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discussed earlier have demonstrated the significant role for VEGF and have been confirmed by multiple subsequent studies, including a study blocking VEGF receptor signaling (58).
The emerging use of anti-VEGF therapies in patients has provided further suggestive evidence corroborating the important role of VEGF in proliferative diabetic retinopathy. For instance, a retrospective analysis was performed in a clinical trial evaluating the anti-VEGF aptamer, pegaptanib, for the treatment of diabetic macular edema (for further details, see Chap. 17). A subset of the study participants exhibited retinal neovascularization in the study eye at baseline. Eight of 13 patients receiving pegaptanib injection (including one which also received laser photocoagulation) had subsequent regression of neovascularization, compared with 0 of 3 in the sham treatment group. Notably, 4 of the 13 pegaptanib-treated patients also had neovascularization in the fellow (untreated) eye that did not regress. Although the study clearly had a small sample size, and indeed was not designed to directly address anti-VEGF and retinal neovascularization, it supports a direct effect of anti-VEGF treatment upon retinal neovascularization (59). The strongest clinical evidence demonstrating a causative role for VEGF in ocular neovascularization comes from clinical trials demonstrating dramatic efficacy of anti-VEGF therapy for choroidal neovascularization in age-related macular degeneration (60). Based on the body of experimental and clinical evidence, anti-VEGF treatments have emerged as a clinical option for the treatment of proliferative diabetic retinopathy, for instance in the context of neovascular glaucoma or vitreous hemorrhage.
BASIC VEGF BIOLOGY
The importance of VEGF as a stimulator of angiogenesis, both in ocular and systemic conditions, has driven intensive research efforts into its basic biology, including its mechanisms of action. In addition to improving our understanding of angiogenesis, these insights into VEGF biology have provided an array of targets for therapeutic manipulation. VEGF (also referred to as VEGF-A) is part of a gene family whose members include placental growth factor (PlGF) (61), VEGF-B (62), VEGF-C (63, 64), and VEGF-D (65). Each of these family members can interact with one or more of three VEGF receptors (Fig. 4).
VEGF has four primary isoforms, generated by alternative splicing of VEGF RNA, which contain 121, 165, 189, and 206 amino acids. These isoforms are referred to, respectively, as VEGF121, VEGF165, VEGF189, and VEGF206 (66, 67). Of these, VEGF165 is the predominant isoform. An important distinguishing property of the VEGF isoforms is their ability to bind heparin, conferred by heparin-binding peptides in exons 6 and 7 of the VEGF gene. VEGF121 lacks both exons, does not bind heparin, and is freely diffusible. In contrast, VEGF189 and VEGF206 contain both exons and are almost completely bound by heparin-like moieties in the extracellular matrix. VEGF165, which contains exon 7 but not 6, has intermediate properties.
RECEPTORS
There are two related high-affinity receptor tyrosine kinases for VEGF: VEGFR-1 (fms-like tyrosine kinase-1 or Flt-1) and VEGFR-2 (kinase insert domain-containing receptor or KDR). Both have seven extracellular immunoglobulin-like domains, a single
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Fig. 4. VEGF receptors and their ligands. VEGF (also referred to as VEGF-A) binds two related receptor tyrosine kinases (RTKs), VEGFR-1 (also known as Flt-1) and VEGFR-2 (also known as KDR). Both VEGFR-1 and VEGFR-2 have an extracellular domain containing seven immunoglobulin-like loops (ovals), a single transmembrane region, and a cytoplasmic domain consisting of a single kinase domain (rectangles) interrupted by a non-catalytic region. VEGF-C and VEGF-D also bind to VEGFR2. Placental growth factor (PlGF) and VEGF-B bind only to VEGFR1. VEGFR3 (also known as Flt-4) is a member of the same family of receptor tyrosine kinases and binds VEGF-C and VEGF-D.
hydrophobic transmembrane domain, and a conserved intracellular tyrosine kinase domain which is interrupted by a kinase insert domain (68, 69). Both VEGFR1 and VEGFR2 are autophosphorylating tyrosine kinases with binding affinities for VEGF in the low picomolar range. VEGFR-2 is known to be the major mediator of VEGF’s mitogenic, angiogenic, and permeability-stimulating effects (70). VEGFR-3 (fms-like- tyrosine kinase-4 or Flt-4) is also a member of the VEGFR family which is a receptor for VEGF-C and VEGF-D, but not VEGF (64, 71) (Fig. 4).
In addition to VEGFR1 and VEGFR2, neuropilin-1 (Npn-1) and neuropilin-2 (Npn-2) serve as coreceptors for VEGF. Neuropilin-1 (72) and neuropilin-2 (73) bind VEGF165
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with high affinity, but do not bind VEGF121. The binding of VEGF165 to these receptors is heparin-dependent. When coexpressed in cells with VEGFR2, neuropilin-1 enhances the binding of VEGF165 to VEGFR2 as well as the stimulation of chemotaxis by VEGF165. In addition, the inhibition of VEGF165 binding to neuropilin-1 inhibits its binding to VEGFR2 as well as its mitogenic activity for endothelial cells (72). These and other studies indicate that the neuropilins function in the enhancement of VEGF signaling and activation of endothelial cells.
