Post-stroke inhibition of induced NADPH oxidase type 4 prevents oxidative stress and neurodegeneration.

Post-Stroke Inhibition of Induced NADPH Oxidase Type 4
Prevents Oxidative Stress and Neurodegeneration
Christoph Kleinschnitz1*, Henrike Grund2, Kirstin Wingler2,3,4,5, Melanie E. Armitage3,5, Emma Jones3,
Manish Mittal2, David Barit6, Tobias Schwarz1, Christian Geis1, Peter Kraft1, Konstanze Barthel7,
Michael K. Schuhmann1,8, Alexander M. Herrmann1,8, Sven G. Meuth1,8, Guido Stoll1, Sabine Meurer3,
Anja Schrewe9, Lore Becker9,10, Vale´rie Gailus-Durner9, Helmut Fuchs9, Thomas Klopstock10, Martin
Hrabe´ de Angelis9,11, Karin Jandeleit-Dahm6, Ajay M. Shah12, Norbert Weissmann2, Harald H. H. W.
Schmidt2,3,4,5*
1Neurologische Klinik und Poliklinik, Universita¨t Wu¨rzburg, Wu¨rzburg, Germany, 2 Rudolf-Buchheim-Institut fu¨r Pharmakologie & Medizinische Klinik, Justus-Liebig-
Universita¨t, Gießen, Germany, 3Department of Pharmacology and Centre for Vascular Health, Monash University, Melbourne, Australia, 4Department of Pharmacology
and Toxicology and Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, The Netherlands, 5National Stroke Research Institute, Florey
Neuroscience Institutes, Melbourne, Australia, 6 Baker IDI Heart and Diabetes Institute, Juvenile Diabetes Research Foundation (JDRF) International Center for Diabetic
Complications Research, Melbourne, Australia, 7Abteilung Neurologie, Georg-August Universita¨t Go¨ttingen, Go¨ttingen, Germany, 8Universita¨tsklinik Mu¨nster, Klinik und
Poliklinik fu¨r Neurologie—Entzu¨ndliche Erkrankungen des Nervensystems und Neuroonkologie, Mu¨nster, Germany, 9 Institute of Experimental Genetics, Helmholtz
Zentrum Mu¨nchen, German Research Center for Environmental Health, Mu¨nchen, Germany, 10 Friedrich-Baur-Institut an der Neurologischen Klinik, Klinikum der Ludwig-
Maximilians-Universita¨t Mu¨nchen, Mu¨nchen, Germany, 11 Lehrstuhl fu¨r Experimentelle Genetik, Technische Universita¨t Mu¨nchen, Freising-Weihenstephan, Germany,
12 King’s College London School of Medicine, The James Black Centre, Cardiovascular Division, London, United Kingdom
Abstract
Ischemic stroke is the second leading cause of death worldwide. Only one moderately effective therapy exists, albeit with
contraindications that exclude 90% of the patients. This medical need contrasts with a high failure rate of more than 1,000
pre-clinical drug candidates for stroke therapies. Thus, there is a need for translatable mechanisms of neuroprotection and
more rigid thresholds of relevance in pre-clinical stroke models. One such candidate mechanism is oxidative stress.
However, antioxidant approaches have failed in clinical trials, and the significant sources of oxidative stress in stroke are
unknown. We here identify NADPH oxidase type 4 (NOX4) as a major source of oxidative stress and an effective therapeutic
target in acute stroke. Upon ischemia, NOX4 was induced in human and mouse brain. Mice deficient in NOX4 (Nox42/2) of
either sex, but not those deficient for NOX1 or NOX2, were largely protected from oxidative stress, blood-brain-barrier
leakage, and neuronal apoptosis, after both transient and permanent cerebral ischemia. This effect was independent of age,
as elderly mice were equally protected. Restoration of oxidative stress reversed the stroke-protective phenotype in Nox42/2
mice. Application of the only validated low-molecular-weight pharmacological NADPH oxidase inhibitor, VAS2870, several
hours after ischemia was as protective as deleting NOX4. The extent of neuroprotection was exceptional, resulting in
significantly improved long-term neurological functions and reduced mortality. NOX4 therefore represents a major source
of oxidative stress and novel class of drug target for stroke therapy.
