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Isoform specific functions of Nox protein-derived reactive oxygen species in the vasculature
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Isoform specific functions of Nox protein-derived reactive oxygen species in the vasculature Katrin Schro¨der The family of NADPH oxidases consists of seven members that are all producing reactive oxygen species (ROS). In the cardiovascular systems three NADPH oxidases are expressed: Nox1, Nox2 and Nox4, which all exhibit different physiological functions such as regulating blood pressure, mediating growth factor signaling and controlling cell proliferation. In this article the recent research concerning the specificity and function mediated by NADPH oxidases will be reviewed. Address Institut fu¨r Kardiovaskula¨re Physiologie, Fachbereich Medizin der Goethe-Universita¨t, Theodor-Stern Kai 7, 60596 Frankfurt am Main, Germany Corresponding author: Schro¨der, Katrin ([email protected])
Current Opinion in Pharmacology 2010, 10:122–126 This review comes from a themed issue on Cardiovascular and renal Edited by Ralf P Brandes Available online 10th February 2010 1471-4892/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coph.2010.01.002
Introduction Reactive oxygen species (ROS) have been attributed potential dangerous molecules as they can oxidize lipids and DNA and limit the availability of NO. In recent years this view changed substantially and it became clear that ROS are important second messengers. Several sources of ROS, such as mitochondria, xanthine oxidase, NO synthase and cytochrome P450 monooxygenases have all been shown to be of relevance for vascular ROS production but NADPH oxidases, potentially because of the availability of knockout mice, recently received most attention. The family of NADPH oxidases consists of seven members, Nox1–Nox5 and Doux1 and Doux2. Among these, Nox1, Nox2 and Nox4 are of relevance in the cardiovascular system. The information concerning Nox5, which is due to gene deletion not expressed in rodents, is still limited and thus, although potentially important, Nox5 will not be addressed in this article. The classic leukocyte NADPH oxidase consists of the large eponymous subunit Nox2 and the small membraneCurrent Opinion in Pharmacology 2010, 10:122–126
bound subunit, p22phox, which is obligatory for the function and stability of Nox1–Nox4. While Nox4 is independent of additional cytosolic proteins, Nox1 and Nox2 require subunits for their activation (for detail see [1]). During the process of activation a cytosolic activator protein, p67phox or NoxA1, translocates to the membrane-bound Nox subunit with the aid of an organizer protein (p47phox or NoxO1). This process is constitutive for NoxO1, whereas p47phox has to be phosphorylated on serine residues by protein kinase C and others to allow its interaction with the plasma membrane and subsequently the Nox subunit. Activation of Nox2 also requires interaction with the active form of the small GTPase Rac. A putative Rac-binding site is also conserved in Nox1, but was not found in Nox4, indicating a Rac-independent activity of the latter [2]. So far, the production of ROS is the only known function of NADPH oxidases. Interestingly different types of ROS are produced by NADPH oxidases. Nox4 predominantly generates hydrogen peroxide (H2O2), whereas superoxide anions (O2 ) are produced by Nox1 and Nox2. Due to the low stability of many types of ROS, the site of production is important for the specificity of ROSmediated signaling, because physical proximity increases the efficiency of substrate oxidation. Accordingly, a focus of research has been the localization of NADPH oxidases, which differs among the Nox homologues at cellular and subcellular level. In rodents, the predominant expression pattern is that endothelial cells express Nox2 and Nox4 whereas VSMC express Nox1 and Nox4. Nox1 and Nox2 are thought to be primarily expressed at the plasma membrane. Nox4 is attached to intracellular membrane compartments [3,4]. Acute agonist activation of NADPH oxidases has been shown for a broad range of stimuli such as cytokines, growth factor, toll-like receptor ligand, physical stimuli and cardiovascular risk factors. These factors may also affect the expression of the respective Nox protein. In analogy to the situation of NO synthases, control of activity through modulation of protein expression has however been predominantely attributed to Nox4, which appears to be the only true inducible NADPH oxidase. Reported inducers of Nox4 are transforming growth factor b1 (TGF-b1), high glucose and oxidized lipids. In the following sections individual Nox homologues will be discussed in more detail to explain the rational not to block general ROS production or action and NADPH www.sciencedirect.com
Isoform specific functions of Nox protein-derived reactive oxygen species in the vasculature Schro¨der 123
Figure 1
Scheme of NADPH oxidase homologues.
oxidases. A pharmacological approach rather should inhibit individual NADPH oxidases involved in specific diseases without affecting others, which are necessary for untroubled cellular or somatic function (Figures 1 and 2).
in blood pressure and ROS production in vascular smooth muscle cells (VSMC) [5,6].
