Hypoxia-dependent regulation of nonphagocytic NADPH oxidase subunit NOX4 in the pulmonary vasculature.

ISSN: 1524-4571
Copyright © 2007 American Heart Association. All rights reserved. Print ISSN: 0009-7330. Online
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Circulation Research is published by the American Heart Association. 7272 Greenville Avenue, Dallas,
DOI: 10.1161/CIRCRESAHA.107.148015
published online Jun 21, 2007; Circ. Res.
Norbert Weissmann
andFink, Jörg Hänze, Werner Seeger, Friedrich Grimminger, Harald H.H.W. Schmidt
Kwapiszewska, Wolfgang Kummer, Walter Klepetko, Mir Ali Reza Hoda, Ludger
Anne-Christin Selbitz, Ralph Theo Schermuly, Hossein Ardeschir Ghofrani, Grazyna
Manish Mittal, Markus Roth, Peter König, Simone Hofmann, Eva Dony, Parag Goyal,
NOX4 in the Pulmonary Vasculature
Hypoxia-Dependent Regulation of Nonphagocytic NADPH Oxidase Subunit
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Hypoxia-Dependent Regulation of Nonphagocytic NADPH
Oxidase Subunit NOX4 in the Pulmonary Vasculature
Manish Mittal, Markus Roth, Peter Ko¨nig, Simone Hofmann, Eva Dony, Parag Goyal,
Anne-Christin Selbitz, Ralph Theo Schermuly, Hossein Ardeschir Ghofrani, Grazyna Kwapiszewska,
Wolfgang Kummer, Walter Klepetko, Mir Ali Reza Hoda, Ludger Fink, Jo¨rg Ha¨nze, Werner Seeger,
Friedrich Grimminger, Harald H.H.W. Schmidt, Norbert Weissmann
Abstract—Nonphagocytic NADPH oxidases have recently been suggested to play a major role in the regulation of
physiological and pathophysiological processes, in particular, hypertrophy, remodeling, and angiogenesis in the
systemic circulation. Moreover, NADPH oxidases have been suggested to serve as oxygen sensors in the lung. Chronic
hypoxia induces vascular remodeling with medial hypertrophy leading to the development of pulmonary hypertension.
We screened lung tissue for the expression of NADPH oxidase subunits. NOX1, NOXA1, NOXO1, p22phox, p47phox,
p40phox, p67phox, NOX2, and NOX4 were present in mouse lung tissue. Comparing mice maintained for 21 days under
hypoxic (10% O2) or normoxic (21% O2) conditions, an upregulation exclusively of NOX4 mRNA was observed under
hypoxia in homogenized lung tissue, concomitant with increased levels in microdissected pulmonary arterial vessels. In
situ hybridization and immunohistological staining for NOX4 in mouse lungs revealed a localization of NOX4 mRNA
and protein predominantly in the media of small pulmonary arteries, with increased labeling intensities after chronic
exposure to hypoxia. In isolated pulmonary arterial smooth muscle cells (PASMCs), NOX4 was localized primarily to
the perinuclear space and its expression levels were increased after exposure to hypoxia. Treatment of PASMCs with
siRNA directed against NOX4 decreased NOX4 mRNA levels and reduced PASMC proliferation as well as generation
of reactive oxygen species. In lungs from patients with idiopathic pulmonary arterial hypertension (IPAH), expression
levels of NOX4, which was localized in PASMCs, were 2.5-fold upregulated. These results support an important role
for NOX4 in the vascular remodeling associated with development of pulmonary hypertension. (Circ Res.
2007;101:000-000.)
