Akt and eNOS phosphorylation by unknown mechanisms.166 Thus, diabetes-associated over-activation of NAD(P)H oxidase may contribute to disturbing proper angiogenic growth factor signaling and function in ECs and EPCs, which may result in impaired angiogenesis/vasculogenesis in response to ischemic injury.

6. ROS as Signaling Molecules in Angiogenesis

Signal transduction by ROS, “redox signaling” is a rapidly growing area of investigation. NAD(P)H oxidase is activated by numerous stimuli including growth factors such as VEGF, transforming growth factor-β1, cytokines, shear stress, hypoxia and G-protein coupled receptor agonists including Ang II in ECs.9,175 ROS produced via activation of NAD(P)H oxidase stimulate diverse redox signaling pathways leading to angiogenesis (Fig. 3). VEGF binds to two tyrosine kinase receptors, VEGF receptor-1 (VEGFR1, also termed Flt-1) and VEGFR2 (also termed KDR/Flk1) in ECs. The mitogenic and chemotactic effects of VEGF in ECs are mediated mainly through VEGFR2.2,176,177 VEGFR2 is activated through ligand-stimulated receptor dimerization and transphosphorylation (autophosphorylation) of tyrosine residues in the cytoplasmic kinase domain. At present, tyrosine residues 951 and 996 in the kinase insert domain, and 1054 and 1059 in the kinase catalytic domain have been identified as autophosphorylation sites for VEGFR2 in a bacterial expression system.178 This event is followed by activation of diverse downstream signaling pathways such as mitogen-activated protein kinases (MAPKs), Akt/protein kinase B and endothelial NOS (eNOS), which are essential for VEGFinduced EC migration and proliferation.179−182 ROS are important mediators for VEGF-mediated angiogenic signaling including VEGF receptor autophosphorylation, cSrc activation and VE-cadherin phosphorylation in ECs.20,76,88,108 Antioxidant green tea catechins suppress VEGF-induced tube formation in EC through inhibiting VE-cadherin tyrosine phosphorylation and Akt activation.183 We have demonstrated that VEGF-induced VEGFR2 autophosphorylation is inhibited by the thiol antioxidant NAC, various NAD(P)H oxidase inhibitors, dominant negative Rac1 and Nox2 antisense oligonucleotides.20 These

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results suggest that VEGFR2 is one of the proximal molecular targets of ROS derived from the Nox2-containing NAD(P)H oxidase in cultured EC (Fig. 3). Moreover, Tie-2 receptors (Tie-2 R) are RTKs activated by angiopoietin-1 (Ang-1) and selectively expressed in ECs. They play an important role in embryonic development and promote differentiation, tube formation, migration, adherence and survival of ECs. Harfouche et al.21 demonstrated that ROS derived from Rac1-dependent, Nox2based NAD(P)H oxidase mediate Ang-1/Tie-2 R signaling linked to EC chemotaxis. However, ROS are not involved in Ang-1-induced Tie-2 R autophosphorylation, suggesting that mechanisms of autorphosphorylation of VEGFR2 and Tie-2 R are different.

A role of ROS in tyrosine phosphorylation of RTKs has been previously reported. We showed that Ang II induces ROS-dependent tyrosine phosphorylation of the epidermal growth factor (EGF) receptor (EGF-R) in VSMCs.24 Rao184 reported that H2O2 induces tyrosine phosphorylation of the EGF-R, which in turn recruits the Shc-Grb2- Sos adaptor protein complex to the receptor to activate subsequent signaling cascades. Furthermore, EGF-, PDGFand insulin-induced autophosphorylation of their receptors are inhibited by catalase, which scavenges intracellular H2O2,185−187 suggesting that endogenouslyproduced H2O2 plays an important role in RTK phosphorylation. We have shown that ROS derived from NAD(P)H oxidase are involved in Ang II-induced transmodulation of EGF-R,23 which serves as a scaffold for various signaling molecules and plays an important role in cross-talk between G-protein coupled receptor and RTK signaling in VSMCs.188 Of note, Ang II-induced angiogenesis has been shown to be mediated through induction of VEGF and angiopoietin expression via heparin-binding EGF-like growth factor (HB-EGF)-mediated EGF-R transmodulation in ECs.189 Thus, it is likely that ROS derived from NAD(P)H oxidase may play a role in Ang II-induced angiogenesis via regulating EGF-R phosphorylation.