Figure 2
In a transgenic hypertensive model (human renin gene, human angiotensinogen gene) the hypertensive effect was potentiated by Nox1 overexpression. Unexpectedly, knockout of Nox1 failed to affect blood pressure suggesting that either the model is too robust for the modulatory effect of Nox1 or that a down-regulation of the antioxidative defense in a life-long hypertensive situation occurs [7].
Scheme of the function of the NADPH oxidase homologues Nox1, Nox2 and Nox4. All homologues are necessary to maintain normal physiological function of cells as well as of the whole organism. Overactivation or failure of function of the NADPH oxidases results in detrimental alterations of cellular functions and subsequently in pathophysiological changes of the organism.
The effect of Nox1 on blood pressure appears to expend beyond a pure scavenging of NO by ROS: Nox1 deficient cells exhibit a mislocalization of the angiotensin II receptor resulting from a declined phosphorylation of caveolin [8]. Moreover, the angiotensin II-mediated induction of Nox1 depends on mitochondrial integrity [9], suggesting crosstalk between mitochondria and Nox1 by means of a positive feedback loop. ROS generated by either source cause Ca2+ influx by inhibition of voltage-dependent potassium-channels and stimulation of ryanodine receptors, leading to a Nox1-dependent contraction of pulmonary artery SMCs [10]. Finally, Nox1-derived ROS target phosphatases: down-regulation of this oxidase leads to a reduced oxidative inactivation of the phosphatases SHP-2 and thereby to an impaired AngII-stimulated phosphorylation of kinases [11]. Obviously, this has an impact on a broad spectrum of cellular functions such as proliferation and migration: neointima formation in mice after vascular injury is Nox1-dependent [12]. The impaired Nox1-induced migration and proliferation of VSMC involves an attenuated JNK-mediated phosphorylation of paxillin [13]
Nox1 mediates agonist-induced ROS production in vascular smooth muscle cells Experiments in knockout mice revealed that Nox1 is involved in the angiotensin II (AngII)-induced increase
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and decreased p21-PAK-mediated phosphorylation of cofilin [12]. On the basis of the above-mentioned observations and studies on gene expression, a role for Nox1 has primarily been attributed to VSMC. Indeed, it has been suggested that situations of excessive cellular stress, such as oscillatory flow are required to induce significant level of Nox1 in endothelial cells [14]. The different stress situations in which this effect becomes relevant still need to be determined in vivo. In contrast to the overwhelming importance of Nox1 in rodents, the contribution of this oxidase in human vascular pathology is still uncertain. Different groups failed to detect Nox1 expression in human arteries [15,16] and the total number of studies on Nox1 in human vascular tissue is still very limited.
Nox2 is the predominant NADPH oxidase in endothelial cells Different to Nox1, the outstanding importance of Nox2 in the control of vascular function in humans is indisputable. Patients carrying mutations of Nox2 exhibit enhanced endothelium-dependent flow-mediated vaso-relaxation and impaired markers of vascular aging and oxidative stress [17]. This suggests that Nox2, which is expressed not only in circulating blood cells such as leukocytes and monocytes but also in endothelial cells, importantly limits NO availability in man. As similar observations were made in isolated vessels from wildtype and Nox2deficient mice [18], it appears appropriate to conclude that endothelial Nox2 at least in part contributes to this phenomenon. Interestingly with respect to signaling, Nox2 apparently has a dual function in vasculature: whereas this oxidase is essentially involved in physiological signal transduction under normal conditions, Nox2-derived ROS are central mediators of oxidative stress in vascular pathology. Under physiological conditions, the signal transduction of a large panel of cytokines, hormones and growth factors has been shown to involve Nox2: for example, the VEGF-induced signal transduction and the subsequent VEGF-mediated pro-angiogenic response to mild ischemia is attenuated in Nox2 deficiency [19,20,21]. We recently have provided evidence that also the signal transduction of erythropoietin (EPO) in endothelial cells and bone marrow cells requires Nox2: Nox2-derived ROS inactivate the phosphatase SHP-2 and thereby allow EPO-mediated mobilization of endothelial progenitor cells and vascular repair [22].
