Angiogenesis, a process of new blood vessel formation, is a key process involved in normal development and wound repair as well as in the various pathophysiologies such as ischemic heart and limb diseases and atherosclerosis. Reactive oxygen species (ROS) such as superoxide and H2O2 function as signaling molecules in many aspects of growth factor-mediated responses including angiogenesis. Vascular endothelial growth factor (VEGF) is a key angiogenic growth factor and stimulates proliferation, migration, and tube formation of endothelial cells (ECs) primarily through the VEGF receptor type2 (VEGR2, KDR/Flk1). VEGF binding initiates autophosphorylation of VEGFR2, which results in activation of downstream signaling enzymes including ERK1/2, Akt, and eNOS in ECs, thereby stimulating angiogenesis. The major source of ROS in EC is a NADPH oxidase which consists of Nox1, Nox2 (gp91phox), Nox4, p22phox, p47phox, p67phox and the small G protein Rac1. The endothelial NADPH oxidase is activated by angiogenic factors including VEGF and angiopoietin-1. ROS derived from this enzyme stimulate diverse redox signaling pathways leading to angiogenesis-related gene induction as well as EC migration and proliferation, which may contribute to postnatal angiogenesis in vivo. The aim of this review is to provide an overview of the recent progress on the emerging area of the role of ROS derived from NADPH oxidase and redox signaling in angiogenesis. Understanding these mechanisms may provide insight into the NADPH oxidase and redox signaling components as potential therapeutic targets for treatment of angiogenesis-dependent cardiovascular diseases and for promoting angiogenesis in ischemic limb and heart diseases.
Reactive oxygen species
Angiogenesis, the formation of new blood vessels from the pre-existing vasculature, is involved in physiological processes including embryonic development and wound repair as well as in various pathologies such as ischemic heart and limb disease, cancer, diabetic retinopathy, or chronic inflammation including atherosclerosis . This tightly regulated process involves the degradation of extracellular matrix, disruption of cell–cell contacts, migration and proliferation, and capillary tube formation of endothelial cells (ECs). Vascular endothelial growth factor (VEGF), a key angiogenic growth factor, stimulates proliferation, migration and tube formation of ECs primarily through the VEGF receptor type2 (VEGR2, KDR/Flk1) . ECs generate reactive oxygen species (ROS) such as O2. − and H2O2 which play a role in physiological and pathophysiological responses. High concentrations of ROS cause apoptosis and cell death and oxidative stress is associated with the cardiovascular diseases including hypertension, heart failure, atherosclerosis and diabetes . Low levels of ROS, which are produced in response to growth factor, tissue ischemia/hypoxia or ischemic preconditioning, function as signaling molecules to mediate EC proliferation and migration [4–6], which may contribute to angiogenesis in vivo [7–11].
Signal transduction by ROS, “redox signaling,” has been an emerging area of investigation. The major source of ROS in ECs is a NADPH oxidase. Recently, Nox homologues to Nox2 (gp91phox) of phagocyte cytochrome b588 have been identified in non-phagocytic cells including ECs . NADPH oxidase is activated by numerous stimuli including growth factors such as VEGF, angiopoietin-1, cytokines, shear stress, hypoxia and G-protein coupled receptor agonists including angiotensin II (Ang II) in ECs [3,13,14]. ROS produced via activation of NADPH oxidase stimulate diverse redox signaling pathways leading to angiogenic responses in ECs as well as postnatal neovascularization in vivo [5,8,14–17]. The aim of this review is to briefly summarize the recent progress and information on the redox signaling in angiogenesis with the focus on NADPH oxidase.
