This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Schiffrin, E. L. Articles by Touyz, R. M. Search for Related Content PubMed PubMed Citation Articles by Schiffrin, E. L. Articles by Touyz, R. M.

(Arteriosclerosis, Thrombosis, and Vascular Biology. 2003;23:707.)
© 2003 American Heart Association, Inc.

Editorial

Inflammation and Vascular Hypertrophy Induced by Angiotensin II

Role of NADPH Oxidase-Derived Reactive Oxygen Species Independently of Blood Pressure Elevation?

Ernesto L. Schiffrin; Rhian M. Touyz

From the Multidisciplinary Research Group on Hypertension, Clinical Research Institute of Montreal, University of Montreal, Canada.

Correspondence to E.L. Schiffrin MD, PhD, Clinical Research Institute of Montreal, 110 Pine Ave West, Montreal, H2W 1R7, Canada. E-mail Ernesto.Schiffrin{at}ircm.qc.ca

Evidence from the last few years has suggested that increased oxidative stress plays a pathophysiological role in cardiovascular disease, including atherosclerosis, hypertension, and heart failure.^1,2 At the same time, emerging data have implicated inflammation as a process involved in the initiation and progression of atherosclerosis, but it is also present in hypertension, diabetes mellitus, and other conditions associated with vascular damage.³ A large body of data has suggested that the renin-angiotensin system mediates part of its physiological and pathophysiological actions via generation of reactive oxygen species (ROS) and stimulation of inflammation.^3,4 In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology, Liu et al⁵ show that angiotensin II (Ang II)–enhanced NADPH oxidase activity contributes to adhesion molecule (ICAM) expression, leukocyte infiltration, and vascular growth in rats, independently of its effects on blood pressure. These important findings expand our knowledge on some of the pleiotropic actions of Ang II, and the mechanisms whereby Ang II-induced inflammation and growth may be mediated by ROS.

See page 776

ROS, which include superoxide anion and hydrogen peroxide among others, are signaling molecules that participate in the regulation of the tone and structure of blood vessels.^6,7 Superoxide anion in the vascular wall may be produced primarily in the endothelium and the adventitia as well as in vascular smooth muscle cells and fibroblasts. The sources of ROS, and specifically of superoxide anion, have been the subject of debate and numerous studies in the past few years, and major strides have been made in identifying the mechanisms of their generation. Several enzymes such as nitric oxide synthase, xanthine oxidase, and myeloperoxidase may generate free radicals, but in the vasculature it appears that the most important enzyme responsible for superoxide anion generation is NAD(P)H oxidase.¹ The production of superoxide anion occurs when oxygen is reduced by an electron with NAD(P)H functioning as the electron donor. NAD(P)H oxidase was originally found in neutrophils. The neutrophil/macrophage NAD(P)H oxidase is composed of five subunits: p40phox (phox for PHagocyte OXidase), p47phox, p67phox, p22phox, and gp91phox (Figure).⁸ p40phox, p47phox, and p67phox exist in the cytosol, whereas p22phox and gp91phox are located in the cell membrane as an heterodimeric flavoprotein, cytochrome b558. When cells are stimulated, p47phox is phosphorylated, and the cytoplasmic complex translocates to the membrane and associates with cytochrome b558 to generate the active oxidase.⁹ Activation requires the participation of two low–molecular weight guanine nucleotide-binding proteins, Rac 2 (or Rac 1) and Rap1A.

View larger version (20K): [in this window] [in a new window]   Schematic demonstrating possible cellular sources of Ang II-stimulated NAD(P)H oxidase-derived reactive oxygen species (ROS) in the vascular wall in the absence (A) and presence of gp91ds-tat (B). Ang II induces complex formation and translocation of cytosolic NAD(P)H oxidase subunits (p47phox, p67phox and p40phox), which associate with membrane-bound subunits (p22phox and gp91phox/nox) resulting in activation of NAD(P)H oxidase and generation of ·O2- from molecular O2 (A). Increased production of ROS from vascular cells or from infiltrating macrophages activate redox-sensitive signaling pathways leading to inflammation and cell growth, important factors contributing to vascular damage in hypertension. Inhibition of Ang II-induced NAD(P)H oxidase activation by gp91ds-tat, which blocks p47phox-gp91phox interaction, results in reduced formation of ·O2- and consequent decreased inflammation, cell growth and vascular injury (B). , increase effect; , decrease effect.