system with Nox2-derived ROS lead to oxidative stress and cellular dysfunction. Thus, genetic deletion of Nox2 has been shown to attenuate ischemia-perfusion injury in various organs [23,24] but also to restore angiogenic responses in the hind-limb ischemia model in the presence of confounding vascular risk factors such as diabetes or smoke-induced vascular dysfunction [25,26].
The constitutively active Nox4 is ubiquitously expressed in the vascular system Nox4 differs from the other vascular NADPH oxidases in its mode of activation, the type of ROS release, the subcellular localization and the requirements for cytosolic activator proteins. These facts may suggest that Nox4 has unique functions in the vascular system. Indeed, increased expression of Nox4 has been reported in differentiated cells and the process of differentiation itself has been linked to this NADPH oxidase [27–29]. Outside the vascular system, the insulin-induced differentiation into adipocytes is Nox4-dependent [29] and this hormone but also hyperglycemic conditions are known to induce Nox4 [30]. Whether Nox4 contributes to diabetesassociated vascular complications is uncertain but cell culture experiments revealed that a part of the vascular resistance to NO under hyperglycemic conditions is mediated by Nox4 [30]. The constant ROS production by Nox4 has also been linked to aging. Replicative senescence was indeed accelerated by overexpression of Nox4 [31], whereas down-regulation of the protein in endothelial cells prolonged the duration till the onset of senescence [32]. Basal ROS levels are also required for proliferation and thus Nox4, by altering the basal activity of MAP kinases, has also been linked to this process in different types of vascular cells [33–35]. This colorful picture of a broad set of differential effects of Nox4 illustrates that a continuous production of ROS impacts on a large spectrum of cellular characteristics. Interestingly, recent reports indicate a possible agonistinduced activation of Nox4; this has been suggested for transforming growth factor b1, for agonists on toll like receptors [36,37] and Fcg-receptor ligands as well as several oxidized lipids [38]. The mechanisms underlying acute activation of Nox4 are largely unknown but may involve interaction with binding proteins, such as Poldip2 [39], translocation in membrane compartments such as lipid rafts [40] and potentially even an interaction with activating GTPases such as Rac1 [38].
Concluding remarks Under disease conditions, such as severe ischemia or diabetes, Nox2 however makes a rather deleterious contribution to the outcome of the models. The over-activation of the enzyme and a subsequent flooding of the Current Opinion in Pharmacology 2010, 10:122–126
In the vasculature all present homologues of the NADPH oxidase permit different functions and the absence of individual NADPH oxidases results in different phenotypes. Pharmacological interference with the function of www.sciencedirect.com
Isoform specific functions of Nox protein-derived reactive oxygen species in the vasculature Schro¨der 125
specific NADPH oxidases might be helpful in special situations: Nox1 inhibition could prevent the development of hypertension. An inhibition of Nox2 on the other hand may modulate angiogenesis. Nox4 might be a drug target in developing fibrosis but at the moment there is no evidence that blocking Nox4 will improve vascular function.
Acknowledgements This study was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB815/TP1), the excellence cluster cardiopulmonary system (ECCPS) and the Goethe-University.
Conflict of interest The author declares that she has no conflict of interest relating to this work.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
2.
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Helmcke I, Heumuller S, Tikkanen R, Schro¨der K, Brandes RP: Identification of structural elements in Nox1 and Nox4 controlling localization and activity. Antioxid Redox Signal 2009, 11:1279-1287. Herein the authors link functional differences between Nox1 and Nox4 to specific molecular features of both enzymes.
3.