2 Role of ROS in angiogenesis in ECs
Exogenous ROS stimulate induction of VEGF by various cell types including vascular smooth muscle cells (VSMCs)  and ECs , and promote cell proliferation and migration [4,6,20], cytoskeletal reorganization  and tubular morphogenesis  in ECs. Hypoxia/reoxygenation and adhesion of activated polymorphonuclear leukocytes to ECs cause ROS production, which results in capillary tube formation [23,24]. Angiogenesis growth factors such as VEGF and angiopoietin-1 induce EC migration and/or proliferation through an increase in ROS [5,14–16]. Ethanol stimulates actin cytoskeletal reorganization, cell motility and tube formation in an ROS-dependent manner in ECs . Leptin, a circulating adipocytokine, upregulates VEGF mRNA and stimulates cell proliferation through an increase in ROS in ECs . Antioxidants such as green tea catechins and vitamin E as well as natural polyphenols from red wine and green tea inhibit angiogenic responses in ECs . Pigment epithelium-derived factor (PEDF), a natural inhibitor of angiogenesis , blocks leptin-induced ROS production in ECs , suggesting that it may function as an endogenous antioxidant factor. These reports suggest that many angiogenenic-related responses are ROS-dependent in cultured ECs.
3 Role of NADPH oxidase in angiogenesis in ECs
ROS are generated from a number of sources including the mitochondrial electron transport system, xanthine oxidase, the cytochrome p450, the NADPH oxidase and nitric oxide synthase (NOS). NADPH oxidase is one of the major sources of ROS in vasculature . NADPH oxidases in phagocytic cells consist of the membrane-bound cytochrome b558 comprising the catalytic gp91phox subunit and the p22phox subunit, and cytosolic components including p47phox, p67phox and the small Rho GTPase Rac1. Recently, novel gp91phox (also termed Nox2) homologues including Nox1, Nox3, Nox4 and Nox5 have been identified in non-phagocytic cells [12,29]. The neutrophil NADPH oxidase releases large amounts of O2− in bursts, whereas the vascular NADPH oxidase(s) continuously produce low levels of O2· − intracellularly in basal state, yet it can be further stimulated acutely by various agonists and growth factors . In ECs, all the phagocytic NADPH oxidase subunits including Nox2, p22phox, Rac1, p47phox as well as Nox1 and Nox4 are expressed [30,31] (Fig. 1).
Structure of NADPH oxidase in ECs. gp91phox (Nox2) and its homologues (Nox1 and Nox4) and cytosolic components p47phox, p67phox and small GTPase Rac1 have been identified in ECs. ROS derived from endothelial NADPH oxidase function as signaling molecules.
3.1 Nox2 and p22phox
Accumulating evidence suggests that Nox2 is a critical component of ROS-generating NADPH oxidase activated by various stimulants in ECs [5,32–35]. VEGF and angiopoietin-1 stimulate EC migration via activation of Nox2-containing NADPH oxidase, suggesting an essential role of Nox2 in angiogenesis in ECs [5,14]. We have shown that Nox2 binds to actin and IQGAP1, an actin- and Rac1-binding scaffold protein, at the leading edge in migrating ECs . IQGAP1 seems to tether Nox2 to actin cytoskeleton at the leading edge to direct ROS production and EC migration, which may contribute to angiogenesis.
By contrast, its binding partner p22phox is required for Ang II- and thrombin-stimulated ROS formation in ECs [37,38], while its role in angiogenesis has not been investigated.
Overexpression of active form of Rac1 induces loss of cell–cell adhesion  and cytoskeletal reorganization  through increase of H2O2, which are required for EC migration. VEGF- and angiopoietin-1-stimulated ROS production and EC migration are inhibited by a dominant-negative Rac1 [5,14]. Rac1 activity is highest at the leading edge in wound-induced migrating cells  and endogenous H2O2 accumulates at the membrane ruffles in actively migrating ECs [36,42]. Given that EC migration is a critical component of angiogenesis, Rac1 may play an important role in angiogenesis in ECs.
Phorbol 12-myristate 13-acetate (PMA)-, TNFα-, Ang II- and oscillatory shear-induced O2− production is inhibited in ECs isolated from p47phox− / − mice [43–45]. Gu et al.  reported that p47phox localizes to the cytoskeletal elements and plays a role in TNFα-induced c-terminal Jun kinase activation in ECV304 cells. After agonist stimulation or during directed cell migration, p47phox translocates from the perinucleus to the membrane ruffles through binding to the WAVE1 and to the leading edge or focal complexes through binding to the adaptor TRAF4 and Hic-5, a focal contact scaffold, in ECs [47,48]. Thus, p47phox is a functional component of NADPH oxidase associated with actin cytoskeleton in ECs, implicating its role in angiogenesis.
