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Artículo destacado 
Drug News & Perspectives LOOKING AHEAD Inhibitors of vascular NAD(P)H oxidase, identified as the major source of vascular superoxide, may provide a novel therapy in cardiovascular disease. Vascular NAD(P)H Oxidase as a Novel Therapeutic Target in Vascular Disease by Julia Brosnan Summary Oxidative stress, involving elevated levels of reactive oxygen species such as superoxide and peroxynitrite, has been implicated in the pathogenesis of several, if not most, forms of cardiovascular disease. Recent studies using viral-mediated gene transfer of genes that redress oxidative stress in animal models of cardiovascular disease have suggested that targeting sources of superoxide would provide a novel therapeutic strategy in cardiovascular disease. Identification of a vascular form of the NAD(P)H oxidase as the major source of superoxide has resulted in a search for effective inhibitors. This review summarizes the developments in the area of vascular NAD(P)H oxidase as a novel therapeutic target in vascular disease. © 2004 Prous Science. All rights reserved. Oxidative stress has been implicated in the pathogenesis of many cardiovascular diseases including hypertension, atherosclerosis, hyperlipidemia, diabetes, arteriosclerosis, unstable angina, vasculitis, myocarditis and restenosis.1,2 Genetic polymorphisms that may predispose to oxidative stress are already being uncovered and have been implicated in some of these disease states.3,4 In addition, recognition of the in-creased atherogenicity of oxidized low-density lipoprotein (LDL) over LDL has contributed to the recognition of the importance of oxidative stress as a therapeutic target in cardiovascular medicine.5 What is oxidative stress? Oxidative stress encompasses a reduction in removal and/or an increase in production of a variety of reactive oxygen species (ROS). ROS are reactive chemical entities classified into either free radicals (species with one or more unpaired electrons) or nonradical derivatives. The free radicals include the superoxide anion (SO), hydroxyl radical (OH*), nitric oxide (NO), peroxynitrite (ONOO-) and lipid peroxide (ROO*). The nonradical derivatives include hydrogen peroxide (H2O2) and singlet oxygen (1O2). ROS promote lipid oxidation, stimulate smooth muscle cell growth and initiate expression of proinflammatory genes. ROS activate matrix metalloproteinases, which may lead to plaque instability and rupture. A particularly important consequence of oxidant stress is the loss of endothelium-derived NO, which reacts with SO and other radicals at near diffusion-limited rates. Many of the important vasoprotective properties of NO, such as inhibition of cell growth, platelet adherence and aggregation, and leukocyte adhesion are compromised by oxidative inactivation of SO into the deleterious ONOO-. The reaction between ROS and components critical to vascular homeostasis such as LDL and NO underlies the growing importance attributed to oxidative stress (Fig. 1).
Fig. 1. The generation and role of superoxide in cardiovascular disease. In recent years the tools of molecular biology, in particular the ability to generate transgenic and knockout animal models, have provided us with a powerful means of assessing the contributions of biological pathways to pathological events. Several of the genes encoding subunits of the NAD(P)H oxidase have been knocked out in mice and the vascular effects determined.6-8 Recently, using genetically modified mice that lack the p47phox cytoplasmic subunit, it has been demonstrated that NAD(P)H oxidases are a major source of O2*- and contribute to hypertension caused by angiotensin II.9 Barry-Lane et al.6 showed that p47phox is required for atherosclerotic lesion progression in ApoE mice. In addition, gene transfer technology has confirmed the beneficial effects of antioxidative stress genes in animal models of hypertension, diabetes and atherosclerosis.10-13 The evolving importance of the deleterious effects of ROS in the vasculature has meant the inevitable quest for therapeutic agents designed to re-dress the oxidative balance. This has traditionally involved agents that eliminate ROS, but, increasingly, the field is leaning to preemptively block SO production. Vascular NAD(P)H oxidase Griendling and others, have de-fined a vascular form of the phagocytic NAD(P)H oxidase as the major source of SO in blood vessels.14,15 This enzyme differs from that found in the phagocyte NAD(P)H oxidase in that it is constitutively active, although at a much lower level. However, similar to the phagocytic form, the vascular NAD(P)H oxidase is a multimeric complex consisting of membrane-bound and cytoplasmic subunits. The membrane-bound component b558 is made up of gp91phox (or one of a number of homologues)16-19 and p22phox. The cytoplasmic subunits are p47phox, p40phox, p67phox and a small rac1 or rac2 and Rap1A. Stimulation, for example, by angiotensin II acting via AT1 receptors (Fig. 2) may stimulate protein kinase C, phospholipase D or Src to activate NAD(P)H oxidase.20 The SO thus generated