It has become increasingly evident that subunits of the leukocyte NAD(P)H oxidase system are also present in nonphagocytic cells, including cells of the vasculature.¹⁰ All subunits of the neutrophil NAD(P)H oxidase are found in endothelial cells.¹¹ Of critical importance in the generation of endothelial-derived superoxide are gp91phox and p47phox.^12,13 gp91phox, p22phox, p47phox, and p67phox have also been detected in adventitial fibroblasts.¹⁴ The situation in vascular smooth muscle cells appears to be more complicated, and it is still unclear which of the neutrophil NAD(P)H oxidase subunits are present and functionally active in these cells. In rat conduit arteries, mRNA for gp91phox is barely detectable in smooth muscle cells.^15,16 However, homologues of gp91phox, nox1, and nox4 (for Nonphagocytic NADPH OXidase),^15–17 as well as p22phox, p47phox, and rac1, are expressed in rat aortic smooth muscle cells (Figure).¹⁸ Although initial studies suggested that nox1 is a subunit-independent low-capacity superoxide-generating enzyme involved in the regulation of mitogenesis,^15,16 recent data indicate that nox1 requires p47phox and p67phox and that it is regulated by NoxO1 (Nox organizer 1) and NoxA1 (Nox activator 1).¹⁹ In vascular smooth muscle cells derived from human resistance arteries, we recently demonstrated that gp91phox and nox4 are present, whereas nox1 is undetectable.²⁰ Furthermore gp91phox plays a major role in superoxide production in these cells.²⁰ p22phox and p47phox have also been shown to be major subunits involved in agonist-mediated superoxide generation in vascular smooth muscle cells.^18,20 Most studies were performed in cells from large arteries from experimental animal models. There may be differences in the expression of nox homologues, as well as the other major NAD(P)H oxidase subunits in peripheral resistance arteries, as we recently demonstrated.

Mechanisms of vascular NAD(P)H oxidase stimulation by Ang II and the potential implication of NAD(P)H oxidase-derived ROS in signaling in vascular cells have received considerable attention. Ang II via AT₁ receptors may stimulate PKC, PLD, or Src^21,22 to activate NAD(P)H oxidase. In turn, superoxide anion generated by NAD(P)H oxidase will stimulate JNK and p38 MAP kinase, which will mediate the effects of increased oxygen free radicals.^23,24

In the study of Liu et al⁵ in this issue, the involvement of activation of NAD(P)H oxidase is shown to lead to growth and inflammation. In brief, an inhibitor of the nox subunit of NADPH oxidase (gp91ds-tat peptide) co-infused with Ang II into rats resulted in reduction in the increased production of superoxide (as measured by formation of nitrotyrosine), inflammation (infiltration of macrophages), upregulation of inflammatory mediators (ICAM-1), and growth of the wall of the aorta, in absence of blood pressure lowering. The authors conclude that stimulation by Ang II of endothelial and adventitial NADPH-derived ROS results in an inflammatory and remodeling vascular response in rats that is independent of blood pressure. The importance of this article lies in the clear-cut demonstration of a NAD(P)H oxidase-mediated effect on growth and inflammation in vivo by use of a specific selective inhibitor of the enzyme. In fact, the inhibitor used is quite ingenious. It is based in the use of short amino acid sequences involved in the docking of phophorylated p47phox to gp91phox (hence the name gp91docking sequence or gp91ds). These are then coupled to a 9–amino acid peptide from the HIV viral coat "tat" which is internalized by all cells, allowing the gp91ds-tat to reach and inhibit gp91phox with reasonable chance of being highly specific. A limitation of the study is that the selectivity of this inhibitor has not been definitively demonstrated to be absolute, and studies using inhibitors almost always leave such questions unanswered. Furthermore, it is unclear which of the specific nox homologues are targeted by the inhibitor. It would have been useful to use a control that includes a scrambled docking sequence (scrambled gp91ds) to ensure the specificity of the blockade by gp91ds, which unfortunately was not done in this study. However, use of the peptide inhibitor may be superior to others, such as apocynin, and certainly provides greater understanding of mechanism than the use of an antioxidant.

Liu et al⁵ attribute the ROS formation in response to Ang II to endothelium and adventitial production. However, the media smooth muscle cells which are known to possess functionally active nox-containing NAD(P)H oxidase and where significant staining for nitrotyrosine could be observed may also have participated in the response described. Furthermore, neutrophils and macrophages that infiltrated the adventitia may have also functioned as an important source of NAD(P)H-derived ROS that would be inhibited by gp91ds-tat. Thus, the source of ROS is not unambiguously demonstrated by the study, and the differences in degree of upregulation of ICAM cited by the authors are not definitive proof of the site of generation of ROS.

