at position 402 in CFH, which increases the risk of AMD by 2.45- to 5.57-fold.^36,37 A CFH polymorphism in which tyrosine is replaced with histidine at position 402 of CFH (the CFH Y402H polymorphism) is implicated as a key risk factor for neovascular AMD, CNV, and geographic atrophy,^38,39 and it has also been associated with the presence of complement in choroidal capillaries and other vessels.⁴⁰ Position 402 of CFH is in a region that binds heparin and C-reactive protein (CRP),⁴⁰ which is important given that CRP haplotypes influence the expression of CFH.⁴¹ The CFH Y402H polymorphism may place individuals at risk of developing AMD if it occurs alongside factors that stimulate the complement cascade.⁴¹ CFH Y402H genotypes have also been shown to be associated with differences in the size of CNV lesions and in responses to treatment.⁴²

The important role of complement in the pathogenesis of neovascular AMD has raised interesting possibilities for the development of new treatments. A number of agents are under investigation as potential complement inhibitors, including TA106 (a monoclonal antibody against complement 3; Taligen Therapeutics, Aurora, CO), POT-4 (a peptide that binds to complement 3; Potential Pharmaceuticals, Louisville, KY), and ARC1905 (an anti-complement 5 aptamer; Ophthotech, Princeton, NJ).

Inflammatory Cells

There is increasing evidence that immunologic events are important in the development and progression of AMD, with macrophages playing a key role.⁴³ There is also evidence that the activation of monocytes may be a marker of increased risk of neovascular AMD.⁴⁴ Several studies have revealed the presence of macrophages and similar cells in specimens taken from individuals with AMD, with the cells often localized in areas of RPE atrophy, Bruch's membrane damage, and CNV.^9,45-47 Oxidized lipoproteins have been shown to occur within CNV, with macrophages expressing cell surface receptors for these lipoproteins.⁴⁸ Experimental models have indicated that the macrophages occurring in association with CNV appear to be derived from bone marrow and are recruited from the blood.⁴⁹ Migration of macrophages from the vasculature into adjacent ocular tissue may result from alterations or deficiencies in chemokines, especially CCL2 and its receptor CCR2.⁴³

There is also evidence for the involvement of a wider variety of inflammatory cells in neovascular AMD pathogenesis, including lymphocytes, fibroblasts, and myofibroblasts.⁵⁰These may be participants in a chronic local inflammatory response centered on degenerated RPE cells involved in drusen formation.²⁹ VEGF upregulation was shown to precede macrophage infiltration in an animal model of CNV,⁵¹ although macrophages and RPE cells are also associated with subsequent additional increases in VEGF.

Targeting leucocytes or the adhesion molecules involved in their recruitment may provide potential for new treatments. For example, the blockade of vascular adhesion protein-1, an endothelial cell adhesion molecule involved in leucocyte recruitment, has been shown to reduce CNV size, fluorescein leakage, and macrophage accumulation while reducing the expression of inflammatory mediators such as tumor necrosis factor-α, monocyte chemoattractant protein-1, and intercellular adhesion molecule-1.⁵²

Platelet-derived Growth Factor

Platelet-derived growth factor may also play a role in CNV and acts as a mitogen for pericytes, smooth muscles cells, fibroblasts, and other mesenchymal cells. Pericytes may be critical for the maintenance of established blood vessels and may be important in the maturation process in CNV.

There is evidence that, in newly formed vessels, there is a period during which endothelial cells depend on VEGF⁵³ and are responsive to the withdrawal of PDGF.⁵⁴ In contrast, established mature vessels may exhibit less dependence on VEGF and may be associated with resistance to anti-VEGF or with rapid recurrence of CNV due to AMD. These observations are consistent with evidence that blockade of both VEGF-A and PDGF was more effective at suppressing neovascularization in experimental models than the blockade of VEGF-A alone.⁵⁵