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20. Tojo T, Ushio-Fukai M, Yamaoka-Tojo M, Ikeda S, Patrushev N, Alexander RW: Role of gp91phox (Nox2)-containing NAD(P)H oxidase in angiogenesis in response to hindlimb ischemia. Circulation 2005, 111:2347-2355.
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Basset O, Deffert C, Foti M, Bedard K, Jaquet V, Ogier-Denis E, Krause KH: NADPH oxidase 1 deficiency alters caveolin phosphorylation and angiotensin II-receptor localization in vascular smooth muscle. Antioxid Redox Signal 2009, 11:23712384.
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Wosniak J Jr, Santos CX, Kowaltowski AJ, Laurindo FR: Crosstalk between mitochondria and NADPH oxidase: effects of mild mitochondrial dysfunction on angiotensin II-mediated increase in Nox isoform expression and activity in vascular smooth muscle cells. Antioxid Redox Signal 2009, 11:1265-1278.
10. Wang YX, Zheng YM: ROS-dependent signaling mechanisms for hypoxic Ca2+ responses in pulmonary artery myocytes. Antioxid Redox Signal 2010, 12:611-623. In this study the authors suggest a mechanism how ROS may influence smooth muscle cell (SMC) contractility. ROS inhibit voltage-dependent www.sciencedirect.com
18. Jung O, Schreiber JG, Geiger H, Pedrazzini T, Busse R, Brandes RP: gp91phox-containing NADPH oxidase mediates endothelial dysfunction in renovascular hypertension. Circulation 2004, 109:1795-1801.
21. Urao N, Inomata H, Razvi M, Kim HW, Wary K, McKinney R, Fukai T, Ushio-Fukai M: Role of nox2-based NADPH oxidase in bone marrow and progenitor cell function involved in neovascularization induced by hindlimb ischemia. Circ Res 2008, 103:212-220. In this study mild hind-limb ischemia was performed. VEGF mediated mobilization of EPCs and subsequently enhanced angiogenesis in the ischemic tissue in a Nox2-dependent manner. Compare with reference number [25]. 22. Schro¨der K, Kohnen A, Aicher A, Liehn EA, Buchse T, Stein S, Weber C, Dimmeler S, Brandes RP: NADPH oxidase Nox2 is required for hypoxia-induced mobilization of endothelial progenitor cells. Circ Res 2009, 105:537-544. In this study the authors show that Nox2 deficiency resulted in an abrogated EPC-mobilization in response to hypoxia and erythropoietin-injection. Nox2 deficiency results in an abrogated inhibition of the Current Opinion in Pharmacology 2010, 10:122–126
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phosphatase SHP-2 leading to an abbrogation of erythropoietin-indiced signaling. 23. Al-Mehdi AB, Zhao G, Dodia C, Tozawa K, Costa K, Muzykantov V, Ross C, Blecha F, Dinauer M, Fisher AB: Endothelial NADPH oxidase as the source of oxidants in lungs exposed to ischemia or high K+. Circ Res 1998, 83:730-737. 24. Kahles T, Luedike P, Endres M, Galla HJ, Steinmetz H, Busse R, Neumann-Haefelin T, Brandes RP: NADPH oxidase plays a central role in blood–brain barrier damage in experimental stroke. Stroke 2007, 38:3000-3006. 25. Haddad P, Dussault S, Groleau J, Turgeon J, Michaud SE, Menard C, Perez G, Maingrette F, Rivard A: Nox2-containing NADPH oxidase deficiency confers protection from hindlimb ischemia in conditions of increased oxidative stress. Arterioscler Thromb Vasc Biol 2009, 29:1522-1528. This publication indicates beneficial effects of Nox2 deficiency for angiogenisis in a model of severe hind-limb ischemia. Both in vitro and in vivo positive effects on neovascularization were lost after treatment with the NO inhibitor L-NAME. Please compare with reference number [21]. 26. Ebrahimian TG, Heymes C, You D, Blanc-Brude O, Mees B, Waeckel L, Duriez M, Vilar J, Brandes RP, Levy BI et al.: NADPH oxidase-derived overproduction of reactive oxygen species impairs postischemic neovascularization in mice with type 1 diabetes. Am J Pathol 2006, 169:719-728. 