Together with previous data showing the proinflammatory effect of Ang II–induced ROS leading to NF-B and AP-1 upregulation,^3,25,26 and recent publications, which, by using DNA microarray technology, identified the genes stimulated in response to Ang II,²⁷ demonstrating upregulation of inflammatory mediators such as osteopontin, PAI-1, MCP-1, and tissue factor, the present study significantly contributes to our knowledge of the pleiotropic actions of Ang II that participate in the pathophysiology of cardiovascular disease.

References

1. Griendling KK, Sorescu D, Ushio-Fukai M. NADPH oxidase: role in cardiovascular biology and disease. Circ Res. 2000; 86: 494–501.[Abstract/Free Full Text]
2. Wilcox CS. Reactive oxygen species: roles in blood pressure and kidney function. Curr Hypert Rep. 2002; 4: 160–166.
3. Brasier AR, Recinos A, Eledrisi MS. Vascular inflammation and the renin-angiotensin system. Arterioscler Thromb Vasc Biol. 2002; 22: 1257–1266.[Abstract/Free Full Text]
4. Touyz RM, Schiffrin EL. Signal transduction mechanisms mediating the physiological and pathophysiological actions of angiotensin II in vascular smooth muscle cells. Pharmacol Rev. 2000; 52: 639–672.[Abstract/Free Full Text]
5. Liu J, Yang F, Yang X-P, Jankowski M, Pagano PJ. NAD(P0H oxidase mediates angiotensin II-induced vascular macrophage infiltration and medial hypertrophy. Arterioscler Thromb Vasc Biol. 2003; 23: 776–782.[Abstract/Free Full Text]
6. Droge W. Free radicals in the physiological control of cell function. Physiol Rev. 2001; 82: 47–95.
7. Sauer H, Wartenberg M, Heschler J. Reactive oxygen species as intracellular messengers during cell growth and differentiation. Cell Phys Biochem. 2001; 11: 173–186.[CrossRef][Medline] [Order article via Infotrieve]
8. Babior BM, Lambeth JD, Nauseef W. The neutrophil NADPH oxidase. Arch Biochem Biophys. 2002; 397: 342–344.[CrossRef][Medline] [Order article via Infotrieve]
9. Fontayne A, Dang PM, Gougerot-Pocidalo MA, El-Benna J. Phosphorylation of p47phox sites by PKC alpha, beta II, delta and zeta: effect on binding to p22phox and on NADPH oxidase activation. Biochemistry. 2002; 41: 7743–7750.[CrossRef][Medline] [Order article via Infotrieve]
10. Griendling KK, Minieri CA, Ollerenshaw JD, Alexander RW. Angiotensin II stimulates NADH and NADPH oxidase activity in cultured vascular smooth muscle cells. Circ Res. 1994; 74: 1141–1148.[Abstract/Free Full Text]
11. Meyer JW, Holland JA, Ziegler LM, Chang MM, Beebe G, Schmitt ME. Identification of a functional leukocyte-type NADPH oxidase in human endothelial cells: a potential atherogenic source of reactive oxygen species. Endothelium. 1999; 7: 11–22.[Medline] [Order article via Infotrieve]
12. Li JM, Mullen AM, Yun S, Wientjes F, Brouns GY, Thrasher AJ, Shah AM. Essential role of the NADPH oxidase subunit p47(phox) in endothelial cell superoxide production in response to phorbol ester and tumour necrosis factor-alpha. Circ Res. 2002; 90: 143–150.[Abstract/Free Full Text]
13. Ushio-Fukai M, Tang Y, Fukai T, Dikalov SI, Ma Y, Fujimoto M, Quinn MT, Pagano PJ, Johnson C, Alexander RW. Novel role of gp91(phox)-containing NAD(P)H oxidase in vascular endothelial growth factor-induced signaling and angiogenesis. Circ Res. 2002; 91: 1160–1167.[Abstract/Free Full Text]
14. Rey FE, Pagano PJ. The reactive adventitia: fibroblast oxidase in vascular function. Arterioscler Thromb Vasc Biol. 2002; 22: 1962–1971.[Abstract/Free Full Text]
15. Suh Y, Arnold RS, Lassegue B, Shi J, Xu X, Sorescu D, Chung AB, Griendling KK, Lambeth JD. Cell transformation by the superoxide-generating oxidase mox 1. Nature. 1999; 401: 79–82.[CrossRef][Medline] [Order article via Infotrieve]
16. Lasssègue B, Sorescu D, Szocs K, Yin Q, Akers M, Zhang Y, Grant SL, Lamberth JD, Griendling KK. Novel gp91phox homologues in vascular smooth muscle cells: nox 1 mediates angiotensin II-induced superoxide formation and redox-sensitive signaling pathways. Circ Res. 2001; 88: 888–894.[Abstract/Free Full Text]
17. Lambeth JD. Nox/Duox family of nicotinamide adenine dinucleotide (phosphate) oxidases. Curr Opin Hematol. 2002; 9: 11–17.[CrossRef][Medline] [Order article via Infotrieve]
18. Mollnau H, Wendt M, Szocs K, Lassegue B, Schulz E, Oelze M, Li H, Bodenschatz M, August M, Kleschyov AL, Tsilimingas N, Walter U, Forstermann U, Meinertz T, Griendling K, Munzel T. Effects of angiotensin II infusion on the expression and function of NAD(P)H oxidase and components of nitric oxide/cGMP signaling. Circ Res. 2002; 90: E58–E65.
19. Banfi B, Clark RA, Steger K, Krause K-H. Two novel proteins activate superoxide generation by the NADPH oxidase Nox1. J Biol Chem. 2003; 278: 3510–3513.[Abstract/Free Full Text]
20. Touyz RM, Chen X, He G, Quinn MT, Schiffrin EL. Expression of a gp91phox-containing leukocyte-type NADPH oxidase in human vascular smooth muscle cells–modulation by Ang II. Circ Res. 2002; 90: 1205–1213.[Abstract/Free Full Text]
21. Touyz RM, Schiffrin EL, Increased generation of superoxide by angiotensin II is mediated via PLD-dependent, NADPH oxidase-sensitive pathways in vascular smooth muscle cells from hypertensive patients. J Hyperten. 2001; 19: 1245–1254.[CrossRef][Medline] [Order article via Infotrieve]
22. Seshiah PN, Weber DS, Rocic P, Valppu L, Taniyama Y, Griendling KK. Angiotensin II stimulation of NAD(P)H oxidase activity: upstream mediators. Circ Res. 2002; 91: 406–413.[Abstract/Free Full Text]
23. Touyz RM, Cruzado M, Tabet F, Yao G, Salomon S, Schiffrin EL. 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: 159–167.[CrossRef][Medline] [Order article via Infotrieve]
24. Viedt C, Soto U, Krieger-Brauer HI, Fei J, Elsing C, Kübler W, Kreuzer J. Differential activation of MAP kinases in smooth muscle cells by Ang II: involvement of p22phox and reactive oxygen species. Arterioscler Thromb Vasc Biol. 2000; 20: 940–948.[Abstract/Free Full Text]
25. Ruiz-Ortega M, Ruperez M, Esteban V, Egido J. Molecular mechanisms of Ang II-induced vascular injury. Curr Hyperten Rep. 2003; 5: 73–79.
26. 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.[Abstract/Free Full Text]
27. Campos AH, Zhao Y, Pollman MJ, Gibbons GH. DNA microarray profiling to identify angiotensin-responsive genes in vascular smooth muscle cells: potential mediators of vascular disease. Circ Res. 2003; 92: 111–118.[Abstract/Free Full Text]