3.4 Nox1 and Nox4
Nox1 is upregulated by oscillatory shear stress, mediating ROS-dependent leukocyte adhesion to ECs  and regulates apoptosis and stimulates branching morphogenesis in sinusoidal ECs . Given that leukocyte adhesion to ECs causes disruption of cell–cell junctions and morphogenesis is a critical component of angiogenesis process, Nox1 may be involved in angiogenesis. Nox4 is expressed more abundantly compared to other Nox proteins in ECs, and seems to be involved in basal superoxide production . Kuroda et al.  demonstrated that Nox4 preferentially localizes to the nucleus in human ECs, which is involved in basal- and PMA-stimulated NADPH oxidase activity in nuclear fraction as well as oxidative stress responsive gene expression. Thus it is tempting to speculate that Nox4 might act as a sensor for nuclear redox , which may contribute to gene expression linked to cell growth, differentiation, senescence or apoptosis. The role of Nox4 in angiogenesis remains unknown.
4 NADPH oxidase-derived ROS as signaling molecules in angiogenesis
NADPH oxidase is activated by numerous stimuli including VEGF, EGF, cytokines, shear stress, hypoxia and G-protein coupled receptor agonists including Ang II in ECs . ROS produced via activation of NADPH oxidase stimulate diverse redox signaling pathways linked to angiogenesis. VEGF binds to two tyrosine kinase receptors, VEGF receptor-1 (VEGFR1, Flt-1) and VEGFR2 in ECs. The mitogenic and chemotactic effects of VEGF in ECs are mediated mainly through VEGFR2  which is activated through autophosphorylation of tyrosine residues in the cytoplasmic kinase domain. This event is followed by activation of downstream signaling pathways such as mitogen-activated protein kinases, Akt and eNOS, which are essential for EC migration and proliferation . VEGF stimulation increases ROS production via activation of Rac1-dependent NADPH oxidase in ECs [5,15,16,54–56] (Fig. 3). We and others have shown that ROS are involved in VEGF-induced VEGFR2 autophosphorylation in ECs [5,16,55] (Fig. 2). Angiopoietin-1 also stimulates ROS production through activation of Rac1-dependent, Nox2-based NADPH oxidase through Tie-2 receptor (Tie-2 R), which is required for EC chemotaxis . ROS are also important for VEGF-induced cSrc activation, phosphorylation of VE-cadherin and Akt, thereby regulating angiogenesis in ECs [15,39,56] (Fig. 2). However, underlying molecular mechanisms are incompletely understood.
Role of gp91phox (Nox2)-based NADPH oxidase in angiogenesis in response to hindlimb ischemia. Hindlimb ischemia was induced by the right femoral artery ligation. (A and B) Immunocytochemical analysis of Nox2 (red) and lectin (green) which stains ECs of capillaries (A) and dihydroethydium (DHE) staining which detects PEG-SOD (500 U/ml)-inhibitable O2− (B) in non-ischemic and ischemic hindlimb tissue sections on day 7 after operation. (C) Laser Doppler blood flow (LDBF) analysis. The ischemic/non-ischemic LDBF ratio in the wild-type (closed circle) and Nox2− / − (open circle) mice before and on post-operative days 0, 3 and 7 (n=7, *P<0.05 vs. wild-type). Figures are taken from ; used with permission.
Role of ROS derived from NADPH oxidase in VEGF signaling linked to angiogenesis. VEGF binding to VEGFR2 leads to the activation and translocation of the small GTPase Rac1 into the plasma membrane, which stimulates the NADPH oxidase in ECs. ROS derived from this oxidase may oxidize and inactivate protein tyrosine phosphatases (PTPs) which negatively regulates VEGFR2, thereby enhancing VEGFR2 autophosphorylation, and subsequent redox signaling linked to angiogenic responses such as EC proliferation and migration.