VEGF’S MULTIPLE ACTIONS ON RETINAL ENDOTHELIAL CELLS
Consistent with its critical role in stimulating angiogenesis, VEGF stimulates multiple steps in the angiogenic process, including survival, migration, proliferation, tubulogenesis, and vascular permeability (70, 74). These effects have been demonstrated in retinal microvascular endothelial cells in addition to numerous other endothelial cell types. Notably, retinal endothelial cells express cell surface VEGF receptors at a higher density than many other endothelial cell types (75). VEGF has been demonstrated to stimulate retinal endothelial cell proliferation (28), migration (76), survival (77, 78), and tubulogenesis (79). In addition, VEGF stimulates retinal endothelial cell permeability (80, 81). VEGF’s vasopermeability properties in the retina are discussed in greater detail in Chap. 14.
MAIN SIGNALING PATHWAYS
The ability of VEGF to stimulate angiogenesis is dependent on its coordinate regulation of multiple endothelial cell activities. This is dependent on VEGF’s ability to stimulate a network of intracellular signaling pathways. In endothelial cells, VEGFR-2 is the major mediator of VEGF signaling. Upon VEGF binding, VEGFR2 dimerizes, with one receptor, trans(auto)-phosphorylating tyrosine residues in the cytoplasmic domain of its partner (74). The phosphorylated tyrosine residues can bind intracellular signaling molecules and initiate a cascade of signaling events leading to multiple cell responses promoting angiogenesis and vascular permeability.
Although VEGF activates multiple signaling pathways in endothelial cells, extensive research has focused on a few pathways that are thought to play particularly important roles (Fig. 5). VEGF stimulates endothelial cell proliferation primarily through stimulation of extracellular-signal-regulated protein kinases (ERK) 1 and 2, also known as p42/44 mitogen-activated protein (MAP) kinase. VEGF activation of VEGFR2 leads to tyrosine-phosphorylation of phospholipase C-γ (PLC-γ) (82), which leads to the generation of inositol 1,4,5-trisphosphate and diacylgycerol (DAG). DAG activates protein kinase C, which in turn activates the Raf/MEK/ERK pathway, which plays a central role in endothelial cell mitogenesis (82).
Activation of protein kinase C (PKC) is essential for VEGF’s mitogenic effects on endothelial cells. The PKC family of serine-threonine kinases consists of multiple PKC isoforms, which differ in their regulatory and biochemical properties. Intravitreal administration of VEGF activates protein kinase C (PKC) in the retina, inducing membrane translocation of PKC isoforms α, βII, and δ (83). PKC inhibitors block VEGFinduced activation of ERK1/2 (84, 85), and endothelial cell proliferation (86). Although
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Fig. 5. VEGF signaling pathways. Most, and possibly all, of the biologically relevant VEGF signaling are mediated by VEGFR-2. Upon binding its ligand, VEGFR-2 undergoes receptor dimerization and autophosphorylation at multiple tyrosine residues in the intracellular domain. This leads to the activation of multiple signaling molecules, notably Akt, PKC, and ERK1/2. VEGF promotion of endothelial cell survival is largely dependent on PI 3-kinase (PI3K)-mediated activation of the anti-apoptotic kinase Akt. VEGF stimulates endothelial cell proliferation primarily through activation of ERK1/2. Binding of VEGF to VEGFR-2 leads to activation of PLC-γ, leading to generation of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) and subsequent activation of PKC, which in turn mediates activation of ERK1/2. PKC (particularly PKC-β) also has an important role in VEGF’s vasopermeability effects.
other PKC isoforms are likely to be important as well, particular attention has been placed on PKC-β. In bovine aortic endothelial cells, pharmacologic inhibition of PKC-β using the isoform selective inhibitor ruboxistaurin (LY333531) inhibited VEGF’s mitogenic effect (86). In addition to its role in VEGF’s mitogenic effects, PKC-β appears to have an important role in VEGF’s vasopermeability effects. Administration of ruboxistaurin strongly inhibited VEGF-induced retinal vascular permeability in vivo (83). This effect was supported by an in vitro study, demonstrating that expression of a dominant negative PKCβII mutant significantly blocked VEGF-induced permeability of cultured retinal endothelial cells (81).
The phosphatidylinositol 3′-kinase (PI3-kinase)/Akt signaling pathway is particularly important for VEGF’s ability to promote endothelial cell survival. Activation of VEGFR2 leads to phosphorylation and activation of Akt/protein kinase B (87), an antiapoptotic kinase which mediates the promotion of cell survival by a variety of growth