Skip main navigation

NAD(P)H Oxidase–Dependent Self-Propagation of Hydrogen Peroxide and Vascular Disease

Originally published Research. 2005;96:818–822


    Excessive production of reactive oxygen species in the vasculature contributes to cardiovascular pathogenesis. Among biologically relevant and abundant reactive oxygen species, superoxide (O2·−) and hydrogen peroxide (H2O2) appear most important in redox signaling. Whereas O2·− predominantly induces endothelial dysfunction by rapidly inactivating nitric oxide (NO·), H2O2 influences different aspects of endothelial cell function via complex mechanisms. This review discusses recent advances establishing a critical role of H2O2 in the development of vascular disease, in particular, atherosclerosis, and mechanisms whereby vascular NAD(P)H oxidase–derived H2O2 amplifies its own production. Recent studies have shown that H2O2 stimulates reactive oxygen species production via enhanced intracellular iron uptake, mitochondrial damage, and sources of vascular NAD(P)H oxidases, xanthine oxidase, and uncoupled endothelial nitric oxide synthase (eNOS). This self-propagating phenomenon likely prolongs H2O2-dependent pathological signaling in vascular cells, thus contributing to vascular disease development. The latest progress on Nox functions in vascular cells is also discussed [Nox for NAD(P)H oxidases, representing a family of novel NAD(P)H oxidases].

    Vascular NAD(P)H oxidase–dependent overproduction of reactive oxygen species contributes to pathogenesis of cardiovascular diseases.1–5 Among biologically relevant and abundant reactive oxygen species, superoxide (O2·−) and hydrogen peroxide (H2O2) appear most important in redox signaling. Whereas O2·− primarily modulates vascular function by rapidly inactivating NO· (reviewed by Cai and Harrison),6 H2O2 impacts on vascular function via complex mechanisms. Ambient production of H2O2 at low levels, likely maintained by pre-assembled NAD(P)H oxidases,3 is necessary for endothelial cell growth and proliferation (reviewed by Griendling and Harrison; Eyries and colleagues).7,8 Under pathological conditions, however, agonists-provoked activation of vascular NAD(P)H oxidases produces H2O2 in large quantities, which in turn amplifies its own production, resulting in compensatory or detrimental consequences. For instance, H2O2 is either compensatorily responsible for endothelium-dependent vasodilatation in hypertension where NO· is substantially reduced,9 or over the long term detrimentally involved in vascular smooth muscle cell proliferation and hypertrophy.10–12 At biochemical levels, H2O2 signals by oxidizing low pKa cysteine residues in protein phosphatases (reviewed by Rhee et al).13,14 The current brief review complements previous reviews to discuss for the first time recent advances establishing the critical role of H2O2 in vascular disease development and mechanisms whereby vascular NAD(P)H oxidase-derived H2O2 amplifies its own production.

    Hydrogen Peroxide and Vascular Disease

    Though reactive oxygen species are clearly involved in vascular pathogenesis, the specific, individual reactive oxygen species that is most important in pathological signaling remains to be identified. Nevertheless, selectively overproducing or removing H2O2 in rodents was found highly influential of atherosclerotic development. Mice overexpressing NAD(P)H oxidase subunit p22phox (first developed by Dr David Harrison’s group at Emory University) had markedly increased atheroma formation in a carotid ligation model.15 This response was associated with enhanced H2O2 production in the vessel wall, and was abolished by scavenging H2O2 with ebselen, implicating a critical role of H2O2 in atherogenesis.15 Parallel studies using different animal models and catalase scavenging of H2O2 from another group confirmed the same notion,16 offsetting the concern that ebselen also removes peroxynitrite. Yang et al cross bred transgenic mice overexpressing Cu/Zn-SOD or catalase with mice deficient in apolipoprotein E (apoE−/−), to examine a specific role of H2O2 versus O2·− in atherogenesis.16 They found that whereas overexpressing Cu/Zn-SOD had no effect on atherosclerotic lesion formation in apoE−/− mice, overexpression of catalase or cooverexpression of catalase and Cu/Zn-SOD markedly retarded atherosclerosis in many aspects including lesion severity, lesion size, and area of affection throughout the aortic tree.16 These observations were consistent with the findings by Tribble et al that overexpression of Cu/Zn-SOD failed to prevent atherosclerosis in high-fat diet–fed apoE−/− mice.17 Taken together, these data indicate that H2O2 is more atherogenic than O2·−. Of interest, the protective effects of catalase overexpression were found independent of plasma lipids.16 One may argue that Cu/Zn-SOD is intracellular, and that the scavenging of O2·− by extracellular SOD (ecSOD) to prevent NO· degradation during its trafficking to vascular smooth muscle is more relevant to atheroprotection. Indeed, evidence gained from ecSOD-null mice and adenovirus-mediated overexpression of ecSOD supports that ecSOD is the main determinant of NO· bioavailability in the vessel wall and is thus involved in blood pressure regulation.18,19 However, the impact of ecSOD overexpression on atherosclerosis is not yet reported. On the other hand, Sentman and colleagues found that mice deficient in ecSOD developed similar atherosclerotic lesions compared with wild-type mice.20 Therefore, whereas O2·− is important in directly modulating NO· bioavailability and serving as the precursor for H2O2,6 relatively lasting H2O2 seems more important in mediating atherogenic signaling.