27. Sorescu D, Weiss D, Lassegue B, Clempus RE, Szocs K, Sorescu GP, Valppu L, Quinn MT, Lambeth JD, Vega JD et al.: Superoxide production and expression of nox family proteins in human atherosclerosis. Circulation 2002, 105:1429-1435. 28. Clempus RE, Sorescu D, Dikalova AE, Pounkova L, Jo P, Sorescu GP, Schmidt HH, Lassegue B, Griendling KK: Nox4 is required for maintenance of the differentiated vascular smooth muscle cell phenotype. Arterioscler Thromb Vasc Biol 2007, 27:42-48. 29. Schroder K, Wandzioch K, Helmcke I, Brandes RP: Nox4 acts as a switch between differentiation and proliferation in preadipocytes. Arterioscler Thromb Vasc Biol 2009, 29:239-245. 30. Tong X, Schro¨der K: NADPH oxidases are responsible for the failure of nitric oxide to inhibit migration of smooth muscle cells exposed to high glucose. Free Radic Biol Med 2009, 47:1578-1583. 31. McCrann DJ, Yang D, Chen H, Carroll S, Ravid K: Upregulation of Nox4 in the aging vasculature and its association with smooth muscle cell polyploidy. Cell Cycle 2009, 8:902-908. This publication deals with polyploidization of VSMC, as a biomarker for aging which is increased in cells overexpressing Nox4. This effect raises from a decreased expression of survivin, a chromosome passenger protein whose absence and mislocalization is assoziated with VSMC polyploidy.
Current Opinion in Pharmacology 2010, 10:122–126
32. Lener B, Koziel R, Pircher H, Hutter E, Greussing R, HerndlerBrandstetter D, Hermann M, Unterluggauer H, Jansen-Durr P: The NADPH oxidase Nox4 restricts the replicative lifespan of human endothelial cells. Biochem J 2009, 423:363-374. 33. Petry A, Djordjevic T, Weitnauer M, Kietzmann T, Hess J, Gorlach A: NOX2 and NOX4 mediate proliferative response in endothelial cells. Antioxid Redox Signal 2006, 8:1473-1484. 34. Menshikov M, Plekhanova O, Cai H, Chalupsky K, Parfyonova Y, Bashtrikov P, Tkachuk V, Berk BC: Urokinase plasminogen activator stimulates vascular smooth muscle cell proliferation via redox-dependent pathways. Arterioscler Thromb Vasc Biol 2006, 26:801-807. 35. Peshavariya H, Dusting GJ, Jiang F, Halmos LR, Sobey CG, Drummond GR, Selemidis S: NADPH oxidase isoform selective regulation of endothelial cell proliferation and survival. Naunyn Schmiedebergs Arch Pharmacol 2009, 380:193-204. 36. Maloney E, Sweet IR, Hockenbery DM, Pham M, Rizzo NO, Tateya S, Handa P, Schwartz MW, Kim F: Activation of NFkappaB by palmitate in endothelial cells: a key role for NADPH oxidase-derived superoxide in response to TLR4 activation. Arterioscler Thromb Vasc Biol 2009, 29:1370-1375. 37. Simon F, Fernandez R: Early lipopolysaccharide-induced reactive oxygen species production evokes necrotic cell death in human umbilical vein endothelial cells. J Hypertens 2009, 27:1202-1216. 38. Lee S, Gharavi NM, Honda H, Chang I, Kim B, Jen N, Li R, Zimman A, Berliner JA: A role for NADPH oxidase 4 in the activation of vascular endothelial cells by oxidized phospholipids. Free Radic Biol Med 2009, 47:145-151. In human aortic endothelial cells stimulated with oxidized 1-palmitoyl-2arachidonyl-sn-glycerol-3-phosphocholine (Ox-PAPC) Nox4 intercedes the transcription of inflammatory and sterol regulatory genes. Although unexpected interesting findings were provided: the authors report a VEGF-R2 transactivation and a subsequent recruitment of rac1 to the membrane necessary for Nox4 activation but no change in Nox4 expression in response to Ox-PAPC stimulation. 39. Lyle AN, Deshpande NN, Taniyama Y, Seidel-Rogol B, Pounkova L, Du P, Papaharalambus C, Lassegue B, Griendling KK: Poldip2, a novel regulator of Nox4 and cytoskeletal integrity in vascular smooth muscle cells. Circ Res 2009, 105:249-259. 40. Han W, Li H, Villar VA, Pascua AM, Dajani MI, Wang X, Natarajan A, Quinn MT, Felder RA, Jose PA, Yu P: Lipid rafts keep NADPH oxidase in the inactive state in human renal proximal tubule cells. Hypertension 2008, 51:481-487.
