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
E
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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.
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 endothelialderived 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 oxidasederived ROS in signaling in vascular cells have received
considerable attention. Ang II via AT1 receptors may stimu-
vidence 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/
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 [email protected]
(Arterioscler Thromb Vasc Biol. 2003;23:707-709.)
© 2003 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
DOI: 10.1161/01.ATV.0000069907.12357.7E
707
708
Arterioscler Thromb Vasc Biol.
May 2003
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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 䡠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 䡠O2⫺ and consequent decreased inflammation,
cell growth and vascular injury (B). 1, increase effect; 2, decrease effect.
late PKC, PLD, or Src21,22 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 gp91dstat. 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.
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Inflammation and Vascular Hypertrophy Induced by Angiotensin II: Role of NADPH
Oxidase-Derived Reactive Oxygen Species Independently of Blood Pressure Elevation?
Ernesto L. Schiffrin and Rhian M. Touyz
Arterioscler Thromb Vasc Biol. 2003;23:707-709
doi: 10.1161/01.ATV.0000069907.12357.7E
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