stimulates JNK and p38 mitogen-activated protein kinase (MAPK) and mediates the effects of increased ROS.21,22 ROS produced by the oxidase regulate the expression of several proinflammatory mediators including monocyte chemoattractant 1, vascular cell adhesion molecule and interleukin 6. Activation of nuclear factor-κB, extracellular signal-regulated kinase 1,2 (ERK1,2), protein tyrosine kinases and the Janus-activated kinase 2-STAT (signal transducers and activators of transcription) pathway contributes to these signaling events. Activation of p38 MAPK, phosphatidylinositol 3- kinase, Akt and ERK1,2 by angiotensin II might contribute to hypertrophy. In the endothelium, H2O2 can stimulate NO* production by endothelial nitric oxide synthase (eNOS) via ERK1,2 and Akt. H2O2 also stimulates angiogenesis by increasing the expression of hypoxia-induced factor 1, which leads to an increase in the expression of vascular endothelial growth factor and subsequent growth of vascular cells and angiogenesis. H2O2-dependent activation of the vascular endothelial growth factor receptor 2 (Flk1/KDR) also contributes to angiogenesis. These mechanisms may at least partially account for the cardiovascular diseases induced by angiotensin II. The plethora of important effects mediated by this activation underlies the efforts to look for effective and specific inhibitors.
Fig. 2. Vascular NAD(P)H oxidase and currently available inhibitors. Antioxidant vitamins Traditionally, the antioxidative vitamins have been viewed as the universal panacea for reducing ROS. However, the initial enthusiasm for the cardiovascular protective effects has been dampened by the largely disappointing results, with several recent trials showing no effect of vitamin E on cardiovascular events. Vitamin E had no benefit over placebo in 9,500 high-risk subjects in the Heart Out-comes Prevention Evaluation (HOPE) trial. In the GISSI prevention trial, survivors of myocardial infarction were randomized to receive vitamin E, n-3 polyunsaturated fatty acids, both or neither. Although the polyunsaturated fatty acid therapy proved beneficial, the effects of vitamins were insignificant. In the Study to Evaluate Carotid Ultrasound changes in patients treated with Ramipril and vitamin E (a substudy of HOPE), vitamin E was found to have no effect on the progression of carotid intima-media thickness (CIMT), whereas ramipril markedly reduced progression. It seems likely that the biochemistry of these vitamins may yield scientific explanations for the disappointing effects. For example, in the case of vitamin E, the rate constant for reactions between vitamin E and SO is 5 x 103 M-1 x sec-1, which is six orders of magnitude less than the rate constant for the reactions of SO with NO. Given the modest increases in plasma and tissue levels of vitamin E during oral therapy, it is possible that oral treatment with vitamin E has no effect on many important biological processes. Second, many of the oxidative events important in atherosclerosis occur in the cytoplasm and in the extracellular space and would not be modulated by lipid-soluble antioxidants. Third, up-on scavenging a radical, vitamin E becomes the tocopheroxyl radical, which can enhance lipid peroxidation. There has been substantial interest in using cocktails of antioxidants: for example, vitamins E and C together. Indeed, in the recent Antioxidant Supplementation in Atherosclerosis Prevention trial, the combination of vitamins E and C produced a substantial reduction in CIMT progression, although when used alone, these vitamins were ineffective. It seems probable that use of antioxidant vitamins will never prove to be the best approach to limit vascular oxidant stress, and this further underpins the need for the development of other classes of therapeutic agents. Clinical agents The inhibitors of NAD(P)H oxidase fall into two groups, pharmacological or genetic agents. Of the pharmacological agents, two widely accep- ted modes of treatment for atherosclerosis, the statins and angiotensin-converting enzyme (ACE) inhibitors, have been shown to potently inhibit production of ROS by vascular cells. Statins represent a well-established class of drugs that effectively lower serum cholesterol levels and are widely prescribed for the treatment of hypercholesterolemia by competitively inhibiting 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting step of cholesterol synthesis. Statins thereby reduce cholesterol levels and may also lower levels of isoprenoids such as farnesyl pyrophosphate and geranylgeranylpyrophosphate. Isoprenoids are needed for the prenylation of a variety of proteins, thus anchoring them to the cell surface. Clinical trials demonstrating that statins reduce cardiovascular-related morbidity and mortality in patients with and without elevated cholesterol suggest that statins may have effects not directly related to their cholesterol lowering activity. Statins may have direct antiinflammatory activities, al-though it is difficult to differentiate between the beneficial effects of lipid-lowering and direct antiinflammatory effects (Fig. 3).