    Of note, different from O2·− that is charged, hardly permeable, and extremely short-lived, H2O2 produced either intracellularly, within mitochondria, or at extracellular space is uncharged, relatively longer-lived, and freely diffusible. As for NO·, this property makes H2O2 an ideal signaling molecule. On the other hand, intracellular scavenging of H2O2 with ebselen or catalase could have removed H2O2 from all these sources. It thus remains unclear whether localized production of H2O2 at certain cellular compartment or vascular space is required in atherogenic signaling.

    Interestingly, besides intracellular autocrine signaling, the capacity of diffusing among adjacent cells enables H2O2 for paracrine signaling. Of note, H2O2 produced by vascular smooth muscle can diffuse to endothelium to regulate endothelial cell function. For example, Laude et al recently showed that H2O2, produced in vivo in mice overexpressing p22phox in vascular smooth muscle, upregulates eNOS gene expression,21 confirming our previous in vitro observations that H2O2 potently upregulates eNOS expression.22,23 These data also indicate that H2O2, derived from adjacent vascular cells, is able to modulate endothelial function, further supporting a unique signaling role of H2O2 in the vasculature.

    Hydrogen Peroxide Signaling and Vascular Function

    Numerous signaling cascades are activated by H2O2 to mediate changes in vascular function including endothelial overgrowth,7,8 angiogenesis,24 smooth muscle proliferation and hypertrophy,25 endothelial barrier dysfunction and cytoskeleton reorganization,26,27 endothelial apoptosis,28 induction of inflammatory proteins,29 endothelium–leukocyte interaction, and vascular remodeling.30,31 H2O2 potently activates MAPK members ERK1/2, p38MAPK, JNK, and ERK5 in both vascular endothelial and smooth muscle cells.32–36 Our recent studies indicate that H2O2 activation of ERK1/2 and p38MAPK in endothelial cells requires CaMKII.37 Receptor tyrosine kinases such as those for EGF, PDGF, FGF, VEGF,38 and non-receptor tyrosine kinases such as JAK2,22,39 Src,33 Cas,35 FAK, and Pyk2,40,41 are responsive to H2O2 in vascular cells and often lie upstream of MAPK. Axl is a novel receptor tyrosine kinase identified in vascular smooth muscle, and its activation by H2O2 mediates neointima formation after vascular injury.42,43 In addition, mitochondrial function was recently found necessary for H2O2-induced growth factor transactivation.44 Redox-sensitive transcriptional factors including NFκB, AP-1, and HIF-1α are often activated via MAPK to modulate changes in gene expression and cellular function.3,28 Phosphorylation-dependent posttranslational regulation of proteins also occurs in response to H2O2. For example, we and others have shown that H2O2 induces PI3-Kinase/Akt-dependent phosphorylation of eNOS, leading to a compensatory, transient increase in NO· production,36,45 which may serve as an intermediate step for long-term detrimental consequences.46 Of note, many protein kinases are indirectly activated, subsequent to H2O2 inactivation of protein phosphatases.13,14

    Mechanisms Underlying Hydrogen Peroxide Self-Propagation

    Emerging evidence has demonstrated that uniquely, H2O2 is able to amplify its own production in vascular cells, and this phenomenon likely contributes to its long-lasting pathological effects. To date, at least 5 different mechanisms potentially underlie self-propagation of H2O2 (Figure). Earlier studies demonstrated that extra-mitochondrial H2O2 can induce mitochondrial DNA damage, destroying respiratory enzymes to produce reactive oxygen species.47 Secondly, transferrin receptor (TfR)-dependent endothelial iron uptake is augmentable by H2O2, amplifying intracellular H2O2 formation to induce apoptosis.48 Mitochondrial iron uptake can also be upregulated by H2O2.49 Thirdly, in vascular smooth muscle and fibroblasts, NAD(P)H oxidase–derived H2O2 is capable of feed-forwardly activating NAD(P)H oxidase itself.50 In endothelial cells, H2O2 was recently found capable of upregulating p22phox expression.51 Likewise, McNally et al recently showed that in endothelial cells, oscillatory shear stress activation of NAD(P)H oxidases lies upstream of xanthine oxidase–dependent production of H2O2.52 Last but not least; endothelial NAD(P)H oxidase–derived H2O2 mediates agonists-provoked tetrahydrobiopterin deficiency to induce eNOS uncoupling.52a This seems consistent with earlier findings that uncoupled eNOS lies downstream of vascular NAD(P)H oxidases in hypertension, and likely also, in diabetes.9,53 Thus H2O2, originated by vascular NAD(P)H oxidases, propagates its own production via enhanced intracellular iron uptake, and sources of mitochondria, NAD(P)H oxidases, xanthine oxidase, and uncoupled eNOS. These feed-forward mechanisms form a vicious circle to amplify and sustain H2O2 production in large quantities, contributing to pathological signaling.