The signaling properties of ROS are due, in part, to reversible oxidation of redox-sensitive target proteins including protein tyrosine phosphatases (PTPs) [57,58] and lipid phosphatase PTEN . Several PTPs including SHP-1, SHP-2 and LMW-PTP (HCPTPA) inducibly associate with VEGFR2 after VEGF stimulation [60–62]. Although a role of SHP-1 in VEGF signaling remains unclear, TNF-α inhibits VEGF signaling and cell proliferation by facilitating recruitment of SHP-2 to the VEGFR2 in ECs . Overexpression of HCPTPA inhibits VEGF-induced VEGFR2 autophosphorylation, EC migration and proliferation . High cell density-enhanced PTP1 (DEP-1)/CD148 attenuates phosphorylation of VEGFR2 in contact-inhibited confluent ECs . A small molecule inhibitor of PTP1B enhances VEGF-induced VEGFR2 autophosphorylation, migration and proliferation of EC as well as neovascularization in a mouse matrigel model . Angiopoietin-1 stimulates association of SHP-2 to the phosphorylated Tie-2 R in ECs , which in turn inhibits PI3 kinase-dependent signaling pathways involved in EC migration. Thus, it is important to determine which PTPs are reversibly oxidized and inactivated by angiogenesis growth factor-induced ROS, thereby promoting RTK-mediated redox signaling linked to angiogenesis (Fig. 2).
Nitric oxide (NO) also plays an important role in VEGF signaling and postnatal angiogenesis . A cross-talk between NADPH oxidase and eNOS in EC growth has been demonstrated . Exogenous H2O2 or Ang II-induced H2O2 derived from NADPH oxidase activates eNOS, and thus promotes NO production in ECs . H2O2 stimulates cell proliferation, migration and cGMP production in ECs, while antioxidants prevent these responses by inhibiting eNOS activity . Using transgenic mice overexpressing human catalase in the vascular endothelium, Lauer et al.  demonstrated that endogenous H2O2 is involved in the endothelial adaptation to exercise by upregulation of eNOS in vivo. Tamarat et al.  provided evidence that eNOS lies downstream from Ang II-induced angiogenesis in ischemic hindlimbs. Thus these findings suggest an importance of NADPH oxidase–eNOS pathway in postnatal angiogenesis in vivo, which is the subject of future investigation.
5 Role of ROS and NADPH oxidase in angiogenesis in vivo
Growing evidence reveals that NADPH oxidase is involved in postnatal angiogenesis in vivo. There is strong correlation between ROS production, neovascularization and VEGF expression in eyes of diabetics [72–74] and in balloon injured arteries . ROS are increased during the reperfusion of the ischemic retina, which upregulates VEGF mRNA . Short periods of ischemia/reperfusion induce an increase in ROS, thereby stimulating myocardial angiogenesis in the ischemic noninfarcted heart . Brief coronary artery occlusion/reperfusion in dogs causes ROS production, which contributes to coronary collateral development . Moreover, ROS are involved in wound healing and repair processes in vivo . Antioxidants such as pyrrolidine dithiocarbamate , epigallocatechin-3-gallate [80,81], and the natural compounds in red wine and grapes [82,83] inhibit neovascularization in the mouse model. The ROS and NADPH oxidase inhibitors reduce angiogenesis in the chick embryo chorioallantoic membrane (CAM) model . It has been shown that anti-angiogenic therapy reduces plaque growth and intimal neovascularization  and that antioxidants vitamins C and E reduce vascular VEGF and VEGFR-2 expression in apolipoprotein-E deficient (ApoE− / −) mice . ROS are also involved in neovascularization during tumor growth . The thiol antioxidant, N-acetylcysteine (NAC) attenuates EC invasion and angiogenesis in a tumor model in vivo . PEDF which has antioxidant properties inhibits angiogenesis and melanoma growth .
