Vascular endothelial growth factor in eye disease

Abstract

Collectively, angiogenic ocular conditions represent the leading cause of irreversible vision loss in developed countries. In the US, for example, retinopathy of prematurity, diabetic retinopathy and age-related macular degeneration are the principal causes of blindness in the infant, working age and elderly populations, respectively. Evidence suggests that vascular endothelial growth factor (VEGF), a 40 kDa dimeric glycoprotein, promotes angiogenesis in each of these conditions, making it a highly significant therapeutic target. However, VEGF is pleiotropic, affecting a broad spectrum of endothelial, neuronal and glial behaviors, and confounding the validity of anti-VEGF strategies, particularly under chronic disease conditions. In fact, among other functions VEGF can influence cell proliferation, cell migration, proteolysis, cell survival and vessel permeability in a wide variety of biological contexts. This article will describe the roles played by VEGF in the pathogenesis of retinopathy of prematurity, diabetic retinopathy and age-related macular degeneration. The potential disadvantages of inhibiting VEGF will be discussed, as will the rationales for targeting other VEGF-related modulators of angiogenesis.

Introduction

Vascular endothelial growth factor (VEGF), a dimeric glycoprotein of approximately 40 kDa, is a potent, endothelial cell mitogen that stimulates proliferation, migration and tube formation leading to angiogenic growth of new blood vessels. It is essential for angiogenesis during development; the deletion of a single allele arrests angiogenesis and causes embryonic lethality (Ferrara et al., 1996). In mammals, the VEGF family consists of seven members: VEGF-A (typically, and hereafter, referred to as VEGF), VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F and PlGF (placental growth factor) (Fig. 1A). Alternative splicing results in several VEGF variants. In humans, these include the relatively abundant VEGF₁₂₁, VEGF₁₆₅, VEGF₁₈₉ and VEGF₂₀₆, and several less-abundant forms (Fig. 1B). The solubility of these splice variants (collectively referred to as VEGF_XXX) is dependent on heparin-binding affinity. VEGF₂₀₆ and VEGF₁₈₉ bind very tightly to heparin and, thus, remain sequestered in the extracellular matrix. VEGF₁₆₅ binds heparin with less affinity, but also can be associated with the matrix, and VEGF₁₂₁ lacks heparin-binding capacity, rendering it highly soluble. Investigations in mice genetically engineered to express less than the full complement of splice variants confirm that the relative solubility of VEGF splice variants strongly affects their specific bioactivities (Takahashi and Shibuya, 2005). Moreover, plasmin and various metalloproteinases can cleave VEGF₁₆₅, resulting in an N-terminal 113-amino acid peptide that is non-heparin binding, but retains its bioactivity (Keyt et al., 1996; Ferrara et al., 2003). Our understanding of the relative expression of the different VEGF isoforms under normal or pathological conditions and the molecular regulators of VEGF alternative splicing is relatively limited.

Recently, discovery of the so called “VEGF_XXXb isoforms” has sparked new interest in the molecular events that regulate VEGF expression (for review, see Ladomery et al., 2007). The VEGF_XXXb isoforms share approximately 94–98% homology with the corresponding VEGF_XXX isoforms and have the same length. However, due to alterations in the C-terminus they bind to VEGF receptors, but do not fully activate them and act as “dominant negative splice variants” (Bates et al., 2002). Studies showing that VEGF_165b is downregulated in angiogenic tissues (Ladomery et al., 2007) suggest a primary role for this isoform in controlling VEGF activity in health and disease. Administration of VEGF_165b has been shown to inhibit retinal angiogenesis in the mouse model of oxygen-induced retinopathy (Konopatskaya et al., 2006).

VEGFR-1/Flt-1 (fms-like tyrosine kinase) and VEGFR-2/KDR/Flk-1 (kinase insert domain-containing receptor/fetal liver kinase), along with structurally related receptors, Flt-3/Flk-2 and VEGFR-3/Flt-4, belong to the receptor tyrosine kinase family (Fig. 1A) (Hanks and Quinn, 1991; Blume-Jensen and Hunter, 2001). VEGFR-1 and -2 are primarily involved in angiogenesis (Yancopoulos et al., 2000) whereas Flt-3 and Flt-4 are involved in hematopoiesis and lymphangiogenesis (Jussila and Alitalo, 2000). The VEGFRs contain an approximately 750-amino-acid-residue extracellular domain, which is organized into seven immunoglobulin-like folds. Adjacent to the extracellular domain is a single transmembrane region, followed by a juxtamembrane domain, a split tyrosine kinase domain that is interrupted by a 70-amino-acid kinase insert and a C-terminal tail.