    Mechanisms underlying NAD(P)H oxidase–dependent self-propagation of H2O2 in vascular cells. M1, H2O2 causes mitochondrial damage to produce reactive oxygen species; M2, H2O2 promotes transferrin receptor-dependent intracellular iron uptake to potentiate its own production; M3, H2O2 feed-forwardly stimulates reactive oxygen species generation from vascular NAD(P)H oxidases; M4, endothelial NAD(P)H oxidase is required for xanthine oxidase oxidation and activation, and subsequently H2O2 amplification in response to oscillatory shear stress; M5, uncoupled eNOS lies downstream of vascular NAD(P)H oxidases to propagate H2O2 production.

    Vascular NAD(P)H Oxidases Origination of Hydrogen Peroxide

    As discussed above, activation of vascular NAD(P)H oxidases is rate-limiting in H2O2 amplification of its own production.1–5,54 Molecular activation of NAD(P)H oxidases in vascular smooth muscle has been elegantly reviewed.1–5,54 In endothelial cells, though much to be learned, p47phox is confirmed to be critical in modulating enzymatic activity by interacting with catalytic unit gp91phox (Nox2, Nox for NAD(P)H oxidases, representing a family of novel NAD(P)H oxidases).55–58 Studies using deficient mice or inhibitory peptide (gp91ds-tat) targeting Nox2 have established an essential role of Nox2 in producing reactive oxygen species in endothelial cells.56,57,59,60 Functions of other newly identified gp91phox homologues (Nox1, Nox4 and Nox5), however, remain obscure but are under intensive investigation. A recent study reported that Nox4 is more abundantly expressed in endothelial cells compared with other Nox proteins, representing the major catalytic unit of the endothelial NAD(P)H oxidase that is activated by growth halting.61,62 Nox1, on the other hand, was upregulated by oscillatory shear stress, mediating reactive oxygen species–dependent leukocyte adhesion to endothelium.63 In addition, VEGF receptor–dependent activation of Nox1 was angiogenic, responsible for tube formation of endothelial cells.64 The observations that Nox1 mediates growth signaling whereas Nox4 is growth suppressive in endothelial cells seems similar to what has been observed in vascular smooth muscle.65,66 It is puzzling why the same reactive oxygen species–producing Nox proteins mediate different cellular responses. One possibility is that each Nox protein functions specifically based on their unique subcellular localization and tight regulation by different agonists.1 For example, in vascular smooth muscle cells, Nox1 localizes to caveolae whereas Nox4 is found in focal adhesions.67 In endothelial cells, however, Nox4 was found at endoplasmic reticulum62 whereas Nox2 is localized to peri-nuclear cytoskeletal structure.56

    Novel homologues of Nox-regulating proteins p47phox and gp67phox have been identified in epithelial cells (p41phox and p51phox, respectively), serving as potent positive regulators for Nox1.68–71 Duox1 and Duox2 are longer Nox proteins with peroxidase tails,72,73 which have been shown to produce H2O2 in epithelial cells.74,75 These proteins are studied for their presence and function in endothelium and vascular smooth muscle. Besides Nox, p22phox presents the only other membrane component of the vascular NAD(P)H oxidases. Overexpression of p22phox led to upregulation of Nox1 and Nox4 in vivo, likely via stabilization of proteins.21 Recent studies have elegantly characterized physical interactions between Nox (Nox1 and Nox4) and p22phox, and the functional, physiological consequences of these interactions regarding O2·− production in vascular smooth muscle.76,77 Whether similar interactions occur in endothelial cells remains to be elucidated. Nonetheless, it was recently found that p22phox expression correlates well with expression of Nox4 in human arteries and that of Nox2 in veins.78

    In summary, recent studies have established a critical role of H2O2 in the development of vascular disease, in particular atherogenesis. Uniquely, vascular NAD(P)H oxidase–derived H2O2 self-propagates via enhanced intracellular iron uptake, mitochondrion, vascular NAD(P)H oxidases, xanthine oxidase, and uncoupled eNOS. This phenomenon likely prolongs H2O2-mediated pathological signaling, thus contributing to vascular disease development. Initial activation of vascular NAD(P)H oxidases serves as the rate-limiting step for H2O2 amplification of redox signals. It is of significant importance to further investigate molecular mechanisms underlying vascular activation of Nox family proteins of the novel vascular NAD(P)H oxidases. This knowledge could lead to novel strategies effective in disrupting cascade production of reactive oxygen species, and of therapeutic potential for vascular disease.

    Original received December 22, 2004; revision received March 14, 2005; accepted March 15, 2005.

    The author’s work is supported by an American Heart Association Scientist Development Grant (#0435189N), an American Diabetes Association Research Award, a Career Development Award from the Schweppe Foundation, and a Start-up Fund from the University of Chicago.