Fig. 3. Effects of HMG-CoA reductase inhibitors on rac proteins. Biosynthetic pathway of isoprenoids leading to prenylated proteins. Statins are specific inhibitors of HMG-CoA reductase (the rate-limiting enzyme of cholesterol biosynthesis). The chemical structures of the prenyl groups are shown, and the positions of the protein-prenylating enzymes PFT and PGGT-1 are indicated. After prenylation, the proteins are further modified and will finally move to the cellular membranes, where they exert their signaling function. Inhibition of prenylation results in a reduced activity of the vascular NAD(P)H oxidase. In addition, the NADPH oxidase is potently activated by angiotensin II,23 and strategies to block angiotensin II production or its receptor dramatically lower SO production in both experimental animal models and human vessels.24 Thus statins, ACE inhibitors and angiotensin-receptor antagonists have antioxidant effects not because they scavenge radicals but because they block the production of ROS. Pharmacological inhibitors The currently available pharmacological inhibitors of the NAD(P)H oxidase act in a variety of ways. Berry and co-workers showed that in human internal mammary artery the major source of SO could be inhibited by diphenyleneiodonium (DPI).14 DPI, a widely used inhibitor of enzymes containing flavin oxidases, has received much attention but lacks specificity. On the other hand, neither allopurinol, an inhibitor of xanthine oxidase, nor an inhibitor of eNOS (N-nitro-l-arginine methyl ester) had significant effects in human vessels. However, the broad spectrum of targets for DPI leaves questions. It is relatively nonspecific and inhibits NOS as well as complex I of the mitochondrial respiratory chain; consequently, DPI's effects on levels of oxidative stress are difficult to interpret. Apocynin, the ortho-methoxy-substituted catechol, (4-hydroxy-3-meth-oxyacetophenone) isolated from the traditional medicinal plant Picrorrhiza kurroa, acts as an alternative inhibitor. Apocynin was originally used as an antiinflammatory agent by the Peruvian Indians and interferes with the association between gp91phox and p47phox. Consequently, it is more specific than DPI. Hamilton et al. investigated the effects of apocynin on hu-man blood vessels25 (Fig. 4). In human internal mammary arteries and saphenous veins, apocynin decreased NAD(P)H-stimulated O2- generation and caused vasorelaxation that was endothelium-dependent and reversed with the addition of the NO synthase inhibitor N(G)-nitro-l-arginine methyl ester.25 In addition, it increased NO production from cultured human endothelial saphenous vein cells. Both apocynin and DPI were shown to eliminate the increased production of SO in the aortic tissues in chronically hyperinsulinemic rats.26 There has been some suggestion that the activation event required for apocynin does not occurin vascular tissues. However, more recently, parallel activation systems have been proposed. Indeed, the efficacy in the experimental treatment of several inflammatory diseases such as arthritis, colitis and atherosclerosis has led to the suggestion that apocynin could be a prototype of a novel series of non-steroidal antiinflammatory drugs. So far, apocynin has mainly been used in vitro to block the generation of ROS by the neutrophil NADPH oxidase. Van den Worm showed that an additional methoxy group at position C-5 displayed enhanced antiinflammatory ac-tivity in vitro in neutrophils.27 This approach may also lead to the generation of derivatives of apocynin with a higher efficacy in the vascular system.