Key Words: hypoxia
�
hypoxic pulmonary vasoconstriction
�
NADPH oxidase
�
pulmonary hypertension
�
vascular smooth muscle cell proliferation
The NADPH oxidases are superoxide-generating enzymesthat release superoxide by electron transfer from
NADPH to oxygen. The classical leukocyte NADPH oxidase
plays an important role in host defense against bacterial and
fungal pathogens.1,2 This phagocytic type of NADPH oxidase
consists of 2 membrane-bound subunits, gp91phox and p22phox
which form the flavocytochrome b558 complex, together with
the cyctosolic subunits p40phox, p47phox, and p67phox. Superox-
ide production by this complex is induced by assembly of the
cytosolic and membrane-bound subunits. Such an assembly
can be induced by the phosphorylation of p47phox.3 Rac
GTPases are also involved in this activation process. Re-
cently, several additional isoforms of the membrane-bound
subunit gp91phox have been described. The first described
homolog of gp91phox, called mox1 (later NOX1), is primarily
expressed in the colon and is suggested to be involved in
mitogenic activity.4 Additional homologs, including NOX3,
NOX4 (Renox), NOX5, Duox1, and Duox2, were subse-
quently described.5–8 According to this new nomenclature,
gp91phox is synonymous with NOX2. It was suggested that
each of these homologs can replace gp91phox in the NADPH
oxidase complex, and it has been demonstrated that these
nonphagocytic NADPH oxidases release lower amounts of
superoxide.9,10 However, very recently 2 new isoforms of the
cytosolic subunits p47phox and p67phox have been identified.
These new subunits, NOXO1 and NOXA1, have been dem-
onstrated to interact with NOX1 to generate significant
amounts of superoxide without being activated by protein
kinase C–dependent phosphorylation.11,12 Isoforms of
gp91phox have been identified in different organs and cell
Original received January 8, 2007; revision received April 30, 2007; accepted June 8, 2007.
From the University of Giessen Lung Center (M.M., M.R., S.H., E.D., P.G., A.-C.S., R.T.S., H.A.G., J.H., S.W., F.G., N.W.), Medical Clinic II/V, the
Department of Anatomy and Cell Biology (P.K., W.K.), and the Department of Pathology (G.K., L.F.), Justus-Liebig-University, Giessen, Germany; the
Department of Cardiothoracic Surgery (W.K., M.A.R.H.), University of Vienna, Austria; and the Department of Pharmacology (H.H.H.W.S.), Monash
University, Melbourne, Australia. Present address for P.K.: Institut fu¨r Anatomie, Universita¨t zu Lu¨beck, Germany.
Correspondence to Norbert Weissmann, PhD, University of Giessen Lung Center, Medical Clinic II/V, Klinikstr. 36, D-35392 Giessen, Germany.
E-mail Norbert.Weissmann@uglc.de
© 2007 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.107.148015
1
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types, including the colon, kidney, uterus, testis, liver, vas-
cular smooth muscle cells, fibroblasts, endothelial cells,
pancreatic islets, and lymphocytes.1,3,9,13–15 The NOX2 ho-
mologs have been suggested to be associated with the
development of atherosclerosis, systemic hypertension, dia-
betic vascular disease, and diseases of the brain.16 The 2
vascular isoforms, NOX1 and NOX4, are thought to play a
role in vascular pathology.17,18 However, with respect to the
lung, relatively few reports of expression and regulation of
the recently-identified new vascular NADPH oxidase sub-
units exist. Hoidal and coworkers19 have demonstrated that
NOX4 is the predominant homolog in human airway and
pulmonary artery smooth muscle cells.20 In addition Hohler et
al identified a low output NADPH oxidase in pulmonary
Figure 1. RT-PCR screening for NADPH oxidase subunits in various mouse organs, and real time PCR quantification of NADPH oxi-
dase subunits in homogenized lungs after 3 days and 3 weeks of hypoxia. a, Analysis by RT-RCR of RNA extracts from homogenized
tissue. An ethidium bromide-stained gel is illustrated. b and c, Real-time PCR quantification of NADPH oxidase subunits in the lung ho-
mogenate after 3 days (duplicate measurements from 3 independent lungs, b) or 3 weeks (duplicate measurements from 5 independent
lungs, c) of hypoxia. *Significant difference compared with normoxic controls; boxes, percentiles 25 and 75; black bar, median; whis-
kers, percentiles 0 and 100; O�value is more than 1.5 lengths of a box away from the edge of a box.