PDGF has been shown to stimulate angiogenesis and pericyte recruitment.^7,8 The loss of pericytes from retinal vessels may be associated with vascular abnormalities and instability, including the formation of microaneurysms and vascular permeability. Blocking PDGF appears to result in loss of pericytes and possibly regression of maturing neovascularization.⁵⁴ Continued production of VEGF-A by pericytes under certain conditions may protect endothelial cells from the effects of VEGF-A blockade by anti-VEGF agents.⁵⁶

Integrins

Integrins are cell adhesion molecules involved in the mediation of cell-cell, cell–extracellular matrix, and cell-pathogen interactions.⁵⁷ Structurally, integrins consist of a heterodimer with 1 α and 1 β subunit. In vertebrates, 18 α subunits and 8 β subunits have been identified and are known to form 24 α/β pairs.⁵⁷ Several integrins have been shown to stimulate endothelial cell migration and macrophage recruitment under specific conditions.⁵⁸

There is growing evidence that β1 integrins are involved in angiogenesis, especially with regard to the maturation and organization of neovascularization. Integrin adhesion receptor activation appears to be important in angiogenesis in both the developing embryo and in disease, while in the central nervous system, β1 integrin expression has been shown to be involved throughout the angiogenic process up to the maturation of vessels.⁵⁹

The blockade of integrins has been shown to inhibit endothelial cell proliferation and induce apoptosis,⁶⁰ while several inhibitors of angiogenesis, such as endostatin, can mediate their effects by binding to integrins.⁶¹ Integrins have also been shown to play a role in VEGF-mediated cell adhesion and endothelial cell migration, which can be suppressed by blocking integrin α5β1 with antibodies.⁶²

There has been interest in the role of integrins in neovascular AMD based on evidence that integrin receptors play important roles in tumor angiogenesis.¹⁰ Blockade of integrin α5β1, the most important of the fibronectin receptors, has been shown to result in the suppression of tube formation stimulated by VEGF and in the apoptosis of proliferating (but not quiescent) endothelial cells.⁶⁰ An anti-α5β1 antibody inhibited VEGF-dependent and VEGF-independent angiogenesis in an experimental model, while anti-VEGF inhibited only VEGF-dependent angiogenesis.⁶⁰ Further evidence of the potential anti-angiogenic effects of integrin inhibition was provided by a study of EMD478761, an α5β3/α5β5 integrin antagonist, in a chick chorioallantoic membrane assay and a rat model of laser-induced CNV.⁶³ In this study, EMD478761 significantly reduced angiogenesis induced by administration of basic fibroblast growth factor and suppressed the CNV development.

Pigment epithelium-Derived Factor

Pigment epithelium-derived factor (PEDF) is an antiangiogenic factor that counters the effects of VEGF.⁶⁴ Therefore, administering PEDF could have the potential to exhibit antiangiogenic effects, although high doses appear to have the opposite effect and lead to increased neovascularization.⁶⁵ It may also be possible to increase local production of PEDF by delivering the PEDF gene to the retina using an adenovector (AdPEDF).⁶⁶

Other Potential Targets

A variety of other potential antiangiogenic treatments could merit further investigation in neovascular AMD, including microtubule inhibitors, nucleic acid therapies, peroxisome proliferator-activated receptor agonists, and rapamycin. Further investigation is needed to determine whether these agents have the potential to target angiogenesis and improve outcomes in neovascular AMD.

CONCLUSIONS

Vascular endothelial growth factor is central to the pathogenesis of CNV in AMD, and its inhibition by agents such as ranibizumab has provided important advances in therapy. However, it is important to note that AMD is a complex condition in which many factors play key roles at different stages and that AMD may not be treated optimally with a single agent. Growing awareness of the structural and inflammatory components of angiogenesis and of the numerous factors involved in the angiogenic process may lead to identification of new targets for novel treatments for CNV due to AMD. In particular, several agents have been developed to target complement, integrins, and PDGF and are under investigation in clinical trials in patients with AMD. If these agents are proven effective, they may provide the means to target multiple steps in the angiogenic process when used in combination with anti-VEGF therapy, thereby providing more complete inhibition of neovascularization than can be achieved with monotherapy. RP

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