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Isoform specific functions of Nox protein-derived reactive oxygen species in the vasculature Katrin Schro¨der The family of NADPH oxidases consists of seven members that are all producing reactive oxygen species (ROS). In the cardiovascular systems three NADPH oxidases are expressed: Nox1, Nox2 and Nox4, which all exhibit different physiological functions such as regulating blood pressure, mediating growth factor signaling and controlling cell proliferation. In this article the recent research concerning the specificity and function mediated by NADPH oxidases will be reviewed. Address Institut fu¨r Kardiovaskula¨re Physiologie, Fachbereich Medizin der Goethe-Universita¨t, Theodor-Stern Kai 7, 60596 Frankfurt am Main, Germany Corresponding author: Schro¨der, Katrin ([email protected])
Current Opinion in Pharmacology 2010, 10:122–126 This review comes from a themed issue on Cardiovascular and renal Edited by Ralf P Brandes Available online 10th February 2010 1471-4892/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coph.2010.01.002
Introduction Reactive oxygen species (ROS) have been attributed potential dangerous molecules as they can oxidize lipids and DNA and limit the availability of NO. In recent years this view changed substantially and it became clear that ROS are important second messengers. Several sources of ROS, such as mitochondria, xanthine oxidase, NO synthase and cytochrome P450 monooxygenases have all been shown to be of relevance for vascular ROS production but NADPH oxidases, potentially because of the availability of knockout mice, recently received most attention. The family of NADPH oxidases consists of seven members, Nox1–Nox5 and Doux1 and Doux2. Among these, Nox1, Nox2 and Nox4 are of relevance in the cardiovascular system. The information concerning Nox5, which is due to gene deletion not expressed in rodents, is still limited and thus, although potentially important, Nox5 will not be addressed in this article. The classic leukocyte NADPH oxidase consists of the large eponymous subunit Nox2 and the small membraneCurrent Opinion in Pharmacology 2010, 10:122–126
bound subunit, p22phox, which is obligatory for the function and stability of Nox1–Nox4. While Nox4 is independent of additional cytosolic proteins, Nox1 and Nox2 require subunits for their activation (for detail see [1]). During the process of activation a cytosolic activator protein, p67phox or NoxA1, translocates to the membrane-bound Nox subunit with the aid of an organizer protein (p47phox or NoxO1). This process is constitutive for NoxO1, whereas p47phox has to be phosphorylated on serine residues by protein kinase C and others to allow its interaction with the plasma membrane and subsequently the Nox subunit. Activation of Nox2 also requires interaction with the active form of the small GTPase Rac. A putative Rac-binding site is also conserved in Nox1, but was not found in Nox4, indicating a Rac-independent activity of the latter [2]. So far, the production of ROS is the only known function of NADPH oxidases. Interestingly different types of ROS are produced by NADPH oxidases. Nox4 predominantly generates hydrogen peroxide (H2O2), whereas superoxide anions (O2 ) are produced by Nox1 and Nox2. Due to the low stability of many types of ROS, the site of production is important for the specificity of ROSmediated signaling, because physical proximity increases the efficiency of substrate oxidation. Accordingly, a focus of research has been the localization of NADPH oxidases, which differs among the Nox homologues at cellular and subcellular level. In rodents, the predominant expression pattern is that endothelial cells express Nox2 and Nox4 whereas VSMC express Nox1 and Nox4. Nox1 and Nox2 are thought to be primarily expressed at the plasma membrane. Nox4 is attached to intracellular membrane compartments [3,4]. Acute agonist activation of NADPH oxidases has been shown for a broad range of stimuli such as cytokines, growth factor, toll-like receptor ligand, physical stimuli and cardiovascular risk factors. These factors may also affect the expression of the respective Nox protein. In analogy to the situation of NO synthases, control of activity through modulation of protein expression has however been predominantely attributed to Nox4, which appears to be the only true inducible NADPH oxidase. Reported inducers of Nox4 are transforming growth factor b1 (TGF-b1), high glucose and oxidized lipids. In the following sections individual Nox homologues will be discussed in more detail to explain the rational not to block general ROS production or action and NADPH www.sciencedirect.com
Isoform specific functions of Nox protein-derived reactive oxygen species in the vasculature Schro¨der 123
Figure 1
Scheme of NADPH oxidase homologues.