Identifying the molecular targets of ROS in agonist-stimulated signal transduction is critically important to the understanding of the mechanisms of redox signaling in the vasculature. Accumulating evidence suggests that protein tyrosine phosphatases (PTPs) are direct targets of ROS, and PTPs negatively regulate RTK activity

and downstream signaling.190−192 Most PTPs have low-pKa cysteine residues within their active site, that exist as thiolate anions at neutral pH.193 The reversible oxidative inhibition of active cysteine residues in PTPs by ROS is an important mechanism through which ROS shift the balance of protein tyrosine phosphorylation/dephosphorylation towards enhanced tyrosine phosphorylation in growth factor signaling 190,192,194. Thus, ROS produced after RTK engagement form a feedback loop that, through the oxidative inhibition of PTPs, promotes the tyrosine phosphorylation/activation and the downstream signaling of RTKs. Furthermore, the lipid phosphatase tumor suppressor PTEN,195 membrane phosphatase CD45,196 and transmembrane receptor RPTPα197 are also susceptible to H2O2-dependent oxidative inactivation in vivo. Indeed, Connor et al.198 demonstrated that mitochondrial H2O2 production by MnSOD overexpression induces oxidative inactivation of PTEN, thereby promoting EC sprouting in a 3D in vitro angiogenesis assay as well as in vivo blood vessel formation in CAM assay. It should be noted that MnSOD can serve as a source of the potent signaling molecule, H2O2, from the mitochondria. Of importance, VEGF-induced ROS have been shown to be involved in induction of MnSOD through activation of Rac1-dependent NAD(P)H oxidase in ECs,75 which can represent a feedforward mechanism by which ROS-triggered ROS formation play an important role in angiogenesis.

A reversible oxidation of PTPs such as PTP1B, low molecular weight (LMW)-PTP and SHP-2 during RTK stimulation with EGF, insulin and platelet-derived growth factor187,195,199−201 have been reported. In case of VEGFR2, several PTPs including SHP-1, SHP-2 and LMW-PTP (HCPTPA) have been shown to inducibly associate with VEGFR2 after VEGF stimulation.202−204 Although a role of SHP-1 in VEGF signaling remains unclear, Guo et al.203 reported that TNF-α inhibits VEGF signaling and cell proliferation by facilitating recruitment of SHP-2 to the VEGFR2 in ECs. Huang et al.204 demonstrated that overexpression of HCPTPA inhibits VEGF-induced VEGFR2 autophosphorylation, cell migration and proliferation. High cell density-enhanced PTP1 (DEP-1)/CD148 has been reported to participate in inhibition of VEGFR2 phosphorylation in confluent,

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contact-inhibited ECs.205 A small molecule inhibitor of PTP1B has been shown to enhance VEGF-induced VEGFR2 activation, migration and proliferation of EC as well as neovascularization in matrigel plugs of mice.206 As with VEGFR2, SHP-2 binds to the activated, autophosphorylated Tie-2 R following Ang-1 stimulation in ECs,207 which in turn inhibits phosphatidylinositol 3 kinase-dependent signaling pathways linked to EC migration.208,209 Thus, it will be important to determine which PTPs are reversibly oxidized by VEGFor Ang- 1-induced ROS, thereby promoting RTK-mediated redox signaling in angiogenesis.

In addition to ROS, NO also plays an important role in VEGF signaling and postnatal angiogenesis.210 NO enhances VEGF synthesis in several cell types and is required for execution of VEGF angiogenic effect in ECs. Cross-talk between NAD(P)H oxidase, EC growth as well as eNOS enzyme activity and expression has been demonstrated.211 It is thus important to understand how both ROS and NO regulate VEGF signaling and angiogenesis. We have demonstrated that ROS, but not NO, are involved in VEGF-induced VEGFR2 autophosphorylation in HUVECs.20 Furthermore, exogenous H2O2 or Ang II-stimulated increase of ROS potently activates eNOS, which in turn promotes NO production in ECs.130,175 H2O2 stimulates EC proliferation, migration and cGMP production, which are reversed by a guanylate cyclase inhibitor, while antioxidants block cell proliferation and migration through downregulation of eNOS activity.212 Thus, NAD(P)H oxidasederived H2O2 seems to play a significant role in promoting angiogenic responses such as EC proliferation and migration at least in part via regulating eNOS/NO pathways.213 This notion is further supported by the recent report using an in vivo mice model that exercise training increases both oxidative stress and eNOS expression, and both are inhibited in transgenic mice overexpressing human catalase in the vascular endothelium.214 This result suggests that endogenouslyproduced H2O2 is involved in the endothelial adaptation to exercise by upregulation of eNOS in vivo. Whether similar mechanisms apply to VEGF signaling and postnatal angiogenesis in vivo require further investigation.

