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in mice. By contrast, FGF-induced angiogenesis as well as C6 glioma tumor development are enhanced in Cu/ZnSOD transgenic mice,63 which may be due to the increase in intracellular H2O2 levels through enhanced Cu/ZnSOD expression. Consistent with this, GrzenkowiczWydra et al.64 have shown that gene transfer of Cu/ZnSOD in NIH3T3 fibroblasts enhances VEGF synthesis by activation of hypoxiainducible factor response element (HRE) as well as SP1 recognition site of VEGF promoter through an increase in H2O2.

4.NAD(P)H Oxidase: A Major Source of ROS in the Vasculature

As noted, ROS are generated in mammalian cells from a number of sources including the mitochondrial electron transport system, xanthine oxidase, the cytochrome p450, the NAD(P)H oxidase and nitric oxide synthase.9 The NAD(P)H oxidase (Nox) family of enzymes have now been accepted as one of the major sources for ROS in the vasculature.9,20 Vascular NAD(P)H oxidase is a multi-subunit enzyme complex that differs structurally and biochemically from the phagocytic NAD(P)H oxidase. The phagocytic oxidase releases large amounts of O•2− in bursts, whereas the vascular NAD(P)H oxidase(s) continuously produce low levels of O•2− in unstimulated cells, and which can be stimulated acutely by various agonists and growth factors.9 ECs express NAD(P)H oxidase subunits that are identical to those found in phagocytes, including the membrane bound gp91phox (also known as Nox2) and p22phox, the cytosolic components p40phox, p47phox and p67phox, and Rac1.65,66 Upon stimulation, cytosolic components translocate to the membrane to form a multimeric protein complex, leading to production of ROS.65 Recently, novel gp91phox (Nox2) homologues, termed Nox1, Nox3, Nox4, Nox5.67 have been identified in non-phagocytic cells including vascular cells,68,69 suggesting the presence of multiple NAD(P)H oxidase forms in these cells.

Abid et al. demonstrated that ROS derived from NAD(P)H oxidase are required for EC proliferation and migration.70 Studies using knockout mice, inhibitory peptides or antisense oligonucleotide have established that Nox2 is a critical component of ROS-generating NAD(P)H

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that expression of p22phox is regulated by ROS derived from p22phoxbased NAD(P)H oxidase, thereby increasing a delayed ROS generation in ECs, which represents a positive feedforward mechanism whereby thrombin stimulates sustained ROS production via upregulation of a critical NAD(P)H oxidase component. A constitutively active form of Rac1 induces, through increase of H2O2, loss of cell-cell adhesion88 and cytoskeletal reorganization89 which are required for the migratory responses of ECs. A role of p47phox in PMA-, TNFα- and oscillatory shear-induced O−2 production has been demonstrated using ECs isolated from p47phox−/− mice.90,91 Furthermore, evolutionary more distinct Nox homologues, termed DUOX (dual oxidase) 1 and DUOX2, which have peroxidase activity have also been isolated.92 In addition, isoforms of p47phox and p67phox termed Nox organizer 1 (Noxo1) and Nox activator 1 (Noxa1) have also been characterized and have been shown to regulate Nox1 activity.93−96 Of importance, Ago et al.97 have recently shown that cerebral artery ECs express Nox1, Nox2 and cytosolic components p67phox and, to a lesser extent, p47phox, Noxo1, and Noxa1. These suggest the presence of multiple NAD(P)H oxidase forms in vascular cells.3,98,99

It is possible that the function of each NAD(P)H oxidase component is dependent on its distinctive subcellular localization, and is subject to specific regulations by selective agonists. In unstimulated ECs, NAD(P)H oxidase components exist as pre-assembled complexes in a predominantly perinuclear location associated with the intracellular cytoskeleton.72 In VSMCs, Nox1 localizes to caveolae while Nox4 is found in focal adhesions.100 In ECs; however, Nox 4 localizes at endoplasmic reticulum101 while Nox2 is found at perinuclear cytoskeletal structure.72 Gu et al.102 reported that p47phox plays a role in TNFα- induced c-terminal Jun kinase activation and that p47phox localizes to the cytoskeletal elements in HUVEC cell line ECV304. After agonist stimulation including VEGF, p47phox translocates to the membrane ruffles through association with WAVE1 in ECs, thereby activating NAD(P)H oxidase.101,103 Qian et al.104 showed that arsenic-induced NAD(P)H oxidase activation and EC migration are dependent on the actin cytoskeleton. Using a monolayer scratch assay with confluent ECs, we have demonstrated that ROS production is increased at the margin

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through activation of Cdc42, which promotes actin cytoskeletal reorganization, cell motility and tube formation in ECs, an in vitro model of angiogenesis.110 Of importance, Cdc42 is involved in arsenic-induced NAD(P)H oxidase activation and cell migration through regulating actin reorganization in ECs.104

These in vitro data strongly suggest that NAD(P)H oxidase may play an important role in postnatal angiogenesis in vivo. Expression of VEGF and Nox2 and production of ROS are increased during angiogenesis in ischemic retinopathy, and inhibition of NAD(P)H oxidase blocks VEGF overexpression and neovascularization.18 Similarily, VEGF and Nox2 expression as well as ROS production are increased in ischemic hindlimbs, and post-ischemic neovascularization is impaired in Nox2 knockout mice.12 Nox4 is upregulated and prominently expressed in newly formed capillaries in brain ischemia-induced angiogenesis in mice.111

In cardiomyocytes NAD(P)H oxidase is an important source of ROS.112−115 Interestingly, the expression of Nox2 and p22phox is increased in parallel with the elevation of lipid peroxidation in myocardial infarct sites.116 Krijnen et al.117 reported that Nox2 expression is upregulated in human cardiomyocytes following acute myocardial infarction. Moreover, short periods of ischemia/reperfusion induce monocyte chemoattractant protein (MCP)-1 expression through an increase in ROS, thereby stimulating angiogenesis in the ischemic noninfarcted heart.118 Of note, most recent study by Kimura et al.119 suggest that ROS formation via activation of NAD(P)H oxidase in cardiac myocytes may facilitate mitochondrial ROS production, thereby contributing to Ang II-induced preconditioning effects. Thus, it is possible that both NAD(P)H oxidaseand mitochondrial-derived ROS play an important role in myocardial angiogenesis.