Fig. 4. Effects of apocynin on nitric oxide (NO) bioavailability in endothelial cells. Apocynin blocks the interaction between subunits of vascular NAD(P)H oxidase, thereby limiting the production of superoxide. This protects NO generated by endothelial cells from conversion into peroxynitrite and allows a higher steady state to be available for biological reactions. *p < 0.05. A newer drug, 6,8-diallyl 5,7-dihydroxy 2-(2-allyl 3-hydroxy 4 meth-oxyphenyl)1-H benzo(b)pyrano-4-one (S-17834) that had been previously shown to inhibit ischemia/reperfusion-induced leakage of macromolecules and leukocyte adhesion in postcapillary venules was further investigated by Cayatte et al.28 They investigated the mechanisms by which S-17834 inhibited the stimulation by tumor necrosis factor (TNF) of mRNA and protein expression of vascular intercellular adhesion molecule in human endothelial cells as well as leukocyte adherence mechanisms and found that S-17834 decreased endogenous superoxide production in TNF-stimulated cells. Furthermore, in ApoE knockout mice, treatment with S-17834 reduced SO production by the aorta and the incidence of atherosclerotic lesions. Synthetic peptides An alternative and more specific approach to inhibiting vascular NAD(P)H oxidase, using synthetic peptides, has been applied by the group of Pagano.29,30 Chimeric peptide (gp91ds-tat) consisting of a nine-ami-no acid tat site, derived from the tat peptide of the HIV virus, which directs uptake into the cell, fused to a nine-amino acid fragment of gp91phox, preventing the interaction of p47phox with the gp91phox subunits. gp91ds-tat appears to be the most specific NADPH oxidase inhibitor currently available. As this p47phox-blocking peptide sequence is specific for the NADPH oxidases, it is likely that gp91ds-tat acts specifically on the oxidase, which makes it a unique tool for studying the involvement of the NADPH oxidase, particularly in in vivo models. This strategy was successfully used to lower blood pressure in mice with angiotensin II-in-duced hypertension. More recently,29 it was shown that inhibition of vascular NADPH oxidases using synthetic peptides suppressed angioplasty-induced neointimal hyperplasia in the carotid artery of rats. The substantial reduc-tion in total nitrotyrosine staining supports the ability of the peptide to suppress oxidase activity in the vascular wall. Using gp91ds-tat applied over an extended observation period makes this study one of the few clearly demonstrating involvement of NADPH oxidases in a specific disease state. Moreover, this peptide inhibitor not only prevented neointima formation and inhibited stretch-induced superoxide anion release from distended vessels, as well as peroxynitrite formation after angioplasty, it forged a clear link between neointima formation and NADPH oxidase-dependent radical formation. While this type of chimeric peptide provides a valuable tool in the analysis of oxidative stress in vascular disease, there are limitations. Neutrophils and macrophages containing NADPH oxidases may also be inhibited by gp91ds-tat. Moreover, sensitization to the peptide will most probably lead to the formation of antibodies, limiting the treatment duration to a couple of weeks. Finally, the tat sequence of the peptide may cause side effects affecting cellular activity and signaling, leaving absolute specificity of effects in some doubt. Conclusion Genetic polymorphisms that may predispose to oxidative stress and lead to the development of cardiovascular disease are already being uncovered. Polymorphisms of superoxide dismutase,31 endothelial nitric oxide synthase,32,33 the promoter region of catalase,3 heme oxygenase and glutathione peroxidase have already been identified and may contribute alone or, much more likely, in combination to "high