This article has been cited by other articles:

				S. M. Zemse, R. H. P. Hilgers, and R. C. Webb Interleukin-10 counteracts impaired endothelium-dependent relaxation induced by ANG II in murine aortic rings Am J Physiol Heart Circ Physiol, June 1, 2007; 292(6): H3103 - H3108. [Abstract] [Full Text] [PDF]

				E. A. Ko, F. Amiri, N. R. Pandey, D. Javeshghani, E. Leibovitz, R. M. Touyz, and E. L. Schiffrin Resistance artery remodeling in deoxycorticosterone acetate-salt hypertension is dependent on vascular inflammation: evidence from m-CSF-deficient mice Am J Physiol Heart Circ Physiol, April 1, 2007; 292(4): H1789 - H1795. [Abstract] [Full Text] [PDF]

				C. De Ciuceis, F. Amiri, P. Brassard, D. H. Endemann, R. M. Touyz, and E. L. Schiffrin Reduced Vascular Remodeling, Endothelial Dysfunction, and Oxidative Stress in Resistance Arteries of Angiotensin II-Infused Macrophage Colony-Stimulating Factor-Deficient Mice: Evidence for a Role in Inflammation in Angiotensin-Induced Vascular Injury Arterioscler. Thromb. Vasc. Biol., October 1, 2005; 25(10): 2106 - 2113. [Abstract] [Full Text] [PDF]

				P. Pignatelli, V. Sanguigni, L. Lenti, D. Ferro, A. Finocchi, P. Rossi, and F. Violi gp91phox-Dependent Expression of Platelet CD40 Ligand Circulation, September 7, 2004; 110(10): 1326 - 1329. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Schiffrin, E. L. Articles by Touyz, R. M. Search for Related Content PubMed PubMed Citation Articles by Schiffrin, E. L. Articles by Touyz, R. M.