Citation: Kleinschnitz C, Grund H, Wingler K, Armitage ME, Jones E, et al. (2010) Post-Stroke Inhibition of Induced NADPH Oxidase Type 4 Prevents Oxidative
Stress and Neurodegeneration. PLoS Biol 8(9): e1000479. doi:10.1371/journal.pbio.1000479
Academic Editor: Malcolm McLeod, University of Edinburgh, United Kingdom
Received February 19, 2010; Accepted July 28, 2010; Published September 21, 2010
Copyright: � 2010 Kleinschnitz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the NHMRC, Australia, the Deutsche Forschungsgemeinschaft (DFG), Germany (to HHHWS and CK), and by the
Bundesministerium fur Bildung und Forschung within the framework of the NGFN-Plus and the European Commission (EUMODIC). The funders had no role in
study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: HHHWS and KW declare a potential competing interest as shareholder and previous employee, respectively, of Vasopharm GmbH, which
develops NADPH oxidase inhibitors such as VAS2870. All authors declare that they adhere to all PLoS Biology policies on sharing data and materials as detailed in
the PLoS Biology guide for authors.
Abbreviations: CISS, constructive interference in steady state; KO, knock out; pMCAO, permanent middle cerebral artery occlusion; ROS, reactive oxygen
species; rt-PA, recombinant tissue plasminogen activator; tMCAO, transient middle cerebral artery occlusion; TTC, 2,3,5-triphenyltetrazolium chloride; WT, wild
type.
* E-mail: h.schmidt@farmaco.unimaas.nl (HHHWS); christoph.kleinschnitz@mail.uni-wuerzburg.de (CK)
Introduction
Ischemic stroke has outstanding medical relevance as it is the
second leading cause of death in industrialized countries [1]. Due
to the aging of the population, the incidence of stroke is projected
to rise even further in the future [2]. Despite tremendous research
activity, with more than 100 clinical trials in human stroke patients
[3], only one therapy approved by the United States Food and
Drug Administration is available, i.e., thrombolysis using recom-
binant tissue plasminogen activator (rt-PA). However, the efficacy
of rt-PA on functional outcomes is moderate at best, and more
than 90% of all stroke patients must be excluded from rt-PA
treatment because of over 25 labeled contraindications. Therefore,
there is an unmet need for more effective therapies in acute stroke.
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Although a plethora of drugs for the treatment of acute stroke
are effective in animal models, their translation into clinical
practice has completely failed [3,4]. As a result, many pharma-
ceutical companies have withdrawn from drug discovery in this
area. To overcome this lack of clinically effective neuroprotective
drugs, innovative strategies are urgently needed to identify
pathways that can be targeted with innovative therapies [5].
Higher quality study designs are also required [6,7].
One such high-potential pathway in ischemic stroke may be the
occurrence of oxidative stress, i.e., the increased occurrence of
reactive oxygen species (ROS) above physiological levels. Oxida-
tive stress has been suggested for many years to cause tissue
damage and neuronal death. The toxicity of ROS can be further
increased by nitric oxide to produce reactive nitrogen species such
as peroxynitrite (ONOO2), a molecule that causes oxidation and
nitration of tyrosine residues on proteins [8]. Disappointingly,
there is no conclusive evidence of a causal link between oxidative
stress and the development of disease, and there is no successful
therapeutic application targeting oxidative stress. To date, clinical
attempts to scavenge ROS by applying antioxidants did not result
in clinical benefit [9] or even caused harm [10,11]. However, the
characterization of the relevant enzymatic sources of oxidative
stress may allow therapeutic targeting of oxidative stress by
preventing the formation of ROS in the first place, instead of
scavenging ROS after they have been formed.
A potential source of ROS are NADPH oxidases, the only
known enzyme family that is only dedicated to ROS production
[12]. Four rodent genes of the catalytic subunit NOX, Nox1, Nox2,
Nox3, and Nox4, have been identified, of which Nox1, Nox2, and
Nox4 are expressed in the vasculature. NOX4 is the most
abundant vascular isoform; its expression is even higher in
cerebral than in peripheral blood vessels [13] and, further,
induced in stroke [14]. Therefore, we hypothesized that NOX4 is
the most relevant source of ROS in stroke.
To test this hypothesis, we generated constitutively NOX4-
deficient (Nox42/2) mice and directly compared them to NOX1-
deficient (Nox1y/2) and NOX2-deficient (Nox2y/2) mice. NOX4
has been implicated in the regulation of systemic and hypoxic
vascular responses. Therefore, we had to exclude systemic vascular
effects of NOX4 deletion on blood pressure, which may affect
stroke outcome independent of a specific neuronal or neurovas-
cular mechanism. Finally, to examine the therapeutic potential of
NOX4 as a drug target, we infused the specific NADPH oxidase
inhibitor VAS2870 [15] after ischemia, thus mirroring the clinical
scenario.