    Correspondence to Hua “Linda” Cai, MD, PhD, Section of Cardiology, Department of Medicine, The Division of Biological Sciences and Pritzker School of Medicine, The University of Chicago, 5841 S. Maryland Ave, MC6088, Chicago, IL 60637. E-mail or [email protected]


    • 1 Griendling KK. Novel NAD(P)H oxidases in the cardiovascular system. Heart. 2004; 90: 491–493.CrossrefMedlineGoogle Scholar
    • 2 Cai H, Griendling KK, Harrison DG. The vascular NAD(P)H oxidases as therapeutic targets in cardiovascular diseases. Trends Pharmacol Sci. 2003; 24: 471–478.CrossrefMedlineGoogle Scholar
    • 3 Li JM, Shah AM. Endothelial cell superoxide generation: regulation and relevance for cardiovascular pathophysiology. Am J Physiol Regul Integr Comp Physiol. 2004; 287: R1014–R1030.CrossrefMedlineGoogle Scholar
    • 4 Touyz RM, Schiffrin EL. Reactive oxygen species in vascular biology: implications in hypertension. Histochem Cell Biol. 2004; 122: 339–352.CrossrefMedlineGoogle Scholar
    • 5 Brandes RP. Role of NADPH oxidases in the control of vascular gene expression. Antioxid Redox Signal. 2003; 5: 803–811.CrossrefMedlineGoogle Scholar
    • 6 Cai H, Harrison DG. Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress. Circ Res. 2000; 87: 840–844.CrossrefMedlineGoogle Scholar
    • 7 Griendling KK, Harrison DG. Dual role of reactive oxygen species in vascular growth. Circ Res. 1999; 85: 562–563.CrossrefMedlineGoogle Scholar
    • 8 Eyries M, Collins T, Khachigian LM. Modulation of growth factor gene expression in vascular cells by oxidative stress. Endothelium. 2004; 11: 133–139.CrossrefMedlineGoogle Scholar
    • 9 Landmesser U, Dikalov S, Price SR, McCann L, Fukai T, Holland SM, Mitch WE, Harrison DG. Oxidation of tetrahydrobiopterin leads to uncoupling of endothelial cell nitric oxide synthase in hypertension. J Clin Invest. 2003; 111: 1201–1209.CrossrefMedlineGoogle Scholar
    • 10 Ushio-Fukai M, Zafari AM, Fukui T, Ishizaka N, Griendling KK. p22phox is a critical component of the superoxide-generating NADH/NADPH oxidase system and regulates angiotensin II-induced hypertrophy in vascular smooth muscle cells. J Biol Chem. 1996; 271: 23317–23321.CrossrefMedlineGoogle Scholar
    • 11 Zafari AM, Ushio-Fukai M, Akers M, Yin Q, Shah A, Harrison DG, Taylor WR, Griendling KK. Role of NADH/NADPH oxidase-derived H2O2 in angiotensin II-induced vascular hypertrophy. Hypertension. 1998; 32: 488–495.CrossrefMedlineGoogle Scholar
    • 12 Jin ZG, Melaragno MG, Liao DF, Yan C, Haendeler J, Suh YA, Lambeth JD, Berk BC. Cyclophilin A is a secreted growth factor induced by oxidative stress. Circ Res. 2000; 87: 789–796.CrossrefMedlineGoogle Scholar
    • 13 Rhee SG. Redox signaling: hydrogen peroxide as intracellular messenger. Exp Mol Med. 1999; 31: 53–59.CrossrefMedlineGoogle Scholar
    • 14 Rhee SG, Chang TS, Bae YS, Lee SR, Kang SW. Cellular regulation by hydrogen peroxide. J Am Soc Nephrol. 2003; 14: S211–S215.CrossrefMedlineGoogle Scholar
    • 15 Khatri JJ, Johnson C, Magid R, Lessner SM, Laude KM, Dikalov SI, Harrison DG, Sung HJ, Rong Y, Galis ZS. Vascular oxidant stress enhances progression and angiogenesis of experimental atheroma. Circulation. 2004; 109: 520–525.LinkGoogle Scholar
    • 16 Yang H, Roberts LJ, Shi MJ, Zhou LC, Ballard BR, Richardson A, Guo ZM. Retardation of atherosclerosis by overexpression of catalase or both Cu/Zn-superoxide dismutase and catalase in mice lacking apolipoprotein E. Circ Res. 2004; 95: 1075–1081.LinkGoogle Scholar
    • 17 Tribble DL, Gong EL, Leeuwenburgh C, Heinecke JW, Carlson EL, Verstuyft JG, Epstein CJ. Fatty streak formation in fat-fed mice expressing human copper-zinc superoxide dismutase. Arterioscler Thromb Vasc Biol. 1997; 17: 1734–1740.CrossrefMedlineGoogle Scholar
    • 18 Jung O, Marklund SL, Geiger H, Pedrazzini T, Busse R, Brandes RP. Extracellular superoxide dismutase is a major determinant of nitric oxide bioavailability: in vivo and ex vivo evidence from ecSOD-deficient mice. Circ Res. 2003; 93: 622–629.LinkGoogle Scholar