2 Circulation Research August 3, 2007
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artery endothelial cells.21 Against this background, we
screened the lung for expression of the new NADPH oxidase
isoforms. We sought to assess the regulation of the various
isoforms, including the classical phagocytic NADPH oxidase
subunits by hypoxia because (1) NADPH oxidases recently
have been proposed as possible pulmonary oxygen sensors
for the acute response to lung alveolar hypoxia (hypoxic
pulmonary vasoconstriction), (2) reactive oxygen species are
thought to play a role in the vascular remodeling that occurs
during chronic alveolar hypoxia,22,23 and (3) the phagocytic
NADPH oxidase subunit NOX2 has recently been suggested
to play an important role in hypoxia-induced pulmonary
hypertension.24 The hypoxia-induced vascular remodeling
process is characterized by hypertrophy and de novo muscu-
larization of the vessel media, leading to a decrease in
vascular luminal area, increased vascular resistance, and thus
development of pulmonary hypertension and right ventricular
hypertrophy.25 In essence, we found that NOX4 is the only
subunit prominently upregulated in pulmonary arterial ves-
sels and in smooth muscle cells during chronic hypoxia, both
at the transcriptional and protein level. Cell culture experi-
ments demonstrated a proproliferative activity of NOX4
during hypoxia, because targeted knock-down of NOX4 with
siRNA suppressed pulmonary arterial smooth muscle cell
(PASMC) proliferation. Most interestingly, NOX4 expres-
sion was upregulated in the vessel media of lungs from
patients with idiopathic pulmonary hypertension (IPAH), in
comparison to lungs from healthy donors, suggesting an
important role of this NADPH oxidase subunit in human
IPAH.
Materials and Methods
Chronic Hypoxia Exposure
All animal experiments were approved by local authorities. Mice
(C57BL/6N) of either sex (Charles River Laboratories, Sulzfeld,
Germany; 20 to 22 g) were exposed to normobaric hypoxia [inspira-
tory O2 fraction (FiO2) 0.10] in a ventilated chamber for up to 3
weeks as described previously.26
Figure 2. NOX4 and NOX2 mRNA quantification of microdissected pulmonary arteries by real time PCR during development of hypox-
ia-induced pulmonary hypertension. a and b, Microdissected small pulmonary arteries (�100-�m diameter) from cryosections of mouse
lungs were used for the quantification of NOX4 and NOX2 mRNA. Mice were maintained under normoxic or hypoxic conditions for up
to 21 days. The NOX2 and NOX4 mRNA levels were quantified by real-time PCR normalized to �2-microglobulin mRNA levels. c, Delta
Ct values of NOX4 and NOX2 from microdissected mouse pulmonary arteries of normoxic mice. Values are duplicate measurements of
n�16 vessels from n�3 lungs each. d, Right ventricular hypertrophy after exposure of mice to chronic hypoxia. The right ventricle (RV)
to left ventricle (LV) � septum (S) ratio was quantified from mouse hearts after exposure to chronic hypoxia (10% O2 for 3, 7, and 21
days, respectively, n�5). *Significant differences as compared with normoxic controls.
Mittal et al Regulation of NOX4 in the Pulmonary Vasculature 3
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Mouse Lung Preparation for Laser Assisted
Microdissection and Right Heart
Hypertrophy Assessment
Mouse lungs were prepared as described previously.27 For details see
the supplemental materials (available online at http://circres.
ahajournals.org).
Laser-Assisted Microdissection
Laser-microdissection was performed as described previously.27
RNA-Extraction and RT-PCR
The RNA was extracted from cells using guanidine thiocyanate-acid
phenol (RNAzol B, WAK-Chemie, Germany) or with spin-columns
(RNeasy, Qiagen, Germany). For details see the supplemental
materials.