Results
NOX4 Is Induced during Ischemic Stroke in Mice and
Humans
Because NOX4 mRNA is expressed at higher levels in cerebral
than in peripheral blood vessels [13] and is induced in stroke [14],
we first sought to validate these data not only at the mRNA but
also at the protein level. In all experiments, we followed current
guidelines defining methodological standards for experimental
stroke studies [4,6,7,16,17]. Here we chose a model of acute
ischemic stroke in which mice are subjected to transient middle
cerebral artery occlusion (tMCAO). This disease model is thought
to involve oxidative stress and an induction of Nox4 expression
[18]. Indeed, expression of NOX4 mRNA was significantly higher
12 h and 24 h after tMCAO in the basal ganglia and neocortex of
wild-type mice than in sham-operated controls, in which basal
NOX4 expression was low (Figure 1A). This result was validated
by immunohistochemistry using a specific NOX4 antibody. We
detected a stronger staining in neurons and cerebral blood vessels
in wild-type mice subjected to tMCAO than in sham-operated
controls. Although immunohistochemistry is not quantitative, this
finding suggests higher levels of NOX4 protein (Figure 1B).
Importantly, NOX4 staining was also stronger in brain samples
from stroke patients. Although NOX4 was barely detectable in
healthy brain regions, clear positive labeling of NOX4 was seen in
neurons and vascular endothelial cells from the forebrain cortex of
stroke patients. This finding was confirmed by double labeling for
NeuN (a neuronal marker) or von Willebrand factor (an
endothelial marker) and NOX4 in brain tissue (Figure 1B). These
data indicate that NOX4 protein is induced during brain ischemia
in mice, and this observation would be in agreement with a major
functional role for NOX4 in ischemic stroke. Our limited
observations in a small number of human cases provide some
support to the hypothesis that these processes are also important in
human stroke.
Nox42/2 but Neither Nox1y/2 nor Nox2y/2 Mice Are
Protected in Both Transient and Permanent Ischemic
Stroke
We first subjected 6- to 8-wk-old male Nox42/2 mice to tMCAO
and, after 24 h, assessed infarct volumes by staining brain sections
with 2,3,5-triphenyltetrazolium chloride (TTC) (Figure 2A). Infarct
volumes were significantly smaller, by approximately 75%, in male
Nox42/2 mice than in sex-matched wild-type controls
(25.5614.8 mm3 versus 78.7619.5 mm3, respectively). The smaller
infarct volume was functionally relevant: compared with wild-type
mice, Nox42/2 mice had significantly better overall neurological
function (Bederson score 1.260.7 in Nox42/2 mice versus 3.761.1
in wild-type mice) as well as better basal motor function and
coordination (grip test score 4.360.7 in Nox42/2 mice versus
1.761.3 in wild-type mice) 24 h after tMCAO (Figure 2B). Gender
can significantly influence stroke outcome in rodents [4,16,17].
Therefore, we also subjected female Nox42/2 mice to 60 min of
tMCAO. In line with the results in male mice, Nox4-deficient
female mice also developed significantly smaller infarctions
Author Summary
Stroke is the second leading cause of death worldwide.
Today, only one approved therapy exists—a drug that
breaks down blood clots—the effectiveness of which is
moderate, and it can only be used in about 10% of
patients because of contraindications. New therapeutic
strategies that are translatable to humans and more rigid
thresholds of relevance in pre-clinical stroke models are
needed. One candidate mechanism is oxidative stress,
which is the damage caused by reactive oxygen species
(ROS). Antioxidant approaches that specifically target ROS
have thus far failed in clinical trials. For a more effective
approach, we focus here on targeting ROS at its source by
investigating an enzyme involved in generating ROS,
known as NADPH oxidase type 4, or NOX4. We found that
NOX4 causes oxidative stress and death of nerve cells after
a stroke. Deletion of the NOX4-coding gene in mice, as
well as inhibiting the ROS-generating activity of NOX with
a pharmacological inhibitor, reduces brain damage and
improves neurological function, even when given hours
after a stroke. Importantly, neuroprotection was preserved
in old male and female Nox42/2 mice as well as in Nox42/2
mice subjected to permanent ischemia. NOX4 thus
represents a most promising new therapeutic target for
reducing oxidative stress in general, and in brain injury due
to stroke in particular.
Role of NOX4 in Stroke
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Role of NOX4 in Stroke
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(30.166.7 mm3 versus 89.5622.2 mm3, respectively) and less
severe neurological deficits (p,0.001) than female controls
(Figure 2A and 2B). Histological analysis revealed that all infarcts
in Nox42/2 mice were restricted to the basal ganglia (arrow in
Figure 2A and 2C), whereas in wild-type mice, the neocortex was
also consistently affected. Serial magnetic resonance imaging of
living mice up to 6 d after stroke showed that in Nox42/2 mice the
infarct volume did not increase over time, thus indicating that
deletion of the Nox4 gene provides sustained protection against
stroke (Figure 2C). Moreover, infarcts always appeared hyperin-
tense on blood-sensitive constructive interference in steady state
(CISS) sequences. Hypointense areas, which typically indicate
intracerebral hemorrhage, were absent from Nox42/2 mice and
wild-type controls. This finding excludes the possibility of an
increased rate of bleeding complications caused by Nox4 deficiency.