oxidative stress." It seems likely that in the future we will be able to measure "oxidative stress" reliably and reproducibly as a phenotype in its own right and then be able to target treatment at a very early stage in the clinic. Perhaps the already currently available drugs such as statins, and ACE and angiotensin II antagonists will provide part of the defense against the generation of oxidative stress. However, it seems more likely that in the future, as our knowledge of the part played by prooxidative molecules such as the vascular NAD(P)H oxidase further expands, we should be able to exploit some of the newer and more specific antioxidants to prevent the development of the earliest stages of cardiovascular disease. References 1. Cai, H. and Harrison, D.G. Endothelial dysfunction in cardiovascular diseases--The role of oxidant stress. Circ Res 2000, 87: 840-4. 2. Berry, C., Brosnan, M.J., Fennell, J., Hamilton, C.A. and Dominiczak, A.F. Oxidative stress and vascular damage in hypertension. Curr Opin Nephrol Hypertens 2001, 10: 247-55. 3. Jiang, Z.W., Akey, J.M., Shi, J.X. et al. A polymorphism in the promoter region of catalase is associated with blood pressure levels. Hum Genet 2001, 109: 95-8. 4. Zafari, A.M., Davidoff, M.N., Austin, H. et al. The A640G and C242T p22(phox) polymorphisms in patients with coronary artery disease. Antioxid Redox Signal 2002, 4: 675-80. 5. Witztum, J.L. and Steinberg, D. The oxidative modification hypothesis of atheroscle-rosis: Does it hold for humans? Trends Cardiovasc Medcine 2001, 11: 93-102. 6. Barry-Lane, P.A., Patterson, C., Van der Merwe, M. et al. p47phox is required for atherosclerotic lesion progression in ApoE mice. J Clin Invest 2001, 108: 1513-22. 7. Hsich, E., Segal, B.H., Pagano, P.J. et al. Vascular effects following homozygous disruption of p47(phox)--An essential component of NADPH oxidase. Circulation 2000, 101: 1234-6. 8. Lavigne, M.C., Malech, H.L., Holland, S.M. and Leto, T.L. Genetic demonstration of p47phox-dependent superoxide anion production in murine vascular smooth muscle cells. Circulation 2001, 104: 79-84. 9. Landmesser, U., Cai, H., Dikalov, S. et al. Role of p47 in vascular oxidative stress and hypertension caused by angiotensin II. Hypertension 2002, 40: 511-5. 10. Alexander, M.Y., Brosnan, M.J., Hamilton, C.A. et al. Gene transfer of endothelial nitric oxide synthase but not Cu/Zn superoxide dismutase restores nitric oxide availability in the SHRSP. Cardiovasc Res 2000, 47: 609-17. 11. Alexander, M.Y., Brosnan, M.J., Hamilton, C.A. et al. Gene transfer of endothelial nitric oxide synthase improves nitric oxide-dependent endothelial function in a hypertensive rat model. Cardiovas Res 1999, 43: 798-807. 12. Fennell, J.P., Brosnan, M.J., Frater, A.J. et al. Adenovirus-mediated overexpression of ex-tracellular superoxide dismutase improves endothelial dysfunction in a rat model of hypertension. Gene Ther 2002, 9: 110-7. 13. Lund, D.D., Faraci, F.M., Miller, F.J. and Heistad, D.D. Gene transfer of endothelial nitric oxide synthase improves relaxation of carotid arteries from diabetic rabbits. Circulation 2000, 101: 1027-33. 14. Berry, C., Hamilton, C.A., Brosnan, J. et al. Investigation into the sources of superoxide in human blood vessels--Angiotensin II increases superoxide production in human internal mammary arteries. Circulation 2000, 101: 2206-12. 15. Griendling, K.K., Sorescu, D. and Ushio-Fukai, M. NAD(P)H oxidase--Role in cardiovascular biology and disease. Circ Res 2000, 86: 494-501. 16. Lassegue, B., Sorescu, D., Szocs, K. et al. 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: 888-94. 17. Lassegue, B., Sorescu, D., Chung, A.B. et al. P65-mox, a novel gp91-phox analogue, participates in superoxide generation and cellular proliferation in vascular smooth muscle. Circulation 1999, 100: 224. 