    • 19 Fennell JP, Brosnan MJ, Frater AJ, Hamilton CA, Alexander MY, Nicklin SA, Heistad DD, Baker AH, Dominiczak AF. Adenovirus-mediated overexpression of extracellular superoxide dismutase improves endothelial dysfunction in a rat model of hypertension. Gene Ther. 2002; 9: 110–117.CrossrefMedlineGoogle Scholar
    • 20 Sentman ML, Brannstrom T, Westerlund S, Laukkanen MO, Yla-Herttuala S, Basu S, Marklund SL. Extracellular superoxide dismutase deficiency and atherosclerosis in mice. Arterioscler Thromb Vasc Biol. 2001; 21: 1477–1482.CrossrefMedlineGoogle Scholar
    • 21 Laude K, Cai H, Fink B, Hoch N, Weber DS, McCann L, Kojda G, Fukai T, Schmidt HH, Dikalov S, Ramasamy S, Gamez G, Griendling KK, Harrison DG. Hemodynamic and biochemical adaptations to vascular smooth muscle overexpression of p22phox in mice. Am J Physiol Heart Circ Physiol. 2005; 288: H7–H12.CrossrefMedlineGoogle Scholar
    • 22 Cai H, Davis ME, Drummond GR, Harrison DG. Induction of endothelial NO synthase by hydrogen peroxide via a Ca(2+)/calmodulin-dependent protein kinase II/janus kinase 2-dependent pathway. Arterioscler Thromb Vasc Biol. 2001; 21: 1571–1576.CrossrefMedlineGoogle Scholar
    • 23 Drummond GR, Cai H, Davis ME, Ramasamy S, Harrison DG. Transcriptional and posttranscriptional regulation of endothelial nitric oxide synthase expression by hydrogen peroxide. Circ Res. 2000; 86: 347–354.CrossrefMedlineGoogle Scholar
    • 24 Maulik N, Das DK. Redox signaling in vascular angiogenesis. Free Radic Biol Med. 2002; 33: 1047–1060.CrossrefMedlineGoogle Scholar
    • 25 Griendling KK, Ushio-Fukai M. Reactive oxygen species as mediators of angiotensin II signaling. Regul Pept. 2000; 91: 21–27.CrossrefMedlineGoogle Scholar
    • 26 McQuaid KE, Keenan AK. Endothelial barrier dysfunction and oxidative stress: roles for nitric oxide? Exp Physiol. 1997; 82: 369–376.CrossrefMedlineGoogle Scholar
    • 27 Dalle-Donne I, Rossi R, Milzani A, Di Simplicio P, Colombo R. The actin cytoskeleton response to oxidants: from small heat shock protein phosphorylation to changes in the redox state of actin itself. Free Radic Biol Med. 2001; 31: 1624–1632.CrossrefMedlineGoogle Scholar
    • 28 Taniyama Y, Griendling KK. Reactive oxygen species in the vasculature: molecular and cellular mechanisms. Hypertension. 2003; 42: 1075–1081.LinkGoogle Scholar
    • 29 Griendling KK, Sorescu D, Lassegue B, Ushio-Fukai M. Modulation of protein kinase activity and gene expression by reactive oxygen species and their role in vascular physiology and pathophysiology. Arterioscler Thromb Vasc Biol. 2000; 20: 2175–2183.CrossrefMedlineGoogle Scholar
    • 30 Lessner SM, Galis ZS. Matrix metalloproteinases and vascular endothelium-mononuclear cell close encounters. Trends Cardiovasc Med. 2004; 14: 105–111.CrossrefMedlineGoogle Scholar
    • 31 Pasterkamp G, Galis ZS, de Kleijn DP. Expansive arterial remodeling: location, location, location. Arterioscler Thromb Vasc Biol. 2004; 24: 650–657.LinkGoogle Scholar
    • 32 Sundaresan M, Yu ZX, Ferrans VJ, Irani K, Finkel T. Requirement for generation of H2O2 for platelet-derived growth factor signal transduction. Science. 1995; 270: 296–299.CrossrefMedlineGoogle Scholar
    • 33 Abe J, Takahashi M, Ishida M, Lee JD, Berk BC. c-Src is required for oxidative stress-mediated activation of big mitogen-activated protein kinase 1. J Biol Chem. 1997; 272: 20389–20394.CrossrefMedlineGoogle Scholar
    • 34 Ushio-Fukai M, Alexander RW, Akers M, Griendling KK. p38 Mitogen-activated protein kinase is a critical component of the redox-sensitive signaling pathways activated by angiotensin II. Role in vascular smooth muscle cell hypertrophy. J Biol Chem. 1998; 273: 15022–15029.CrossrefMedlineGoogle Scholar
    • 35 Yoshizumi M, Abe J, Haendeler J, Huang Q, Berk BC. Src and Cas mediate JNK activation but not ERK1/2 and p38 kinases by reactive oxygen species. J Biol Chem. 2000; 275: 11706–11712.CrossrefMedlineGoogle Scholar