Using a carotid injury model of transgenic mice overexpressing p22phox in smooth muscle cells, Khatri et al.120 reported that vascular NAD(P)H oxidase-derived ROS promote VEGF expression and intimal neovascularization. We demonstrated, using a sponge implant and hindlimb ischemic mouse model, that neovessel formation in response to VEGF as well as to ischemia is significantly inhibited both in wild-type mice treated with antioxidants and in Nox2−/− mice.12,20

Moreover, inhibition of NAD(P)H oxidase activity with apocynin and gp91ds-tat blocks ischemia-induced increase in ROS production, VEGF expression and retinal neovascularization in a mouse model of ischemic retinopathy.18 These results suggest that ROS derived from Nox2-containing NAD(P)H oxidase are important in postnatal angiogenesis in vivo. Consistent with our findings, HMG CoA reductase inhibitors, statins, which reduce vascular NAD(P)H oxidase activity through inhibiting Rac1115,121−125 have been shown to inhibit angiogenesis dose-dependently in vivo.126

The renin-angiotensin system has been implicated in angiogenesis. Ang II is a potent stimulator for NAD(P)H oxidase in various cardiovascular cells including VSMCs,22−25,127 ECs,128−132 adventitia,133 cardiac myocytes113,134−136 and isolated hearts subjected to ischemia/reperfusion.137 In vitro AT1 receptor stimulation induces migration of VSMC and monocytes, and promotes EC proliferation.138,139 Ang II potentiates the VEGF-induced tube formation of bovine retinal ECs.140 In primary culture of myofibroblasts isolated from adult rat infarcted heart, Ang II stimulation increases expression of VEGF and the VEGF receptor, which may contribute to angiogenesis at this site.141 In vivo, Ang II has been shown to be involved in ischemiaand VEGF-induced angiogenesis142−145 through upregulation of VEGF or VEGF receptor.140,146−148 Chymase, an alternative Ang II-generating enzyme, is also involved in angiogenesis in a hamster sponge implant model.149 Moreover, AT1 receptor and angiotensin I-converting enzyme play an important role in tumor-associated angiogenesis in a murine model150−152. Ang II has been shown to be involved in coronary capillary angiogenesis at the insulin-resistant stage of a non-insulin diabetes mellitus (NIDDM) rat model.153 Given that Ang II is a potent stimulator for vascular NAD(P)H oxidase, one may speculate that ROS derived from oxidase may play a role in Ang II-induced angiogenesis.

While non-transformed cells respond to growth factors/cytokines with the regulated production of ROS, tumor cells frequently overproduce H2O2. Arbiser et al.154 demonstrated that overexpression of Nox1 into a prostate cancer cell line increases VEGF, VEGF receptor expression and MMP activity through increase of H2O2, which contributes to the vascularization of tumors. Most recently, Lim et al.155 have shown

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that both Nox1 and H2O2 are increased in human prostate cancer tissue and in an animal model.

Endothelial progenitor cells (EPCs) also contribute to postnatal neovascularization.156,157 Recent evidence suggests that ROS derived from NAD(P)H oxidase may also regulate vasculogenesis. Dernbach et al.158 reported that EPCs express higher level of the antioxidant enzymes catalase, glutathione peroxidase and MnSOD than EC to protect against oxidative stress. Indeed, the survival and migratory capacity of EPCs is reduced by knockdown of antioxidant enzymes using siRNA.158 Furthermore, redox state modulates self-renewal and differentiation of EPCs.159,160 Short-term exposure of Ang II, a potent stimulator of vascular NAD(P)H oxidase, potentiates VEGF-induced proliferation and network formation of EPCs,161 while its long-term exposure accelerates senescence of EPC through induction of Nox2 and oxidative stress.162 Thus, the amount of ROS is likely to determine the fate and function of EPCs. Most recently, Sauer et al.163 demonstrated that NAD(P)H oxidase-derived ROS are involved in EC differentiation and angiogenesis of mouse embryonic stem cells after direct current electrical field stimulation, further suggesting an important role of ROS in the function of EPCs.

Hyperglycemia is a primary cause of macroand micro-vascular complications in diabetes. Furthermore, impaired reparative angiogenesis impedes proper post-ischemic healing and wound closure in diabetic patients. This defect was attributed to the shortage of, or insensitivity to, angiogenic growth factors including VEGF.164,165 EPCs, which play a critical role in forming new vessels, are also dysfunctional in hyperglycemia.166 As discussed, low concentrations of ROS acts as signaling molecules and are necessary for reparative angiogenesis and wound healing,53,167 while excess amount of ROS (oxidative stress) contributes to the pathologensis of atherosclerosis and diabetes, in part by inactivating nitric oxide and causing EC dysfunction.168−171 Of importance, antioxidants accelerate diabetic wound healing.172 Evidence suggests that NAD(P)H oxidase is involved in increased production of ROS in diabetic patients and mice as well as ECs cultured under high glucose conditions.9,169,173,174 Hyperglycemia also contributes to an impairment of EPC count and function, at least in part, through inhibition of