Abstract

Modulation of VEGF signaling: the therapeutic hub

Angiogenesis (see Glossary) is a physiological process involving the growth of new blood vessels from pre-existing vessels. In the eye, physiologic angiogenesis is critical during early development and indispensable for normal vision. In contrast, many disease states involving the eye such as diabetic retinopathy, retinal vein occlusions, age-related macular degeneration (AMD), and retinopathy of prematurity (ROP), have been linked to the disrupted regulation of this process leading to pathologic angiogenesis and blindness (Figure 1) [1].

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Figure1

Anti-VEGF treatment of neovascular eye diseases

Fluorescein angiogram of a control eye (a), a patient with wet AMD (b) or proliferative diabetic retinopathy (PDR) (c). Bright hyperfluorescence demonstrates leakage of dye from the choroidal neovascularization (CNV) under the macula (b, arrow) and retinal neovascularization (c, arrows). Anomalous circulation characterized by neovascularization, ischemia and collateral vessel formation can be noted in the retina of PDR patient (c). Optical coherence tomography (OCT) of the central retina of an AMD patient before (d) and after (e) anti-VEGF treatments. Intraocular injections of anti-VEGF neutralizing antibody (e) led to resolution of cystoid macular edema (d, stars) and significant improvement in the CNV (d, arrowheads). OCT of neovascular AMD patient (f) shows a pigment epithelial detachment (PED) overlying the CNV (f, arrow). After intravitreal anti-VEGF treatment (g), there is an increase in the size of the PED (including the CNV complex) as well as the amount of subretinal fluid indicating persistent CNV activity (g, arrows).

Diabetes often results in significant tissue damage as manifested by nephropathy, neuropathy and in the eye, diabetic retinopathy. The systemic changes associated with elevated blood glucose and metabolic syndrome associated with diabetes lead to significant end organ damage in the eye, and especially in the retinal microcirculation [2]. Diabetic retinopathy is the leading cause of blindness in working age adults in the U.S., and it is associated with functional and structural alterations of retinal vasculature that later result in capillary degeneration and retinal ischemia. This is a public health crisis. In the U.S. alone, there are almost 750,000 people with proliferative diabetic retinopathy and over 60,000 new cases are reported each year. The earliest manifestation of diabetic retinopathy is in the form of pericyte loss that leads to instability of the retinal capillaries, formation of capillary microaneurysms, development of ischemic zones within the retina, and capillary leakage with subsequent retinal exudation and edema. Progressive damage to retinal microvascular network leads to tissue ischemia, upregulation of hypoxia inducible factor-1 (HIF-1), stimulation of vascular endothelial growth factor (VEGF) secretion and retinal neovascularization characterized by the development of new retinal and pre-retinal vascular fronds [3].

AMD is multifactorial disease and aging is the main risk factor. There are two forms of AMD; neovascular or wet AMD, and non-neovascular or dry AMD, which precedes the wet form [4]. Advanced AMD is characterized by an alteration of regulatory processes that either affect cell survival or induce cell proliferation, specifically neovascularization. AMD is the leading cause of blindness in people over 50 years of age in the industrialized world. Dry AMD is characterized by the development of lipid rich deposits called drusen underneath the retina and within the macula. The composition of these deposits has been reviewed recently [5]. As the disease progresses, drusen components induce inflammation and alterations in the retinal pigmented epithelium (RPE). In some patients, disease progresses to the advanced stages that can be associated with severe central vision loss. In advanced dry AMD, vision loss is secondary to RPE damage and cell death that subsequently leads to photoreceptor cell death and atrophic changes in the neurosensory retina. In the neovascular form, blindness is caused by pathogenic and proliferative neovascularization underneath the retina (choroidal neovascularization, CNV). The majority of acute vision loss in AMD is from the wet form of disease. These features can be overlapping as patients with advanced dry AMD can develop CNV and vice versa. In addition, as therapeutic agents targeted at factors that promote neovascularization become more efficacious, vision loss from atrophic retinal changes associated with dry AMD are becoming more prevalent [6].

