play a critical role in the transition from nonproliferative to proliferative diabetic retinopathy. The evidence supporting the importance of VEGF in diabetic retinopathy is presented, including clinical, preclinical, and basic research studies. Furthermore, the regulation of VEGF expression in the retina as well as its actions at the cellular and molecular level is discussed in detail. In the light of VEGF’s pathophysiologic importance in DR, the development of therapeutics targeting VEGF and its downstream actions is a promising approach for current and future treatment of proliferative diabetic retinopathy.

Key Words: Angiogenesis; endothelial cell; retinal neovascularization; VEGF.

INTRODUCTION

Despite improvements in medical management of diabetes and treatment of ocular complications, diabetic retinopathy (DR) remains the most common cause of severe visual loss in working-age adults in the United States and other industrialized countries. In the United States, DR results in blindness in over 10,000 individuals with diabetes per year (1). Retinal neovascularization, the formation of new blood vessels from preexisting blood vessels, is a major underlying factor, and can cause severe vision loss from vitreous hemorrhage and tractional retinal detachment.

Significant research advances have been made regarding the mechanisms underlying the development of retinal neovascularization in DR. In particular, the identification of vascular endothelial growth factor (VEGF) as a major stimulus of retinal neovascularization has led to the development of therapies targeting this growth factor, and anti-VEGF treatments are being increasingly used in clinical management of patients with advanced diabetic retinopathy. This chapter is divided into two parts. The first part focuses on the current understanding of the mechanisms of angiogenesis, particularly with respect to diabetic retinopathy. The second part focuses on VEGF’s critical role in retinal neovascularization, as well as its functional and biochemical properties, which provide insights with potentially important implications for anti-VEGF therapy in humans.

PROGRESSION OF NONPROLIFERATIVE TO PROLIFERATIVE

DIABETIC RETINOPATHY

Diabetic retinopathy is clinically divided into two stages, nonproliferative diabetic retinopathy (NPDR) and proliferative diabetic retinopathy (PDR), which is characterized by retinal neovascularization (Figs. 1 and 2). As NPDR progresses, retinal capillary dropout occurs which results in progressive retinal ischemia and hypoxia. Ischemia and hypoxia are thought to play a critical role in the transition from nonproliferative to proliferative diabetic retinopathy. Indeed, the concept of ischemia and hypoxia as stimulators of retinal neovascularization arose over half a century ago (2, 3), supported by clinical observations. For instance, neovascularization commonly occurs at the borders of perfused and nonperfused retina. In addition, retinal neovascularization is more common and severe in eyes with extensive capillary nonperfusion.

Ischemia and hypoxia result in the upregulation of various molecules that promote angiogenesis, including pro-angiogenic growth factors. Specifically, ischemic retinal cells secrete vasoproliferative growth factors which induce the formation of

Fig. 1. Optic nerve head neovascularization in proliferative diabetic retinopathy.

Fig. 2. Early retinal neovascularization in diabetes, with a new vessel extending from the retina into the vitreous. (Courtesy of W. Richard Green, MD.) H&E, original magnification ×160.

new blood vessels in the retina or iris. This neovascularization constitutes the hallmark of proliferative diabetic retinopathy. The new vessels grow along the retinal surface and along the vitreous scaffold of the posterior vitreous hyaloid. These new vessels are fragile and often bleed, resulting in preretinal as well as vitreous hemorrhage. In addition, glial tissue associated with the new vessels can contract, producing traction on the retina and eventually leading to retinal detachment. Vitreous hemorrhage

and traction retinal detachment are the direct cause of most cases of severe vision loss in diabetic retinopathy.

STAGES OF ANGIOGENESIS

Angiogenesis, the formation of new blood vessels from existing vessels, occurs through a multi-step process, including: production of angiogenic growth factors by diseased tissue, binding of angiogenic growth factors to receptors on existing vascular endothelial cells (EC), activation of EC gene expression of pro-angiogenic molecules, EC invasion of surrounding tissue, EC migration and proliferation, formation of vascular tubes by EC, and stabilization of new blood vessels by mural cells. Each of these steps is potentially vulnerable to pharmacologic targeting, and anti-angiogenic therapies directed at various steps are under investigation (4).