Real-Time PCR
Relative quantification of the NADPH oxidase subunits was done
using ABI prism 7700 detection system (Applied Biosystem, Weit-
erstadt, Germany). For details please refer to the supplemental
materials.
In Situ Hybridization
For a detailed description of the in situ hybridization protocol please
refer to the supplemental materials.
Immunohistochemistry for Mouse Lung Sections
Immunohistochemistry was performed as described previously.26
For details see the supplemental materials.
Immunohistochemistry for NOX4 and NOX2 in
Human Lung Sections
Lung tissue samples from healthy individuals and from patients with
IPAH were formalin-fixed, paraffin-embedded, and cut into 3-�m
sections. The immunostaining of the human lung sections was
performed with a custom-made rabbit anti-human NOX4 polyclonal
antibody29 or rabbit anti-human NOX2 polyclonal antibody (Upstate,
Germany) as previously described.26,28
Western Blot of NOX4 in Frozen Human
Lung Tissue
For the detection of NOX4 by Western blot, a custom-made
polyclonal anti-NOX4 antibody raised in rabbits was used.28 For
details see the online supplemental materials.
Cell Culture
Smooth muscle cells from human and murine pulmonary arteries
were isolated and cultured as described previously.29,30 For the
investigation of the effect of hypoxia on NOX4 mRNA levels, cells
were either exposed to 1% O2 (hypoxia) or to 21% O2 (normoxia).
Immunocytochemistry of Murine PASMCs
Isolated PASMCs were cultured on chamber slides, treated as
indicated, fixed in ice cold acetone and methanol (1:1), and blocked
with 3% (m/v) BSA in PBS for 1 hour, followed by overnight
incubation with an anti-NOX4 antibody (1:25) diluted in 3% (m/v)
BSA in PBS.28 Indirect immunofluorescence was obtained by
incubation with a Cy3-conjugated anti-goat antibody (Dako, Den-
mark) diluted 1:100 in PBS for 90 minutes. Nuclear counterstaining
was performed with Hoechst-33258 (1:10 000 dilution in PBS;
Invitrogen, Karlsruhe, Germany) for 10 minutes.
RNA Interference and Proliferation Assay
A detailed description of the siRNA transfection and the proliferation
assay is available in the supplemental materials.
Statistics
Values are given as mean�SEM if not indicated differently. For
statistical analysis a Student t test was used for comparison of 2
groups. For more than 2 groups, ANOVA with LSD posthoc test was
performed. A probability value of less then 0.05 was considered
significant. Empirical assessment of NOX4 immunoreactive vessels
was performed in blinded fashion. Two conditions were evaluated
for assessment of NOX4-immunoreactive vessels: first, the number
of NOX4-immunoreactive vessels different between the groups, and
second, the mean diameter of NOX4-immunoreactive vessels differ-
ent between the groups. Statistical analysis was performed by a
nonparametric variance analysis (Kruskal-Wallis test). If the proba-
bility value in that test was �0.05, a comparison of the groups
between each other was performed using a Mann-Whitney test,
where P�0.05 was regarded as significant. Comparison of groups
was stopped after P�0.05 to prevent �-inflation.
Results
To investigate the role of NADPH oxidases in the development
of hypoxia-induced pulmonary hypertension, expression of the
NADPH-oxidase subunits NOX1, NOX2, NOX4, p22phox,
Figure 3. Localization of NOX4 in mouse lung sections by nonisotopic in situ hybridization (NISH). a and b, Hybridization of the NOX4
antisense probe to mouse lung cryosections (green fluorescence). c and d, The same sections stained with a Cy3-labeled antibody
directed against �-smooth muscle actin (SMA, red fluorescence). e and f, Overlay of the images a and b depicting predominant colo-
calization of NOX4 transcripts with SMA in the smooth muscle cell layer of the pulmonary artery (yellow fluorescence). B indicates
bronchus; PA, pulmonary artery.
4 Circulation Research August 3, 2007
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