Functional role of Nox2 in postnatal angiogenesis in vivo has been appreciated. Neovascularization in response to ischemia or VEGF is inhibited in Nox2− / − mice and in wild-type mice treated with antioxidant ebselen or NADPH oxidase inhibitor apocynin or gp91ds-tat [5,8,17] (Fig. 3). Furthermore, Nox 2 expression is increased in association with ROS production in mice ischemia hindlimb and retinopathy models [8,17] (Fig. 2). Recently, Vallet et al.  reported that Nox4 expression is upregulated in new capillaries in brain ischemia-induced angiogenesis of mice . Khatri et al.  have shown that vascular NADPH oxidase-derived ROS promote VEGF expression and neovascularization of experimental atheroma in transgenic mice overexpressing p22phox in SMCs. HMG-CoA reductase inhibitors, statins which has been shown to inhibit Rac1 activity,  can block angiogenesis in vivo . Arbiser et al.  demonstrated that overexpression of Nox1 increases VEGF and VEGF receptor expression and matrix metalloproteinase (MMP) activity, markers of the angiogenic switch, through increase in ROS, thereby promoting tumor angiogenesis. It is important to investigate a functional role of endogenous Nox1, Nox4, p22phox, p47phox and Rac1 in angiogenesis in vivo using classical and conditional knockout mice in future studies.
6 Angiotensin II is a potent stimulator for NADPH oxidase and angiogenesis
A peptide hormone Ang II is a potent stimulator for vascular NADPH oxidase . The renin–angiotensin system has been implicated in both angiogenesis and pathological vascular growth. In vitro, AT1 receptor stimulation induces migration of VSMC and promotes EC proliferation . Furthermore, Ang II potentiates the VEGF-induced in vitro tube formation of bovine retinal ECs . Moreover, we have shown that ROS derived from NADPH oxidase are involved in Ang II-induced tyrosine phosphorylation (transactivation) of EGF-R . Given that Ang II stimulates induction of VEGF and angiopoietin via heparin binding EGF-like growth factor-mediated EGF-R transactivation in ECs , ROS-dependent EGF-R transactivation by Ang II may be involved in the mechanism of neovascularization.
In vivo, Ang II has been shown to be involved in ischemia-induced angiogenesis [99–102], which is mediated through AT1 receptor-mediated upregulation of VEGF  or VEGF receptor [96,103,104]. Chymase, an alternative Ang II-generating enzyme, is also involved in angiogenesis in a hamster sponge implant model . Moreover, AT1 receptor antagonists and ACE inhibitors have been shown to inhibit tumor-associated angiogenesis in a murine model [106,107]. In myofibroblasts isolated from adult rat infarct site of heart, Ang II stimulation increases expression of VEGF and VEGF receptor, which may contribute to tissue remodeling and angiogenesis . Furthermore, VEGF-mediated angiogenesis is impaired by AT1 receptor blockade in cardiomyopathic hamster hearts . Ang II has been shown to be involved in coronary capillary angiogenesis at the insulin-resistant stage of a diabetes mellitus rat model . These findings suggest that Ang II plays an important role in angiogenesis in vitro and various types of postnatal angiogenesis in vivo including myocardial angiogenesis in which increase of collateral blood flow is required.
7 Effects of superoxide dismutase (SOD) on angiogenesis
Overexpression of extracellular SOD (ecSOD) inhibits tumor vascularization in mice , suggesting that antioxidant ecSOD treatment is useful therapeutic strategy for angiogenesis-dependent diseases. By contrast, FGF-induced angiogenesis and tumor development are enhanced in Cu/ZnSOD transgenic mice  and gene transfer of Cu/ZnSOD in NIH3T3 fibroblasts enhances VEGF synthesis through an increase in H2O2. Connor et al.  have reported that overexpression of MnSOD promotes mitochondrial H2O2 production, thereby stimulating EC sprouting and neovascularization in the CAM assay. Furthermore, VEGF-induced ROS via activation of Rac1 upregulate MnSOD expression in ECs , which could represent a feed-forward mechanism by which ROS-triggered H2O2 formation plays an important role in angiogenesis (Fig. 4). Thus, although Cu/ZnSOD and MnSOD have been considered as antioxidant enzymes in cytosol and mitochondria, respectively, they seem to serve as H2O2-generating, angiogenic enzymes in some settings . These studies indicate that SOD treatment becomes anti-angiogenic and angiogenic depending on its isoform or location.