Under normal conditions, the vasculature is quiescent except during processes such as wound healing and the menstrual cycle, presumably due to a balance between inducers and inhibitors of angiogenesis (5). A critical step in the initiation of angiogenesis arises from changes in the tissue milieu which leads to an imbalance between inducers and inhibitors, either from increased levels of inducers, decreased levels of inhibitors, or both. Hypoxia in the retina is thought to alter this balance largely by increasing levels of pro-angiogenic growth factors. An important mediator of this process is hypoxiainducible factor 1 (HIF-1), which is a hetero-dimer of α and β subunits. HIF-1 is a transcriptional regulator which is induced by hypoxia and which activates the transcription of an array of hypoxia-inducible genes. In the mouse model of oxygen-induced ischemic retinopathy, HIF-1α protein levels were increased in the retina, particularly in the hypoxic inner retina (6). HIF-1 is known to activate the transcription of multiple proangiogenic molecules, including VEGF and erythropoietin. Indeed, intraocular injection of an adenovirus encoding a constitutively-active form of HIF-1α resulted in increased retinal levels of messenger RNAs for various angiogenic growth factors, including VEGF, placental growth factor, angiopoietin-2, and platelet-derived growth factor-B (7).

The binding of pro-angiogenic growth factors to their cognate receptor(s) on preexisting vascular endothelial cells (ECs) results in the activation of these cells, causing an increase in the expression of molecules important for the angiogenic process, including integrins and proteinases. Invasion of endothelial cells through the capillary basement membrane and extracellular matrix is dependent on the production and activation of extracellular proteinases, particularly the serine proteinase, urokinase plasminogen activator (uPA), as well as members of the matrix metalloproteinase (MMP) family. The expression of proteinase genes is induced by angiogenic growth factors including VEGF. In addition, the proteolytic process is induced by activation of pro-proteinases and downregulation of protease inhibitors. A detailed discussion of uPA and MMP’s is provided in Chap. 16.

Dissolution of the capillary basement membrane and surrounding tissue is accompanied by endothelial cell migration. Growth factor-induced activation of endothelial cells leads to increased expression and activation of integrins, including αvβ3 and αvβ5 (see Chap. 16). These cell surface adhesion molecules play an important role in the attachment of endothelial cells to specific ligands in the extracellular matrix, including fibronectin, which serve as a scaffold for the migrating endothelial cells.

Activated endothelial cells proliferate and subsequently form vascular tubes. These immature vessels undergo further remodeling, with subsequent formation of a new basement membrane as well as recruitment of mural cells (pericytes or smooth muscle cells) to form a mature vessel. The recruitment of these mural cells is particularly important for the stabilization of the new blood vessels, and plays a critical role in the development of vessel resistance to regression (8).

More recently, it has become appreciated that in addition to preexisting vascular endothelial cells, endothelial progenitor cells from the circulation may also play a role in retinal neovascularization. When hematopoietic stem cells (HSCs) containing a population of endothelial progenitor cells (EPCs) were administered by intravitreal injection into neonatal mouse eyes, there was stable incorporation of some of these cells into the developing retinal vasculature (9). In addition, systemic administration of donor HSCs in an animal model of retinal venous occlusion resulted in incorporation of a subset of these cells into the retinal neovasculature (10). Therefore, it is possible that EPCs may also play an important part in proliferative diabetic retinopathy, which may have therapeutic implications.

ANIMAL MODELS OF RETINAL NV: THE OXYGEN-INDUCED RETINOPATHY MODEL OF RETINAL NEOVASCULARIZATION

Existing animal models of diabetes have been limited by the absence of advanced lesions of diabetic retinopathy, including preretinal neovascularization. This is likely due in part to the shorter life span of these animals. Consequently, studies of retinal neovascularization have largely focused on animal models of retinopathy of prematurity. One of the most widely used animal models is the mouse model of oxygen-induced retinopathy (OIR) (11).

Development of the retinal vasculature in mice occurs postnatally. In the mouse model of OIR, neonatal mice are exposed to high oxygen tensions (typically around 75%) from postnatal Day 7 (P7) until P12. This hyperoxic exposure results in retinal vessel regression and cessation of normal radial vessel growth. This vaso-obliteration leads to extensive retinal nonperfusion. The mice are then returned to room temperature at P12. The nonperfused retina becomes hypoxic, leading to the elaboration of angiogenic growth factors and retinal neovascularization, which is typically maximal by P17 (12). Although this model clearly has important differences from proliferative diabetic retinopathy, it shares important similarities, most notably the induction of retinal neovascularization by retinal ischemia and hypoxia. The model has proven very useful in allowing the acquisition of insights into the pathogenesis of ischemic retinopathies, including PDR. Many studies have also been performed in related animal models of ROP, including the rat (13).

VASCULAR ENDOTHELIAL GROWTH FACTOR

It has long been known that retinal neovascularization is strongly associated with retinal ischemia, based on clinical observations of ischemic retinopathies including diabetic retinopathy. Retinal capillary nonperfusion precedes neovascularization in these retinopathies (14, 15). The degree of capillary nonperfusion correlates with the