VEGF receptor activation requires dimerization. Guided by the binding properties of the ligands, VEGFRs form both homodimers and heterodimers (Rahimi, 2006). The signal transduction properties of the VEGFR heterodimers, compared with homodimers, remain to be fully elucidated. Dimerization of VEGFR is accompanied by activation of receptor kinase activity, leading to autophosphorylation. Site-directed mutagenesis studies have demonstrated that the Tyr1214 residue, located in the carboxy terminus of VEGFR-2, is required for the ligand-dependent autophosphorylation of the receptor and its ability to activate signaling proteins. Signal transduction is propagated when activated VEGF receptors phosphorylate SH2 domain-containing protein substrates.

In addition to VEGFRs, VEGF serves as a ligand to another family of receptors, the neuropilins. Neuropilins are 120–130-kDa non-tyrosine kinase receptors that mediate critical functions in tumor cells and the nervous and vascular systems. In endothelial cells, neuropilins serve as receptors for the class 3 semaphorins and co-receptors for VEGF family members. The role of Neuropilin-1 (NRP-1) in mediating VEGF activity is now being elucidated. VEGF signaling through NRP-1 stimulates endothelial cell migration and adhesion. The addition of an anti-NRP-1 antibody suppressed the mitogenic effects of VEGF₁₆₅ on bovine retinal endothelial cells (RECs) (Oh et al., 2002). In another in vitro model, the VEGF-dependent differentiation of a subset of human bone marrow-derived cells into vascular precursors, and subsequent proliferation of these cells, required the activation of a VEGFR-2/NRP-1-dependent signaling pathway (Fons et al., 2004). Finally, VEGF promotion of the synthesis and release of prostacyclin (PGI₂), an important mediator of angiogenesis, is thought to be mediated via NRP-1 binding (Neagoe et al., 2005). The angiogenic effects regulated through VEGF binding to NRP-2 are less well characterized and appear to be modulated differently from the effects controlled by NRP-1. For example, VEGF selectively upregulates NRP-1, but not NRP-2, on endothelial cells (Oh et al., 2002).

BIAcore analysis has shown NRP-1 to interact with VEGFR-1, greatly reducing its binding affinity for VEGF₁₆₅ (Fuh et al., 2000). Co-culture systems of endothelial cells and breast carcinoma cells indicate that NRP-1 significantly enhances VEGF₁₆₅ binding to VEGFR-2 (Soker et al., 2002). In aortic endothelial cells, NRP-2 interacted with VEGFR-1, but less is known at present about how this influences VEGF bioactivity (Gluzman-Poltorak et al., 2001). Finally, using multiple in vitro systems, NRP-2 was shown to interact with VEGFR-3, leading to lymphangiogenic activity, but no interaction was seen between NRP-2 and VEGFR-2 (Karpanen et al., 2006).

Few SH2 domain-containing proteins have been shown to interact directly with VEGFR-2. Phospholipase C-γ (PLCγ binds to phosphorylated Tyr1175 (Tyr1173 in the mouse), and mediates the activation of the mitogen-activated protein kinase (MAPK) cascade, leading to proliferation of endothelial cells (Takahashi et al., 2001) (Fig. 2). PLCγ activates protein kinase C via the production of diacylglycerol and increased concentrations of intracellular calcium. A Tyr1173Phe mutation of VEGFR-2 causes embryonic lethality due to vascular defects, mimicking the defects of VEGFR-2^−/− mice (Sakurai et al., 2005). These data demonstrate an essential function of the Tyr1173 residue during vascular development.

