1. Introduction

218 M. Ushio-Fukai & R. W. Alexander

oxidative stress is associated with the pathogenesis of various cardiovascular diseases including hypertension, heart failure and cardiac hypertrophy, atherosclerosis, and diabetes9 Low (micromolar or submicromolar) levels of ROS, which can be produced during tissue ischemia/hypoxia or ischemic preconditioning,10−12 stimulate EC proliferation and migration13,14 as well as angiogenesis in vivo.10−12,14−18 The precise underlying mechanisms, however, are not completely understood.

Although multiple ROS-generating systems have been described, NAD(P)H oxidase (NOX) enzymes are one of the major source of ROS in endothelial and other vascular cells.9 Vascular cells may also be exposed to exogenous ROS produced extrinsically by phagocytes or circulating enzymes such as xanthine oxidase.19 Endogenous ROS derived from NAD(P)H oxidase serve as signaling molecules to activate multiple intracellular signaling pathways leading to cell growth, migration, and modification of the extracellular matrix, which are fundamental responses contributing to angiogenesis. Of importance are angiogenic growth factors such as VEGF and angiopoietin-1, which stimulate, through their receptor tyrosine kinases (RTK), NAD(P)H oxidase-derived ROS production, which are involved in EC migration and proliferation.20,21 We and others showed that NAD(P)H oxidase plays an essential role in vivo in VEGF-mediated postnatal neovascularization,20 hindlimb ischemia,12 as well as ischemic retinopathy.18 Moreover, the peptide hormone angiotensin II (Ang II), a major stimulus for vascular NAD(P)H oxidase,22−25 plays an important role in angiogenesis. In this chapter we shall summarize recent progress that has been made in the rapidly emerging area of redox signaling and the role of ROS in angiogenesis.

2. Reactive Oxygen Species (ROS) in the Vasculature

Macrophages and monocytes have been assumed to be the source of most of the ROS in the vessel wall. However, virtually all vascular cells such as endothelial cells, vascular smooth muscle cells (VSMCs) and adventitial fibroblasts produce ROS, in varying amounts and in response to diverse stimuli.9 Cells also use different enzymes to produce

and scavenge ROS in different circumstances. Ultimately, it is the balance of pro-oxidant and antioxidant enzyme activity that dictates both intracellular and extracellular ROS levels. ROS can then act in an autocrine or paracrine fashion to modulate cellular function. In the vasculature, multiple enzymatic systems produce ROS, including the NAD(P)H oxidases, xanthine oxidase, myeloperoxidase, uncoupled endothelial nitric oxide synthase (eNOS), the cytochrome p450s, cyclooxygenases, and mitochondrial electron transport chain.9

Cellular redox state is an important determinant regulating cell growth, survival, apoptosis and gene expression. One of the most important product of ROS in the vasculature is superoxide (O•2−), which is formed by the univalent reduction of oxygen.4 Although O•2− can itself exert effects on vascular function, at high concentrations its reaction with nitric oxide (NO•) to generate peroxynitrite (ONOO−), a highly reactive ROS, can be particularly deleterious. Superoxide is rapidly dismutated by superoxide dismutase (SOD) which consists of three isoforms, including extracellular SOD (ecSOD), cytosolic copper/zinc SOD (Cu/ZnSOD), and the mitochondrial-restricted manganese SOD (MnSOD), thereby resulting in producing the more stable and diffusible ROS, hydrogen peroxide (H2O2). Of importance, H2O2 functions as a signaling molecule to regulate various biological responses in the vasculature.26 H2O2 is then converted enzymatically into H2O by catalase and glutathione peroxidase (GPx), or undergoes Fenton reaction to form hydroxyl radical (•OH) in the presence of heavy metals, or metabolized by myeloperoxidase (MPO) to form hypochlorous acid (HOCl) (Fig. 1).