Formerly known as retrolental fibroplasia, ROP was originally described in the 1940s by Terry, who first connected disease symptoms with premature birth [7]. At that time, no treatment for ROP was available. Major advances in ROP treatment became available in the 1980s and 1990s, when cryotherapy and laser photocoagulation of the avascular retina were shown to be partially effective in preventing blindness in ROP infants [8]. Although these treatments can reduce the incidence of blindness by 25%, patients are often left with poor visual acuity even after treatment. The pathogenesis of ROP has been associated with the use of high levels of oxygen on premature infants. The hyperoxia of the extra uterine environment, as well as the use of supplemented oxygen given to premature infants, slows or ceases retinal vascular growth that would normally occur in utero, and developed retinal vessels begin to regress [9]. As the infant matures, the non-vascularized retina becomes increasingly metabolically active, and in the absence of adequate vascularization, leads to tissue ischemia and hypoxia. This results in compensatory retinal neovascularization. New vessels form at the junction between the vascularized retina and the avascular zone of the retina. Over time, this pathological growth of vessels produces a fibrous scar extending from the retina to the vitreous gel and lens [9].

VEGF has been identified as one of the critical mediators of pathologic neovascularization. The role of VEGF in angiogenesis has been examined in great detail and is described in several review articles [10, 11]. In the eye, numerous studies have demonstrated the crucial role of VEGF during retinal vascular development [12, 13]. In this highly regulated process, VEGF is essential for the proliferation and migration of endothelial cells. It is also required for proper vascular patterning and tube formation (Figure 2). Additionally, the distinct vascular defects observed in experimental animal models depleted for alternative splice variants of VEGF confirmed the precise role of these isoforms in regulating retinal angiogenesis [14]. VEGF signaling has been attributed a key role in the maintenance of corneal transparency and the avascular state. Imbalance in VEGF related pathway is strongly associated with corneal neovascularization, a common clinical feature in diverse corneal diseases [15, 16]. More recent work demonstrated that VEGF also plays a prominent role in overall ocular development. Indeed, selective inactivation of VEGF expression in RPE resulted in a severe reduction of eyeball size associated with the loss of visual function [17]. RPE derived VEGF is also involved in the formation and maintenance of the choroidal vasculature that provides oxygen and nutrients to the outer retina [18].

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Figure2

Role of VEGF in retinal angiogenesis and homeostasis

Schematic cross-section through the human eye (a) and detailed representation of the retina (b). In the retina, VEGF is produced and secreted by neurons, glia and vascular endothelial cells. Additionally, different cell types also express VEGF receptor. During retinal vascular development, VEGF signaling plays an essential role in the formation and patterning of the vascular network (vascular plexus). The retinal pigmented epithelium (RPE)-derived VEGF is responsible for the development and the maintenance of the choriocapillaries. It also provides neurotrophic support to photoreceptors, retinal bipolar cells (RBC) and retinal ganglion cells (RGC). Retinal VEGF expression is upregulated in response to hypoxia and contributes to pathologic neovascularization. In these conditions, the majority of the VEGF is produced by Müller glial cells and astrocytes.

The role of VEGF in ocular neovascular-related diseases has been characterized in experimental and translational studies [19]. Elevated intraocular VEGF is closely linked to the pathogenesis of several blinding eye diseases. In addition, early findings that the inhibition of VEGF signaling prevented neovascularization in ischemic retinal diseases highlighted the therapeutic relevance of anti-VEGF strategies in these conditions [20]. However, there is now substantial evidence showing that VEGF expression can also be induced through hypoxia-independent mechanisms [19].