In addition to PLCγ, the adaptor molecule, Shb, also binds to phosphorylated Tyr1175. VEGF-induced migration and PI3K activation are inhibited by small interfering RNA (siRNA)-mediated knockdown of Shb in endothelial cells (Holmqvist et al., 2004). The serine/threonine kinase, Akt, is activated downstream of PI3K and mediates endothelial cell survival (Fujio and Walsh, 1999). Akt also regulates nitric oxide (NO) production by direct phosphorylation and activation of endothelial NO synthase (eNOS). Finally, phosphorylated Tyr1175 is known to interact with Sck (Igarashi et al., 1998; Sakai et al., 2000), an adaptor molecule that binds Grb2, and participates in MAPK signaling in the epidermal growth factor pathway (Thelemann et al., 2005).

Another important phosphorylation site in VEGFR-2 is Tyr951 (Tyr949 in the mouse), a binding site for the signaling adaptor VEGF receptor-associated protein (VRAP) (Matsumoto et al., 2005). The phosphorylated Tyr951–VRAP pathway has been shown to regulate endothelial cell migration (Matsumoto et al., 2005; Zeng et al., 2001). Reduced microvessel density and tumor growth in VRAP^−/− mice confirm an essential function for this residue in endothelial cells of the angiogenic phenotype (Matsumoto et al., 2005). VEGF induces the formation of a complex between VRAP and Src (Matsumoto et al., 2005), indicating that VRAP might regulate Src activation and its signaling downstream of VEGFR-2.

Mice that express a Tyr1212Phe (corresponding to the human Tyr1214) VEGFR-2 mutant are viable and fertile (Sakurai et al., 2005). However, phosphorylation of Tyr1212/1214 has been implicated in VEGF-induced actin remodeling through the sequential activation of CDC42 and p38 MAPK (Lamalice et al., 2004). Inhibition of p38 MAPK augments VEGF-induced angiogenesis in the chicken chorioallantoic membrane (CAM) assay (Issbrucker et al., 2003; Matsumoto, T., et al., 2002), without an accompanying increase in vascular permeability (Issbrucker et al., 2003). Moreover, p38 MAPK induces phosphorylation of heat-shock protein-27 (HSP27), a molecular chaperone that positively regulates VEGF-induced actin reorganization and migration (McMullen et al., 2005; Rousseau et al., 1997).

The existence of multiple ligands and receptors provides initial diversity to VEGF bioactivity. Groupings of receptor homo- and heterodimers, activated by both common and specific ligands, further augment the diversity of VEGF signaling. A final level of diversity is provided by activation of distinct signaling intermediates downstream of each VEGF receptor. The combination of these features yields an elaborate signaling network capable of regulating the extremely complex angiogenic cascade.

During hypoxia, VEGF gene expression increases via several different mechanisms (Dor et al., 2001). These mechanisms include increased transcription, mRNA stability, and protein translation using an internal ribosomal entry site (IRES), as well as increased expression of oxygen-regulated protein 150 (ORP 150), a chaperone required for intracellular transport of proteins from the endoplasmic reticulum to the Golgi apparatus prior to secretion (Chen and Shyu, 1995; Forsythe et al., 1996; Levy and Levy et al., 1996, Levy and Chung et al., 1998; Ozawa et al., 2001).

The increase in VEGF transcription is largely mediated via hypoxia inducible factor-1 (HIF-1) (Fig. 3). HIF-1 is a heterodimeric transcription factor composed of two subunits—the constitutively produced HIF-1β subunit and the inducible component, HIF-1α (Wang and Semenza, 1995). Under normoxic conditions, HIF-1α is inactivated and targeted for proteosomal degradation by hydroxylation, whereas under hypoxic conditions the specific hydroxylases are inhibited, resulting in the rescue of HIF-1α from degradation (Schofield and Ratcliffe, 2004). When this occurs, HIF-1α complexes with HIF-1β, translocates into the nucleus and binds to a specific sequence in the 5′ flanking region of the VEGF gene, the hypoxia responsive element (HRE) (Ikeda et al., 1995; Laughner et al., 2001; Shima et al., 1996; Wenger, 2002; Forsythe et al., 1996). The importance of interaction between HIF-1α and the VEGF promoter has been confirmed in studies of HIF-1α^−/− mouse embryonic stem cells, in which basal expression of VEGF mRNA remains low in response to hypoxia (Carmeliet et al., 1998; Iyer et al., 1998).