18. Sorescu, D., Weiss, D., Lassegue, B. et al. Superoxide production and expression of Nox family proteins in human atherosclerosis. Circulation 2002, 105: 1429-35. 19. Cai, H., Griendling, K.K. and Harrison, D.G. The vascular NAD(P)H oxidases as therapeutic targets in cardiovascular diseases. Trends Pharmacol Sci 2003, 24: 471-8. 20. Touyz, R.M., He, G., Wu, X.H., Park, J.B., El Mabrouk, M. and Schiffrin, E.L. Src is an important mediator of extracellular signal-regulated kinase 1/2-dependent growth signaling by angiotensin II in smooth muscle cells from resistance arteries of hypertensive patients. Hypertension 2001, 38: 56-64. 21. Viedt, C., Soto, U., Krieger-Brauer, H.I. et al. Differential activation of mitogen-activat- ed protein kinases in smooth muscle cells by angiotensin II--Involvement of p22phox and reactive oxygen species. Arterioscler Thromb Vasc Biol 2000, 20: 940-8. 22. Touyz, R.M., Cruzado, M., Tabet, F., Yao, G.Y., Salomon, S. and Schiffrin, E.L. Redox-dependent MAP kinase signaling by Ang II in vascular smooth muscle cells: Role of receptor tyrosine kinase transactivation. Can J Physiol Pharmacol 2003, 81: 159-67. 23. Griendling, K.K., Minieri, C.A., Olleren-shaw, J.D. and Alexander, R.W. Angiotensin-II stimulates NADH and NADPH oxidase activity in cultured vascular smooth-muscle cells. Circ Res 1994, 74: 1141-8. 24. Touyz, R.M., Yao, G.Y. and Schiffrin, E.L. Upregulation of Src by Ang II induces oxidative stress in vascular smooth muscle cells from hypertensive patients. Hypertension 2002, 40: 21. 25. Hamilton, C.A., Brosnan, M.J., Al Benna, S., Berg, G. and Dominiczak, A.F. NAD(P)H oxidase inhibition improves endothelial function in rat and human blood vessels. Hypertension 2002, 40: 755-62. 26. Kashiwagi, A., Shinozaki, K., Nishio, Y. et al. Endothelium-specific activation of NAD(P)H oxidase in aortas of exogenous- ly hyperinsulinemic rats. Am J Physiol Endocrinol Metab 1999, 277: E976-83. 27. Van den Worm, E., Beukelman, C.J., Van den Berg, A.J.J., Kroes, B.H., Labadie, R.P. and Van Dijk, H. Effects of methoxylation of apocynin and analogs on the inhibition of reactive oxygen species production by stimulated human neutrophils. Eur J Pharmacol 2001, 433: 225-30. 28. Cayatte, A.J., Rupin, A., Oliver-Krasinski, J. et al. S17834, a new inhibitor of cell adhesion and atherosclerosis that targets NADPH oxidase. Arterioscler Thromb Vasc Biol 2001, 21: 1577-84. 29. Jacobson, G.M., Dourron, H.M., Liu, J.H. et al. Novel NAD(P)H oxidase inhibitor suppresses angioplasty-induced superoxide and neointimal hyperplasia of rat carotid artery. Circ Res 2003, 92: 637-43. 30. Rey, F.E., Cifuentes, M.E., Kiarash, A., Quinn, M.T. and Pagano, P.J. Novel competitive inhibitor of NAD(P)H oxidase assembly attenuates vascular O-2(-) and systolic blood pressure in mice. Circ Res 2001, 89: 408-14. 31. Marklund, S.L., Nilsson, P., Israelsson, K., Schampi, I., Peltonen, M. and Asplund, K. Two variants of extracellular-superoxide dismutase: Relationship to cardiovascular risk factors in an unselected middle-aged population. J Intern Med 1997, 242: 5-14. 32. Hingorani, A.D., Liang, C.F., Fatibene, J. et al. A common variant of the endothelial nitric oxide synthase (Glu(298)→Asp) is a major risk factor for coronary artery disease in the UK. Circulation 1999, 100: 1515-20. 33. Lembo, G., De Luca, N., Battagli, C. et al. A common variant of endothelial nitric oxide synthase (Glu298Asp) is an independent risk factor for carotid atherosclerosis. Stroke 2001, 32: 735-40. Julia Brosnan is Senior Lecturer in the Division of Cardiovascular and Med-ical Sciences, University of Glasgow, Glasgow, G11 6NT Scotland, U.K. E-mail: jbrosnan@ordwayresearch.org Drug News & Perspectives
Vol. 17, No. 7, 2004, pp. 429-434
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