Although VEGF clearly drives pathologic angiogenesis in disease, it also plays a crucial role in cellular homeostasis as a neurotrophic and cell survival factor [21-24]. Numerous cell types in the eye express and secrete VEGF, including endothelial cells, glial cells, retinal neurons, RPE, and immune cells such as monocytes/macrophages [25-28]. In disease states, intraocular VEGF expression is upregulated by hypoxia and inflammation [29]. VEGF-driven neovascularization and increased vascular permeability lead to complications including bleeding, retinal detachment and fibrovascular proliferation, all of which ultimately cause photoreceptor neuron death and blindness [1, 11]. As such, many treatment options for these complications are geared towards modifying VEGF levels within the eye in order to inhibit abnormal angiogenesis (Figure 1d,e). Numerous clinical trials have investigated therapies targeting VEGF and have shown efficacy in controlling disease and preventing vision loss (Table 1) [30-41].

It is important to note that therapeutic VEGF inhibition leads to significant vision recovery in only a subset of patients with the retinal diseases highlighted above [42]. As such, VEGF-driven pathways are only a part of the complex machinery regulating angiogenesis in the eye [42]. This creates a large unmet need for developing novel strategies targeting pathways that regulate ocular angiogenesis that are independent of VEGF (Table 2). In addition, VEGF also plays a critical role in normal homeostasis in the eye in maintaining vascular integrity, as a neurotrophic and neuroprotective factor in the retina and the central nervous system, and indiscriminate or prolonged VEGF inhibition can lead to cell death and vision loss as demonstrated in animal models, cell culture and human randomized clinical trials [24, 42-44]. In this review, we will focus on VEGF-independent pathways involving the immune system that could provide additional insights into the regulation of normal and pathological angiogenesis in the eye.

Innate immunity and ocular angiogenesis

In both physiological and pathological conditions, the innate immune system has been shown to play a determinant role in the vascular remodeling process. The link between inflammation and angiogenesis has been defined in the pathophysiology of several diseases, including cancer, atherosclerosis and eye disorders [1, 5, 45]. In these conditions, activation of innate immunity is often an early event in the disease process that further guides the local tissue microenvironment and ultimately molds cellular and molecular responses. The crucial role of VEGF signaling in ocular angiogenesis has been extensively studied and the molecular response of endothelial cells to VEGF is well documented [1, 10, 11]. There is also a wealth of evidence implicating innate immunity in key processes during angiogenesis, including endothelial cell proliferation, migration and vessel anastomosis [46]. The concomitant activation of an innate immune response, associated with dysregulated blood vessel growth (as observed in many eye diseases), and the implication of inflammatory mediators in VEGF signaling have led to the belief that VEGF plays an omnipotent role in regulating ocular angiogenesis. Although recent translational studies targeting VEGF have confirmed its pivotal role in blinding eye diseases, there is emerging evidence highlighting VEGF-independent pathways in disease pathogenesis that suggest potentially novel approaches involving components of innate immunity that may modulate pathological angiogenesis in the eye.

The role of adaptive immunity in ocular angiogenesis

Recent studies have suggested that the adaptive immune compartment may play a role in promoting pathologic angiogenesis in the eye [72-75]. A number of studies have suggested that Th17 cells play a role in advanced AMD [76-78]. Th17 cells are effector CD4+ T cells that preferentially produce IL-17 upon specific surface markers stimulation or activation by various pathogens. IL-17 is a pro-inflammatory cytokine that mostly targets epithelial, endothelial and stromal cells and defective production of this cytokine is associated with several autoimmune diseases [79, 80]. Complement components such as C5a have been implicated in inducing CD4+ T cells to express IL-17 and IL-22. The level of these cytokines was also increased in serum from patients with AMD [76]. In addition, one study showed that the IL-17RC promoter, a component of the IL-17R complex, was significantly hypomethylated in patients with AMD [81]. This was associated with increased expression of IL-17RC in the peripheral blood, retina and choroid of AMD patients [81]. A subsequent study that carefully examined the methylation status of the IL-17RC promoter using global methylation arrays, bisulfite sequencing and PCR-based methylation assays found no significant difference between AMD patients and age-matched controls in two independent cohorts [82]. These findings were consistent for no difference either in the peripheral blood or in eye-derived tissues such as the retina or choroid [82]. Furthermore, it was demonstrated that IL-17 alone had no impact on vessel growth, although it can potentiate the effect of other angiogenic factors such as VEGF, basic fibroblast growth factor (bFGF) and hepatocyte growth factor (HGF) [72]. Recent findings have shown that combined production of IL-17 from γδT cells and innate lymphoid cells was responsible for the increased neovascularization observed in experimental model of CNV [74]. As such, the precise role of IL-17 in regulating angiogenesis in the eye remains unclear.