Two additional isoforms of HIF, known as HIF-2α and HIF-3α, have been identified by screening for proteins that associate with HIF-1β (Ratcliffe, 2007). HIF-2α appears to be closely related to HIF-1α and can promote HRE-dependent gene transcription. While structurally and functionally similar, HIF-1α and HIF-2α appear to exert different biological functions, as demonstrated in studies using knockout mice (Hu et al., 2003). For example, while HIF-1α antagonizes c-Myc function, inhibiting renal cell carcinoma (RCC) growth, HIF-2α promotes cell cycle progression in hypoxic RCC and many other cell lines (Gordan et al., 2007). Interestingly, the most distantly related isoform, HIF-3α, appears to antagonize HRE-dependent gene expression, suggesting a possible negative influence on hypoxia-induced gene expression. Additional study is needed to determine if HIF-2α or HIF-3α is involved in the regulation of retinal VEGF expression.

Clearly, post-transcriptional events are also important in the regulation of VEGF production in the diseased retina, as underscored by the correlation of polymorphisms within the 5′-untranslated region (UTR) of the VEGF gene with the occurrence of age-related macular degeneration (AMD). The 3′UTR and the 5′UTR of the VEGF gene are important sites of regulation controlling mRNA stability and the rate of translation through the IRES (for review, see Yoo et al., 2006).

Evidence for the increase in VEGF mRNA stability in response to hypoxia comes from in vitro mRNA degradation assays that have led to the identification of adenylate/uridylate-rich elements (AREs) in the 3′ UTR of VEGF mRNA. VEGF mRNA is extremely labile in normoxic conditions, with a half-life of less than 1 h, as compared with the average half-life of 10–12 h for eukaryotic mRNA. During hypoxia, the half-life of VEGF mRNA increases by 2–3-fold (Levy et al., 1996) due to a stabilizing effect of HuR, a 36 kDa RNA-binding protein, which binds with high affinity to AREs in the 3′-UTR of VEGF mRNA, protecting it from degradation by endonucleases (Brennan and Steitz, 2001; Robinow et al., 1988).

Post-transcriptional regulation can also occur in the 5′-UTR of VEGF mRNA. This region contains multiple IRES. These are specific sites of attachment to the ribosomal machinery, which provide sites for initiation of translation alternative to the classical 5′ cap- and elF-dependent translational system (for review, see van der Velden and Thomas, 1999). Several IRES have been identified in the 5′-UTR of VEGF mRNA, and these provide alternative sites of translational control of VEGF expression (Bornes et al., 2004). Notably, evidence suggests that IRES sites in the VEGF 5′-UTR can potentially control the generation of alternatively spliced VEGF (Bornes et al., 2004; Huez et al., 2001).

Another regulatory mechanism consists of increased production of ORP150 in response to hypoxia. Studies using human macrophages transfected with adenovirus coding for ORP150 showed that overexpression of ORP150 resulted in increased VEGF secretion in hypoxia. Evidence suggests that under hypoxic conditions, ORP150 functions as a molecular chaperone to facilitate VEGF protein transport and secretion (Ozawa et al., 2001).

VEGF is not only regulated by hypoxia. VEGF function is also affected by insulin-like growth factor 1 (IGF-1) which plays an important role in retinal vascularization. Several lines of evidence, including in vitro studies, support the notion that IGF-1 is critical for vessel development (King et al., 1985; Grant et al., 1993). Preterm infants with reduced serum levels of IGF-1 have a higher incidence of development of retinopathy (Hellstrom et al., 2003). Mice null for the IGF-1 gene have retarded retinal vascular growth compared to wild-type controls (Hellstrom et al., 2001). However, the action of IGF-1 is not mediated by decreasing VEGF expression, as the amount of VEGF mRNA is similar in knockout and wild-type control mice; instead IGF-1 acts by decreasing VEGF activation of the Akt signaling pathway. Both MAPK and Akt pathways have been shown to be necessary for endothelial cell survival (Smith et al., 1999).