    • 36 Cai H, Li Z, Davis ME, Kanner W, Harrison DG, Dudley SC Jr. Akt-dependent phosphorylation of serine 1179 and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2 cooperatively mediate activation of the endothelial nitric-oxide synthase by hydrogen peroxide. Mol Pharmacol. 2003; 63: 325–331.CrossrefMedlineGoogle Scholar
    • 37 Nguyen A, Chen P, Cai H. Role of CaMKII in hydrogen peroxide activation of ERK1/2, p38 MAPK, HSP27 and actin reorganization in endothelial cells. FEBS Lett. 2004; 572: 307–313.CrossrefMedlineGoogle Scholar
    • 38 Aslan M, Ozben T. Oxidants in receptor tyrosine kinase signal transduction pathways. Antioxid Redox Signal. 2003; 5: 781–788.CrossrefMedlineGoogle Scholar
    • 39 Simon AR, Rai U, Fanburg BL, Cochran BH. Activation of the JAK-STAT pathway by reactive oxygen species. Am J Physiol. 1998; 275: C1640–C1652.CrossrefMedlineGoogle Scholar
    • 40 Vepa S, Scribner WM, Parinandi NL, English D, Garcia JG, Natarajan V. Hydrogen peroxide stimulates tyrosine phosphorylation of focal adhesion kinase in vascular endothelial cells. Am J Physiol. 1999; 277: L150–L158.MedlineGoogle Scholar
    • 41 Frank GD, Mifune M, Inagami T, Ohba M, Sasaki T, Higashiyama S, Dempsey PJ, Eguchi S. Distinct mechanisms of receptor and nonreceptor tyrosine kinase activation by reactive oxygen species in vascular smooth muscle cells: role of metalloprotease and protein kinase C-δ. Mol Cell Biol. 2003; 23: 1581–1589.CrossrefMedlineGoogle Scholar
    • 42 Melaragno MG, Wuthrich DA, Poppa V, Gill D, Lindner V, Berk BC, Corson MA. Increased expression of Axl tyrosine kinase after vascular injury and regulation by G protein-coupled receptor agonists in rats. Circ Res. 1998; 83: 697–704.CrossrefMedlineGoogle Scholar
    • 43 Konishi A, Aizawa T, Mohan A, Korshunov VA, Berk BC. Hydrogen peroxide activates the Gas6-Axl pathway in vascular smooth muscle cells. J Biol Chem. 2004; 279: 28766–28770.CrossrefMedlineGoogle Scholar
    • 44 Chen K, Thomas SR, Albano A, Murphy MP, Keaney JF Jr. Mitochondrial function is required for hydrogen peroxide-induced growth factor receptor transactivation and downstream signaling. J Biol Chem. 2004; 279: 35079–35086.CrossrefMedlineGoogle Scholar
    • 45 Thomas SR, Chen K, Keaney JF Jr. Hydrogen peroxide activates endothelial nitric-oxide synthase through coordinated phosphorylation and dephosphorylation via a phosphoinositide 3-kinase-dependent signaling pathway. J Biol Chem. 2002; 277: 6017–6024.CrossrefMedlineGoogle Scholar
    • 46 Cai H, Li Z, Dikalov S, Holland SM, Hwang J, Jo H, Dudley SC Jr, Harrison DG. NAD(P)H oxidase-derived hydrogen peroxide mediates endothelial nitric oxide production in response to angiotensin II. J Biol Chem. 2002; 277: 48311–48317.CrossrefMedlineGoogle Scholar
    • 47 Lenaz G. Role of mitochondria in oxidative stress and ageing. Biochim Biophys Acta. 1998; 1366: 53–67.CrossrefMedlineGoogle Scholar
    • 48 Tampo Y, Kotamraju S, Chitambar CR, Kalivendi SV, Keszler A, Joseph J, Kalyanaraman B. Oxidative stress-induced iron signaling is responsible for peroxide-dependent oxidation of dichlorodihydrofluorescein in endothelial cells: role of transferrin receptor-dependent iron uptake in apoptosis. Circ Res. 2003; 92: 56–63.LinkGoogle Scholar
    • 49 Dhanasekaran A, Kotamraju S, Kalivendi SV, Matsunaga T, Shang T, Keszler A, Joseph J, Kalyanaraman B. Supplementation of endothelial cells with mitochondria-targeted antioxidants inhibit peroxide-induced mitochondrial iron uptake, oxidative damage, and apoptosis. J Biol Chem. 2004; 279: 37575–37587.CrossrefMedlineGoogle Scholar
    • 50 Li WG, Miller FJ Jr, Zhang HJ, Spitz DR, Oberley LW, Weintraub NL. H(2)O(2)-induced O(2) production by a non-phagocytic NAD(P)H oxidase causes oxidant injury. J Biol Chem. 2001; 276: 29251–29256.CrossrefMedlineGoogle Scholar