oxidases. A pharmacological approach rather should inhibit individual NADPH oxidases involved in specific diseases without affecting others, which are necessary for untroubled cellular or somatic function (Figures 1 and 2).
in blood pressure and ROS production in vascular smooth muscle cells (VSMC) [5,6].
Figure 2
In a transgenic hypertensive model (human renin gene, human angiotensinogen gene) the hypertensive effect was potentiated by Nox1 overexpression. Unexpectedly, knockout of Nox1 failed to affect blood pressure suggesting that either the model is too robust for the modulatory effect of Nox1 or that a down-regulation of the antioxidative defense in a life-long hypertensive situation occurs [7].
Scheme of the function of the NADPH oxidase homologues Nox1, Nox2 and Nox4. All homologues are necessary to maintain normal physiological function of cells as well as of the whole organism. Overactivation or failure of function of the NADPH oxidases results in detrimental alterations of cellular functions and subsequently in pathophysiological changes of the organism.
The effect of Nox1 on blood pressure appears to expend beyond a pure scavenging of NO by ROS: Nox1 deficient cells exhibit a mislocalization of the angiotensin II receptor resulting from a declined phosphorylation of caveolin [8]. Moreover, the angiotensin II-mediated induction of Nox1 depends on mitochondrial integrity [9], suggesting crosstalk between mitochondria and Nox1 by means of a positive feedback loop. ROS generated by either source cause Ca2+ influx by inhibition of voltage-dependent potassium-channels and stimulation of ryanodine receptors, leading to a Nox1-dependent contraction of pulmonary artery SMCs [10]. Finally, Nox1-derived ROS target phosphatases: down-regulation of this oxidase leads to a reduced oxidative inactivation of the phosphatases SHP-2 and thereby to an impaired AngII-stimulated phosphorylation of kinases [11]. Obviously, this has an impact on a broad spectrum of cellular functions such as proliferation and migration: neointima formation in mice after vascular injury is Nox1-dependent [12]. The impaired Nox1-induced migration and proliferation of VSMC involves an attenuated JNK-mediated phosphorylation of paxillin [13]
Nox1 mediates agonist-induced ROS production in vascular smooth muscle cells Experiments in knockout mice revealed that Nox1 is involved in the angiotensin II (AngII)-induced increase
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124 Cardiovascular and renal
and decreased p21-PAK-mediated phosphorylation of cofilin [12]. On the basis of the above-mentioned observations and studies on gene expression, a role for Nox1 has primarily been attributed to VSMC. Indeed, it has been suggested that situations of excessive cellular stress, such as oscillatory flow are required to induce significant level of Nox1 in endothelial cells [14]. The different stress situations in which this effect becomes relevant still need to be determined in vivo. In contrast to the overwhelming importance of Nox1 in rodents, the contribution of this oxidase in human vascular pathology is still uncertain. Different groups failed to detect Nox1 expression in human arteries [15,16] and the total number of studies on Nox1 in human vascular tissue is still very limited.