7.Angiogenesis-Dependent Transcription Factors and Genes Regulated by ROS

In addition to PTPs, transcription factors with low-pKa cysteine residues such as the nuclear factor-κB (NF-κB),215 AP-1,216 hypoxiainducible factor1α (HIF-1α),217 p53218 and p21Ras219 can be oxidized by H2O2. Low concentrations of H2O2 stimulate induction of the transcription factor Ets-1, which is required for EC proliferation and tube formation.34 Inflammation plays an important role in angiogenesis. H2O2 induces an increase in MCP-1 mRNA levels in an AP-1- and NF-κB-dependent manner in ECs.118 In addition, H2O2-induced increase in NF-κB binding to DNA is involved in IL-8 production, which is required for tubular morphogenesis in human microvascular ECs.36 Ang II-induced ROS derived from NAD(P)H oxidase are involved in upregulation of MCP-1 and NF-κB,220 vascular celladhesion molecule-1 (VCAM-1)221 and STAT1.222 It should be noted that all of these redox-sensitive genes and transcription factors are activated by VEGF, raising the possibility that they are regulated by ROS generated after VEGF stimulation (Fig. 3). Consistent with this notion, VEGF-induced ROS have been shown to be involved in induction of NFκB in VSMCs.223

HIF-1 is a heterodimeric basic helix-loop-helix transcription factor composed of HIF-1α and HIF-1β aryl hydrocarbon nuclear translocator subunits (see Chapter 7). HIF-1 expression is induced by hypoxia, growth factors, and activation of oncogenes. In response to hypoxia, HIF-1 activates the expression of many angiogenesis-related genes including VEGF and erythropoietin. Of importance, accumulating evidence suggest that ROS derived from NAD(P)H oxidase are involved in induction of HIF-1α under normoxia and hypoxia in vascular cells.224−230 Under normoxia, agonist-induced ROS may serve as signaling molecules to upregulate HIF-1α possibly by modulating upstream signaling pathways such as hydroxylases or kinases and phosphatases. In contrast, the role of ROS in hypoxia-induced HIF-1α regulation is less well understood. Turcotte et al.231 demonstrated that hypoxia stimulates ROS production, thereby increasing Rho GTPase expression

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which is required for HIF-1α accumulation in renal cell carcinoma. It has been shown that NAD(P)H oxidase or a cytochrome b-type NAD(P)H oxidoreductase or mitochondria may produce ROS under hypoxia. The difference may be due to variabilities in the experimental conditions, cell type, the amount of oxygen available or the measuring techniques.107 Gorlach et al.229 showed that overexpression of Rac1 increases expression of HIF-1 through ROS-dependent mechanisms. These data suggest that Rac1/NAD(P)H oxidase/ROS pathways are important for upregulation of HIF-1 and VEGF expression in response to VEGF and hypoxia (Fig. 3) Of note, the redox protein thioredoxin-1 increases HIF-1α protein expression in cancer cells, thereby promoting VEGF production and tumor angiogenesis.232 It has been shown that anti-angiogenic therapy reduces both plaque growth and intimal neovascularization in apolipoprotein-E deficient (ApoE−/−) mice. Dietary supplementation with antioxidants vitamins C and E reduces vascular VEGF and VEGFR-2 expression in ApoE−/−mice233.

Other important ROS-dependent genes and proteins associated with angiogenesis are urokinase plasminogen activator (uPA) and MMPs (Fig. 3). ROS activate and increase the expression of MMPs.234 Ets-1, which is induced by H2O2,34 regulates the expression of genes involved in extracellular matrix degradation, including uPA and MMP-1. Grote et al..235 have shown that the mechanical stress-induced increase in MMP-2 activation and mRNA expression are inhibited in VSMCs derived from p47phox−/− mice. Furthermore, lysophosphatidylcholine (lysoPC) increases the secretion of MMP-2 through activation of NAD(P)H oxidase in cultured ECs.236 Given that VEGF stimulates induction of MMP-1 and -2 expression in ECs,237 this mechanism may also be mediated through ROS derived from NAD(P)H oxidase. Moreover, HIF-1 mediates upregulation of plasminogen activator inhibitor-1 (PAI-1) expression under hypoxia and ROS have also been implicated in PAI-1 gene expression. Thus, overexpression of Rac1 upregulates PAI-1 expression through an increase in ROS.229 Of note, lysoPC also induces uPA and its cell surface receptor in human macrophages, in a ROS-dependent manner, which may contribute to the intimal neovascularization in atherosclerotic plaque.238