The influence of VEGF in retinal diseases is profound. It has been implicated in a large number of retinal diseases and conditions including, but not limited to, highly prevalent conditions like age-related macular degeneration (AMD) and diabetic retinopathy; less common disorders such as retinopathy of prematurity, sickle cell retinopathy and retinal vascular occlusion; and as a non-causal, but important, secondary influence in neovascular glaucoma (Bock et al., 2007) and inherited retinal dystrophies (Penn et al., 2000). Collectively, these conditions, all of which have critical angiogenic components, account for the vast majority of irreversible vision loss in developed countries.

At least five retinal cell types have the capacity to produce and secrete VEGF. These include the retinal pigmented epithelium (RPE) (Miller et al., 1997), astrocytes (Stone et al., 1995), Müller cells (Robbins et al., 1997), vascular endothelium (Aiello et al., 1995a) and ganglion cells (Ida et al., 2003). These cells differ widely in their responses to hypoxia; in vitro studies show that Müller cells and astrocytes generally produce the greatest amounts of VEGF under hypoxic conditions (Morrison et al., 2007; Aiello et al., 1995a; Hata et al., 1995). To date, the relative capacity of these cells to produce specific splice variants remains unclear, as do the patterns of splice variant production throughout retinal development and aging.

The distinct roles of the different VEGF splice variants in retinal vascular development is being explored, however, in mice expressing only a single variant (Stalmans et al., 2002). Vascular development was normal in the retinas of mice expressing only VEGF₁₆₄ (VEGF^164/164), indicating that this variant is sufficient for directing normal vascular growth and remodeling. In contrast, retinas of VEGF^120/120 mice exhibited severe vascular defects, displaying retarded venous and severely flawed arterial development. VEGF^188/188 mice had normal development of retinal veins but little or no arterial growth.

Evidence for the expression patterns and roles of VEGFR in retinal tissues comes from a variety of species and experimental venues. In the human retina VEGFR-1 and -2 can be expressed by neural, glial and vascular elements. In adults, expression is generally restricted to the inner nuclear layer (Müller cells and amacrine cells), the ganglion cell layer, and the retinal vasculature (Stitt et al., 1998). However, during retinal neurogenesis VEGFR-2 is also expressed by neural progenitor cells (Hashimoto et al., 2006). Notably, neural cell VEGFR-2 can be activated by VEGF in vitro (Yang and Cepko, 1996). In cultured retinal pericytes VEGFR-1, but not -2, is expressed (Takagi et al., 1996), whereas in cultured RPE cells, both receptors are expressed and are induced by oxidative stress (Sreekumar et al., 2006). In the mouse, ganglion cells express both receptors, but only VEGFR-2 is increased by intraocular inoculation with herpesvirus (Vinores et al., 2001). Studies in newborn mice using the VEGFR-specific kinase inhibitor, SU5416, indicate that Müller cell survival or proliferation during retinal development is VEGFR- and MAPK-dependent (Robinson et al., 2001). In a study of patients with diabetic retinopathy, VEGFR-1 expression dominated in normal retina, but was not increased in the diabetic retina, while VEGFR-2 levels were increased, particularly in the vascular elements (Smith et al., 1999). Finally, VEGFR-1 and -2 are found on uterine smooth muscle cells in vivo. When these cells are cultured in vitro, VEGFR-1 can be phosphorylated and is capable of inducing smooth muscle cell proliferation (Brown et al., 1997). To date, neither VEGFR-1 nor -2 has been identified in retinal smooth muscle cells.

This article will review the role of VEGF in angiogenesis related to three blinding conditions: retinopathy of prematurity, diabetic retinopathy and age-related macular degeneration. These conditions constitute the leading causes of irreversible vision loss in infants, and working age and elderly Americans, respectively. That VEGF is believed to play a causal role in all three of these disorders underscores its profound impact in eye disease. VEGF antagonists have already proven their value in tumor angiogenesis and choroidal neovascularization, and new VEGF antagonists are being tested pre-clinically and clinically for other ocular indications. Only with a more complete understanding of VEGF and its retinal and choroidal activities can we hope to develop better strategies to prevent, retard or repair the damage caused by ocular neovascularization.