Nox2 is the predominant NADPH oxidase in endothelial cells Different to Nox1, the outstanding importance of Nox2 in the control of vascular function in humans is indisputable. Patients carrying mutations of Nox2 exhibit enhanced endothelium-dependent flow-mediated vaso-relaxation and impaired markers of vascular aging and oxidative stress [17]. This suggests that Nox2, which is expressed not only in circulating blood cells such as leukocytes and monocytes but also in endothelial cells, importantly limits NO availability in man. As similar observations were made in isolated vessels from wildtype and Nox2deficient mice [18], it appears appropriate to conclude that endothelial Nox2 at least in part contributes to this phenomenon. Interestingly with respect to signaling, Nox2 apparently has a dual function in vasculature: whereas this oxidase is essentially involved in physiological signal transduction under normal conditions, Nox2-derived ROS are central mediators of oxidative stress in vascular pathology. Under physiological conditions, the signal transduction of a large panel of cytokines, hormones and growth factors has been shown to involve Nox2: for example, the VEGF-induced signal transduction and the subsequent VEGF-mediated pro-angiogenic response to mild ischemia is attenuated in Nox2 deficiency [19,20,21]. We recently have provided evidence that also the signal transduction of erythropoietin (EPO) in endothelial cells and bone marrow cells requires Nox2: Nox2-derived ROS inactivate the phosphatase SHP-2 and thereby allow EPO-mediated mobilization of endothelial progenitor cells and vascular repair [22].
system with Nox2-derived ROS lead to oxidative stress and cellular dysfunction. Thus, genetic deletion of Nox2 has been shown to attenuate ischemia-perfusion injury in various organs [23,24] but also to restore angiogenic responses in the hind-limb ischemia model in the presence of confounding vascular risk factors such as diabetes or smoke-induced vascular dysfunction [25,26].
The constitutively active Nox4 is ubiquitously expressed in the vascular system Nox4 differs from the other vascular NADPH oxidases in its mode of activation, the type of ROS release, the subcellular localization and the requirements for cytosolic activator proteins. These facts may suggest that Nox4 has unique functions in the vascular system. Indeed, increased expression of Nox4 has been reported in differentiated cells and the process of differentiation itself has been linked to this NADPH oxidase [27–29]. Outside the vascular system, the insulin-induced differentiation into adipocytes is Nox4-dependent [29] and this hormone but also hyperglycemic conditions are known to induce Nox4 [30]. Whether Nox4 contributes to diabetesassociated vascular complications is uncertain but cell culture experiments revealed that a part of the vascular resistance to NO under hyperglycemic conditions is mediated by Nox4 [30]. The constant ROS production by Nox4 has also been linked to aging. Replicative senescence was indeed accelerated by overexpression of Nox4 [31], whereas down-regulation of the protein in endothelial cells prolonged the duration till the onset of senescence [32]. Basal ROS levels are also required for proliferation and thus Nox4, by altering the basal activity of MAP kinases, has also been linked to this process in different types of vascular cells [33–35]. This colorful picture of a broad set of differential effects of Nox4 illustrates that a continuous production of ROS impacts on a large spectrum of cellular characteristics. Interestingly, recent reports indicate a possible agonistinduced activation of Nox4; this has been suggested for transforming growth factor b1, for agonists on toll like receptors [36,37] and Fcg-receptor ligands as well as several oxidized lipids [38]. The mechanisms underlying acute activation of Nox4 are largely unknown but may involve interaction with binding proteins, such as Poldip2 [39], translocation in membrane compartments such as lipid rafts [40] and potentially even an interaction with activating GTPases such as Rac1 [38].
Concluding remarks Under disease conditions, such as severe ischemia or diabetes, Nox2 however makes a rather deleterious contribution to the outcome of the models. The over-activation of the enzyme and a subsequent flooding of the Current Opinion in Pharmacology 2010, 10:122–126
In the vasculature all present homologues of the NADPH oxidase permit different functions and the absence of individual NADPH oxidases results in different phenotypes. Pharmacological interference with the function of www.sciencedirect.com
Isoform specific functions of Nox protein-derived reactive oxygen species in the vasculature Schro¨der 125
specific NADPH oxidases might be helpful in special situations: Nox1 inhibition could prevent the development of hypertension. An inhibition of Nox2 on the other hand may modulate angiogenesis. Nox4 might be a drug target in developing fibrosis but at the moment there is no evidence that blocking Nox4 will improve vascular function.
Acknowledgements This study was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB815/TP1), the excellence cluster cardiopulmonary system (ECCPS) and the Goethe-University.
Conflict of interest The author declares that she has no conflict of interest relating to this work.
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