Targeting NAD(P)H Oxidase

Reactive Oxygen Species (ROS) have been shown to modulate vascular signaling in endothelium, smooth muscle, and adventitia, regulate vascular hypertrophy, inflammation, remodeling, intracellular calcium, and disturb nitric oxide bioactivity.1,2 Since Griendling et al discovered the activation of NAD(P)H oxidase by angiotensin II (Ang II),3 researches have focused on the regulation of this enzyme in Ang II signaling/Ang II-induced hypertension both in vitro and in vivo. Vascular NAD(P)H oxidase consists of multiple subunits including p22, p40, p47, p67, Rac1, and unique catalytic subunits, Nox isoforms (gp91 homologue).4 The signaling mechanisms for the rapid activation of NAD(P)H oxidase by Ang II have been identified using cultured aortic vascular smooth muscle cells (VSMCs). Ang II rapidly activates PLC to increase intracellular calcium and diacylglycerol levels, which causes the activation of protein kinase C (PKC). PKC phosphorylates p47 and releases ROS from Nox subunits. Subsequently, ROS activates cSrc, EGF-receptor, PI3-kinase, and Rac1, leading to the secondary activation of NAD(P)H oxidase to augment the intracellular ROS levels.4 These events occur within 30 minutes in cultured VSMCs. Considering the ROS generation associated with hypertension in vivo, the latter transcriptional upregulation of NAD(P)H oxidase subunits by Ang II might be more important. Ang II upregulates the expressions of NAD(P)H oxidase subunits after more than 4 hours including p22, Nox2 (gp91), p47, and p67,5 however the mechanisms of transcriptional regulations for NAD(P)H oxidase subunits have not fully been elucidated yet. In this issue of Circulation Research, Ni and colleagues identified that Ets-1 was a critical transcriptional regulator of p47 induced by Ang II in vitro and in vivo.6 Ets-1 has been known as a proto-oncogene transcription factor to induce matrix-degradation proteins such as collagenase, plasminogen activation inhibitor-1(PAI-1), and matrixmetalloproteinases. Ets-1 can be induced by TNF, endothelin-1, prostanoid(s), and platelet-derived growth factor,7–8 suggesting an implication in vascular inflammation. Indeed, this group previously reported that Ets-1 was a critical factor needed to induce cyclin-dependent kinase, PAI-I, vascular cells adhesion molecule-1, and monocyte chemoattractant protein-1 in response to Ang II. In Ets-1 / mice, the vascular inflammation by Ang II infusion, which was represented by the recruitment of T cell and macrophage to vessel wall, was blunted, although hypertensive response was preserved.9 In this article, the authors carefully seek the molecular mechanisms to regulate the expression of NAD(P)H oxidase subunits by Ets-1. The augmentation of superoxide and hydrogen peroxide (H2O2) generations by Ang II were markedly attenuated in aorta from Ets-1 / mice or VSMCs with siRNA for Ets-1. siRNA for Ets-1 also blunted the upregulation of p47 without affecting the expression of Nox1, Nox4, Rac1, p22, and p67 by Ang II. They used gel-shift assay, luciferase reporter assay, and chromatin immunoprecipitation assay with deletion mutant of p47 promoter, and identified the 45 Ets-1–binding promoter region as essential for the induction of p47. They developed peptides to inhibit ETS-1 bindings (DN-Ets-1 peptides) and delivered them to the Ang II–infused mice in vivo. DN-Ets-1 peptides attenuated medial hypertrophy and aortic ROS generation without affecting hypertensive response to Ang II. This article impacts the field of hypertension research in 3 major ways. First, the authors demonstrate the importance of p47 induction for aortic ROS generation in Ang II–induced hypertension. p47 is phosphorylated at S359/S370/S379 by PKC, which causes association with p22. S303/S304 of p47 were also phosphorylated to augment the catalytic activity of NAD(P)H oxidase.4 We expressed S303A/S304A mutant p47 in VSMCs to suppress the redox-sensitive signal by Ang II.10 The posttranslational modifications of p47 by Ang II are critical for the rapid activation of NAD(P)H oxidase. The importance of p47 expression in Ang II–induced hypertension was also shown by Landmesser and colleagues.11 An increase in superoxide generation of aorta by Ang II was blunted in p47 / mice but rose 3-fold in control. Hypertensive response to Ang II was modestly decreased in p47 / mice. Consistent with these observations and the results by Ni and colleagues,6 p47 induction is essential for aortic ROS generation and vascular inflammation in Ang II-infusion, whereas it is not required for the hypertensive response. Because Ets-1 is a critical transcriptional regulator for p47, it is a potential therapeutic target for vascular inflammation. Second, this study showed the importance of transcriptional regulation of the NAD(P)H oxidase. The authors clearly show the specific induction of p47 by Ets-1 without affecting the expressions of other subunits by Ang II. AP-1 was shown to regulate the expression of p67 in The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association. From the Department of Biochemistry and Integrative Medical Biology, School of Medicine, Keio University, Tokyo, Japan. Correspondence to Takeshi Adachi, MD, PhD, Department of Biochemistry and Integrative Medical Biology, School of Medicine, Keio University, Research Park 4N8, 35 Shinanomachi Shinjuku-ku, Tokyo Japan 160-8582. E-mail [email protected] (Circ Res. 2007;101:962-964.) © 2007 American Heart Association, Inc.