    • 51 Djordjevic T, Pogrebniak A, BelAiba RS, Bonello S, Wotzlaw C, Acker H, Hess J, Gorlach A. The expression of the NADPH oxidase subunit p22phox is regulated by a redox-sensitive pathway in endothelial cells. Free Radic Biol Med. 2005; 38: 616–630.CrossrefMedlineGoogle Scholar
    • 52 McNally JS, Davis ME, Giddens DP, Saha A, Hwang J, Dikalov S, Jo H, Harrison DG. Role of xanthine oxidoreductase and NAD(P)H oxidase in endothelial superoxide production in response to oscillatory shear stress. Am J Physiol Heart Circ Physiol. 2003; 285: H2290–H2297.CrossrefMedlineGoogle Scholar
    • 52A Chalupsky K, Nguyen A, Cai H. Endothelial dihydrofolate reductase: critical for tetrahydrobiopterin-NO bioavailability and role in angiotensin II uncoupling of eNOS. Proc Natl Acad Sci U S A. In press.Google Scholar
    • 53 Hink U, Li H, Mollnau H, Oelze M, Matheis E, Hartmann M, Skatchkov M, Thaiss F, Stahl RA, Warnholtz A, Meinertz T, Griendling K, Harrison DG, Forstermann U, Munzel T. Mechanisms underlying endothelial dysfunction in diabetes mellitus. Circ Res. 2001; 88: E14–E22.CrossrefMedlineGoogle Scholar
    • 54 Lassegue B, Clempus RE. Vascular NAD(P)H oxidases: specific features, expression, and regulation. Am J Physiol Regul Integr Comp Physiol. 2003; 285: R277–R297.CrossrefMedlineGoogle Scholar
    • 55 Bayraktutan U, Blayney L, Shah AM. Molecular characterization and localization of the NAD(P)H oxidase components gp91-phox and p22-phox in endothelial cells. Arterioscler Thromb Vasc Biol. 2000; 20: 1903–1911.CrossrefMedlineGoogle Scholar
    • 56 Li JM, Shah AM. Intracellular localization and preassembly of the NADPH oxidase complex in cultured endothelial cells. J Biol Chem. 2002; 277: 19952–19960.CrossrefMedlineGoogle Scholar
    • 57 Frey RS, Rahman A, Kefer JC, Minshall RD, Malik AB. PKCzeta regulates TNF-α-induced activation of NADPH oxidase in endothelial cells. Circ Res. 2002; 90: 1012–1019.LinkGoogle Scholar
    • 58 Li JM, Shah AM. Mechanism of endothelial cell NADPH oxidase activation by angiotensin II: Role of the p47phox subunit. J Biol Chem. 2003; 278: 12094–12100.CrossrefMedlineGoogle Scholar
    • 59 Gorlach A, Brandes RP, Nguyen K, Amidi M, Dehghani F, Busse R. A gp91phox containing NADPH oxidase selectively expressed in endothelial cells is a major source of oxygen radical generation in the arterial wall. Circ Res. 2000; 87: 26–32.CrossrefMedlineGoogle Scholar
    • 60 Furst R, Brueckl C, Kuebler WM, Zahler S, Krotz F, Gorlach A, Vollmar AM, Kiemer AK. Atrial natriuretic peptide induces mitogen-activated protein kinase phosphatase-1 in human endothelial cells via Rac1 and NAD(P)H oxidase/Nox2-activation. Circ Res. 2005; 96: 43–53.LinkGoogle Scholar
    • 61 Ago T, Kitazono T, Ooboshi H, Iyama T, Han YH, Takada J, Wakisaka M, Ibayashi S, Utsumi H, Iida M. Nox4 as the major catalytic component of an endothelial NAD(P)H oxidase. Circulation. 2004; 109: 227–233.LinkGoogle Scholar
    • 62 Buul JD, Fernandez-Borja M, Anthony EC, Hordijk PL. Expression and localization of NOX2 and NOX4 in primary human endothelial cells. Antioxid Redox Signal. 2005; 7: 308–317.CrossrefMedlineGoogle Scholar
    • 63 Sorescu GP, Song H, Tressel SL, Hwang J, Dikalov S, Smith DA, Boyd NL, Platt MO, Lassegue B, Griendling KK, Jo H. Bone morphogenic protein 4 produced in endothelial cells by oscillatory shear stress induces monocyte adhesion by stimulating reactive oxygen species production from a nox1-based NADPH oxidase. Circ Res. 2004; 95: 773–779.LinkGoogle Scholar
    • 64 Kobayashi S, Nojima Y, Shibuya M, Maru Y. Nox1 regulates apoptosis and potentially stimulates branching morphogenesis in sinusoidal endothelial cells. Exp Cell Res. 2004; 300: 455–462.CrossrefMedlineGoogle Scholar
    • 65 Lassegue B, Sorescu D, Szocs K, Yin Q, Akers M, Zhang Y, Grant SL, Lambeth JD, Griendling KK. Novel gp91(phox) homologues in vascular smooth muscle cells : nox1 mediates angiotensin II-induced superoxide formation and redox-sensitive signaling pathways. Circ Res. 2001; 88: 888–894.CrossrefMedlineGoogle Scholar