3. ROS and Angiogenesis

Accumulating evidence reveals that ROS are emerging as important regulators of angiogenesis. Exogenous ROS stimulates induction of VEGF by VSMCs,27 skeletal myotubes,28 fibroblasts,29 keratinocytes,29,30 retinal pigment epithelial cells,31 human U937 macrophage, rat peritoneal macrophages and RAW264.7 cell lines32 as well as ECs.33 High concentrations of ROS inhibit while low concentrations of ROS stimulate EC proliferation.13,14,34 In ECs, H2O2 also induces angiogenic-related responses including cell migration,34 cytoskeletal reorganization35 and

220 M. Ushio-Fukai & R. W. Alexander

Fig. 1. Schema of superoxide generation. See text for details.

tubular morphogenesis.36 H2O2 is also involved in the T-lymphocyte activation and induction of angiogenesis during tumor growth.37

Hypoxia/reoxygenation, which produces ROS, elicits capillary tube formation in human microvascular ECs grown on a 3D reconstituted extracellular matrix (Matrigel).38 Scratch wounding of confluent monolayers of ECs stimulates H2O2 accumulation in actively migrating cells at the wound edge area, which is required for EC migration towards the site of injury.39,40 Adhesion of activated polymorphonuclear leukocytes (PMNs) to ECs promotes angiogenesis through an increase in H2O2.41 Advanced glycation end-products (AGEs) stimulate VEGF expression in RAW264.7 macrophages through an increase in ROS, thereby contributing to the development of angiopathy in diabetes mellitus.42 Leptin, a circulating hormone secreted mainly from adipose tissue, functions as an angiogenic factor43 and increases VEGF mRNA expression and EC proliferation through an increase in ROS.44

Antioxidants, green tea catechins and vitamin E, inhibit angiogenesis-related responses of human microvascular ECs via suppression of IL-8 production.45 Recently, Oak et al.46 reported that natural polyphenols, which have antioxidant properties, inhibit key angiogenic processes such as proliferation and migration of ECs and VSMCs

as well the expression of two major pro-angiogenic factors, VEGF and matrix metalloproteinase (MMP)-2.46 Pigment epithelium-derived factor (PEDF), a potent natural inhibitor of angiogenesis 47 with antioxidant properties, blocks angiogenic effects of leptin through inhibiting ROS production in ECs 44 as well as the H2O2-induced increase in VEGF mRNA in retinal pericytes.48 Thus, PEDF might be a novel therapeutic antioxidant factor for treatment of angiogenesis-dependent pathophysiologies such as diabetic retinopathy and cancer.

In vivo, there is strong correlation between ROS production with neovascularization and VEGF expression in the eyes of diabetics 49−51 and in balloon injured arteries.27 ROS are increased during the reperfusion of the ischemic retina in vivo, which contributes to VEGF mRNA expression through increasing its mRNA stability.31 Short exposure to hypoxia/reoxygenation in hearts produces ROS, which contributes to myocardial angiogenesis.10,11 Of note, arteriogenesis is an important aspect of the development of collateral circulation. Gu et al. reported that brief coronary artery occulusion and reperfusion of dogs cause ROS production which contribute to coronary collateral development.52 Moreover, it has been proposed that ROS play an important role in wound healing and repair processes in vivo.53,54

The antioxidant, pyrrolidine dithiocarbamate55 and the major green tea extract, epigallocatechin-3-gallate (EGCG) which has antioxidant properties,56 inhibit retinal neovascularization in the mouse. Similarly, EGCG prevents the growth of new blood vessels in a murine Matrigel model.57 The natural compounds in red wine and grapes block angiogenesis in the chick embryo chorioallantoic membrane (CAM) or cornea models of mice.46,58 The SOD, its membrane permeable mimetic tempol, catalase, and the NAD(P)H oxidase inhibitors, 4-(2- aminoetyyl)-benzenesulfonyl fluoride (AEBSF) and apocynin, but not the xanthine/xanthine oxidase inhibitor allopurinol, decrease angiogenesis and the expression and activity of iNOS in the CAM model.59 The thiol antioxidant, N-acetylcysteine (NAC) attenuates EC invasion and angiogenesis in a tumor model in vivo.60 Moreover, overexpression of PEDF inhibits angiogenesis and melanoma growth in vivo.61 Wheeler et al.62 demonstrated that overexpression of ecSOD using adenovirus inhibits tumor vascularization and growth of B16 melanomas