Role of ROS derived from NADPH oxidase in induction of transcription factors and genes involved in angiogenesis. Ischemia/hypoxia stimulates VEGF induction and NADPH oxidase, thereby promoting activation of redox signaling events and induction of redox sensitive transcription factors and genes involved in angiogenesis. ROS derived from NADPH oxidase also upregulate MnSOD which increases mitochondrial H2O2 production, which could represent a feed-forward mechanism by which ROS-triggered H2O2 formation plays an important role in angiogenesis.
8 Angiogenesis-dependent transcription factors and genes regulated by ROS
H2O2 stimulates induction of the transcription factor Ets-1, thereby promoting EC proliferation and tube formation , and increases MCP-1 mRNA levels in an AP-1 and NF-κB-dependent manner in ECs . In addition, H2O2-induced increase in NF-κB binding to DNA is involved in IL-8 production, which contributes to tubular morphogenesis in ECs . Ang II-induced ROS derived from NADPH oxidase are involved in upregulation of MCP-1 and NF-κB, VCAM-1 and STAT1 . Given that all of these redox-sensitive transcription factors and genes can be activated by VEGF, they may be regulated by VEGF-induced ROS (Fig. 4). In response to hypoxia, HIF-1 activates the expression of many angiogenesis-related genes including VEGF and erythropoietin. ROS derived from NADPH oxidase are also involved in induction of HIF-1α under normoxia and hypoxia in vascular cells [115–117]. A cytochrome b-type NADPH oxidoreductase or mitochondria also produce ROS under hypoxia. Görlach et al.  showed that overexpression of Rac1 increases HIF-1 expression in an ROS-dependent mechanism. Thus, Rac1/Nox/ROS pathways play an important role for upregulation of HIF-1 and VEGF expression in response to VEGF and hypoxia (Fig. 4).
Other important ROS-dependent genes and proteins associated with angiogenesis are urokinase plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1) and MMPs (Fig. 4). Ets-1, which is induced by H2O2, regulates the expression of uPA and MMP-1. Lysophosphatidylcholine (lysoPC) stimulates induction of uPA in human macrophages through ROS, which may contribute to the intimal neovascularization in atherosclerotic plaque . It also increases the secretion of MMP-2 through activation of NADPH oxidase in ECs . Given that VEGF stimulates MMP-1 and 2 expression in ECs , this mechanism may be mediated through ROS derived from NADPH oxidase. Proteomic analysis in Nox2 and other NADPH oxidase knockout mice is needed to identify the ROS-dependent genes during angiogenesis in vivo.
The effects of ROS derived from ECs are tightly regulated and dependent on the amount and site of production as well as the balance of pro-oxidant and antioxidant enzyme activity. It is likely that low levels of ROS play an important role in reparative angiogenesis in response to ischemia and wound healing [8,78], while excess amount of ROS (oxidative stress) contributes to the various pathologies including atherosclerosis and diabetes. Although our existing data strongly support a critical role of NADPH oxidase in VEGF signaling and angiogenic-related responses in ECs, contribution of ROS derived from inflammatory cells and EPC cannot be eliminated in postnatal angiogenesis in vivo. Moreover, significant additional work on the mechanisms of activation of NADPH oxidases by various angiogenesis factors and identification of molecular targets of ROS in angiogenesis signaling will be required. Understanding these mechanisms may provide insight into the NADPH oxidase and redox signaling components as potential therapeutic targets for treatment of angiogenesis-dependent cardiovascular diseases and for promoting angiogenesis in ischemic limb and heart diseases.
Author's work is supported by NIH grant HL077524, an AHAGrant-in-Aid 0555308B and an AHA National Scientist Development Grant 0130175N (to M.U.-F).
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