    • 66 Geiszt M, Kopp JB, Varnai P, Leto TL. Identification of renox, an NAD(P)H oxidase in kidney. Proc Natl Acad Sci U S A. 2000; 97: 8010–8014.CrossrefMedlineGoogle Scholar
    • 67 Hilenski LL, Clempus RE, Quinn MT, Lambeth JD, Griendling KK. Distinct subcellular localizations of Nox1 and Nox4 in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 2004; 24: 677–683.LinkGoogle Scholar
    • 68 Geiszt M, Lekstrom K, Witta J, Leto TL. Proteins homologous to p47phox and p67phox support superoxide production by NAD(P)H oxidase 1 in colon epithelial cells. J Biol Chem. 2003; 278: 20006–20012.CrossrefMedlineGoogle Scholar
    • 69 Banfi B, Clark RA, Steger K, Krause KH. Two novel proteins activate superoxide generation by the NADPH oxidase NOX1. J Biol Chem. 2003; 278: 3510–3513.CrossrefMedlineGoogle Scholar
    • 70 Takeya R, Ueno N, Kami K, Taura M, Kohjima M, Izaki T, Nunoi H, Sumimoto H. Novel human homologues of p47phox and p67phox participate in activation of superoxide-producing NADPH oxidases. J Biol Chem. 2003; 278: 25234–25246.CrossrefMedlineGoogle Scholar
    • 71 Kawahara T, Kuwano Y, Teshima-Kondo S, Takeya R, Sumimoto H, Kishi K, Tsunawaki S, Hirayama T, Rokutan K. Role of nicotinamide adenine dinucleotide phosphate oxidase 1 in oxidative burst response to Toll-like receptor 5 signaling in large intestinal epithelial cells. J Immunol. 2004; 172: 3051–3058.CrossrefMedlineGoogle Scholar
    • 72 Edens WA, Sharling L, Cheng G, Shapira R, Kinkade JM, Lee T, Edens HA, Tang X, Sullards C, Flaherty DB, Benian GM, Lambeth JD. Tyrosine cross-linking of extracellular matrix is catalyzed by Duox, a multidomain oxidase/peroxidase with homology to the phagocyte oxidase subunit gp91phox. J Cell Biol. 2001; 154: 879–891.CrossrefMedlineGoogle Scholar
    • 73 Lambeth JD. Nox/Duox family of nicotinamide adenine dinucleotide (phosphate) oxidases. Curr Opin Hematol. 2002; 9: 11–17.CrossrefMedlineGoogle Scholar
    • 74 Geiszt M, Witta J, Baffi J, Lekstrom K, Leto TL. Dual oxidases represent novel hydrogen peroxide sources supporting mucosal surface host defense. FASEB J. 2003; 17: 1502–1504.CrossrefMedlineGoogle Scholar
    • 75 Forteza R, Salathe M, Miot F, Conner GE. Regulated H2O2 production by duox in human airway epithelial cells. Am J Respir Cell Mol Biol. In press.Google Scholar
    • 76 Hanna IR, Hilenski LL, Dikalova A, Taniyama Y, Dikalov S, Lyle A, Quinn MT, Lassegue B, Griendling KK. Functional association of nox1 with p22phox in vascular smooth muscle cells. Free Radic Biol Med. 2004; 37: 1542–1549.CrossrefMedlineGoogle Scholar
    • 77 Ambasta RK, Kumar P, Griendling KK, Schmidt HH, Busse R, Brandes RP. Direct interaction of the novel Nox proteins with p22phox is required for the formation of a functionally active NADPH oxidase. J Biol Chem. 2004; 279: 45935–45941.CrossrefMedlineGoogle Scholar
    • 78 Guzik TJ, Sadowski J, Kapelak B, Jopek A, Rudzinski P, Pillai R, Korbut R, Channon KM. Systemic regulation of vascular NAD(P)H oxidase activity and nox isoform expression in human arteries and veins. Arterioscler Thromb Vasc Biol. 2004; 24: 1614–1620.LinkGoogle Scholar


    eLetters should relate to an article recently published in the journal and are not a forum for providing unpublished data. Comments are reviewed for appropriate use of tone and language. Comments are not peer-reviewed. Acceptable comments are posted to the journal website only. Comments are not published in an issue and are not indexed in PubMed. Comments should be no longer than 500 words and will only be posted online. References are limited to 10. Authors of the article cited in the comment will be invited to reply, as appropriate.

    Comments and feedback on AHA/ASA Scientific Statements and Guidelines should be directed to the AHA/ASA Manuscript Oversight Committee via its Correspondence page.