Complement components and lipid peroxidation can induce oxidation specific epitopes that may function as antigens for immune recognition [83]. Carboxyethyl pyrrole (CEP) is an example of such an epitope that is an oxidation fragment of docohexaenoic acid (DHA) [84]. CEP has previously been shown in animals to initiate an immune response that leads to damage to the RPE similar to that seen in non-neovascular AMD [85-87]. In addition, not only is CEP increased in AMD donor eyes, but autoantibodies to CEP and CEP-modified proteins can be identified in plasma from AMD patients [88, 89]. The mechanism by which CEP induces an immune response is thought to be via the activation of T lymphocytes by classically activated M1 macrophages [77]. In murine models, this leads to increased expression of IFN-γ and IL-17 [77]. Despite the association of CEP and similar epitopes with the disease phenotype in dry (non-neovascular) AMD, the link to pathologic angiogenesis and wet AMD remains elusive.

Autoantibody profiling of sera from AMD patients has identified retinal autoantibodies that have the ability to promote endothelial cell growth in vitro. Although underlying mechanisms that lead to endothelial cell proliferation are unknown, these findings suggest that autoantibodies could be involved in the pathogenesis of neovascular AMD and especially in CNV formation [73].

Concluding remarks and future perspectives

It is now apparent that chronic, intraocular anti-VEGF treatments could lead to permanent vision loss due to the pleiotropic effects of VEGF and, more importantly, its neurotrophic potential. Both experimental and translational studies have confirmed the deleterious effects of long-term VEGF inhibition. There are several lines of evidence for VEGF-independent regulation of ocular angiogenesis. Different components of the immune system have been shown to modulate vascularization in the eye. Although some regulatory pathways are specific to the innate or adaptive compartment, the interplay between immune components and retinal cells is necessary for the control of ocular angiogenesis (Figure 3). For instance, macrophages/microglia can directly regulate endothelial cells growth upon activation, and also mediate the effects of the complement system. The impact of immune responses on retinal vasculature can be facilitated or enhanced by surrounding neurons. The latter shows the interconnection between the immune system, retinal vasculature and neurons. Future studies will determine the impact of selective inhibition of ocular angiogenesis on neuron survival and local immune responses (Box 2).

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Figure3

Immune regulation of VEGF-independent angiogenesis

Depending on their activation state, macrophages/microglia can differentially modulate endothelial cell growth. M1 macrophages are anti-angiogenic, whereas M2 macrophages promote angiogenesis. The effect of macrophages on endothelial cells can be contact-dependent or through secretion of angiogenic factors. Complement factor B (cfB), a component of the alternative complement pathway, in combination with the complement inhibitor (CD55), selectively inhibits pathological neovessel formation. In hypoxic conditions, CD55 is downregulated in endothelial cells therefore allowing the specific targeting of neovessels by cfB. Complement components can also modulate macrophage activation. Complement components C3 and C5 polarize macrophages to an M1 anti-angiogenic phenotype. Activation of microglial cells upon exposure to ischemia induces the secretion of IL-1β and subsequent production of Semaphorin-3a (Sema-3A) by retinal neurons. Sema-3A induces degeneration of the retinal vasculature. The adaptive immunity may promote angiogenesis through autoantibodies or secretion of IL-17 by T cells. Ocular angiogenesis can be further regulated through the remodeling of the extracellular matrix (ECM). Activation of ECM-stimulated pathways through neuropilin-1 (NRP1) signaling also promotes angiogenesis in the eye.

Acknowledgments

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