To establish any potential specificity of this function for NOX4
compared to NOX1 and NOX2 in stroke, we carried out identical
experiments in 6- to 8-wk-old Nox1y/2 and Nox2y/2 mice.
However, in contrast to Nox42/2 mice, we observed no protection
in these animals, neither in terms of infarct volumes nor on
functional outcomes on day 1 after tMCAO, even with large
subject sample sizes (n=19 for Nox2y/2 mice, p.0.05; Figure 2A).
Ischemic stroke is usually a disease of the elderly and,
consequently, one should verify any stroke-protective effects
observed in young adult laboratory animals also in an older
cohort [4,16,17]. Indeed, 18- to 20-wk-old Nox42/2 mice also
developed significantly smaller brain infarctions (27.8615.1 mm3
versus 81.8619.0 mm3, respectively) and less severe neurological
deficits than age-matched controls, thereby confirming our results
in young animals (Figure 2B). We also determined the functional
outcome and mortality of 6- to 8-wk-old male Nox42/2 mice and
matched wild-type controls over a longer time period after
ischemic stroke (Figure 2D). Five days after 60 min of tMCAO, 15
of 15 wild-type mice (100%) had died, which is in line with
previous reports [19]. In contrast, seven of ten Nox42/2 mice
(70%) survived until day 5, and five of these were still alive after
1 wk (p=0.0039) (Figure 2D). In line with these findings, Nox4-
deficient mice showed significantly better Bederson scores than
controls over the whole observation period, and neurological
deficits remained low until day 7 (Figures 2D and S4).
According to the current experimental stroke guidelines
[4,16,17], any protective effect also requires evaluation in models
of both transient and permanent ischemia. We therefore subjected
Nox42/2 mice to filament-induced permanent middle cerebral
artery occlusion (pMCAO), a procedure in which no tissue
reperfusion occurs (Figure 2E). In the absence of Nox4, infarct
volumes (66.7628.6 mm3 versus 120.1615.6 mm3, p,0.05) and
neurological deficits (Bederson score 2.361.7 versus 3.460.8,
p,0.05) at day 1 after pMCAO were significantly reduced
compared with those in wild-type controls, although to a lesser
extent than they were in the tMCAO model (Figures 2E and S5).
Brain infarctions following filament-induced pMCAO are large,
and the infarct borders are often not very well defined, which
limits the accuracy of any estimation on infarct volumes. We
therefore used another model of permanent stroke, cortical
photothrombosis, to further verify our findings. Here, the lesions
are restricted to the cortex and highly reproducible in size and
location. Moreover, photothrombosis has been shown to induce
early and profound ROS formation and blood-brain-barrier
leakage [20,21], two key readout parameters of the present
investigation. Importantly, photothrombosis-induced infarct vol-
umes were as reduced in Nox42/2 mice relative to wild-type mice
(3.364.6 mm3 versus 25.0612.8 mm3, respectively, a difference
of 86.8%; Figure 2F) as they were in the tMCAO model.
No Apparent Vascular Phenotype of Nox42/2 Mice Other
Than in Stroke
Based on the physiological distribution of NOX4 in kidney [22],
lung [23], and aorta [24], as well as cell biology data obtained
using small interfering RNA approaches [23], one would predict
basal phenotypes in a Nox42/2 mouse, such as arterial
hypotension, reduced hypoxic pulmonary hypertension, and
altered renal function. Importantly, these effects could potentially
modulate or interfere with stroke outcome even in the absence of a
specific neuronal or neurovascular mechanism. Surprisingly,
systemic elimination of Nox4 did not result in any apparent
abnormal vascular phenotype (Text S1; Figures S1 and S2; Table
S1). In particular, blood pressure was normal, and hypoxic
pulmonary hypertension still occurred despite a 20-fold induction
of NOX4 in wild-type animals [23]. In contrast, Nox1- and
p47phox-deficient mice (a Nox2 subunit) have a lower basal blood
pressure, and their blood-pressure response to angiotensin II is
reduced [25–27]. Our data suggest that any phenotype caused by
deleting Nox4, unlike those caused by deleting Nox1 and Nox2,
would indeed be brain-specific.
Protection from Ischemic Stroke in Nox42/2 Mice Is a
Result of Reduced Oxidative Stress, Neuronal Apoptosis,
and Blood-Brain-Barrier Leakage
Next we sought to elucidate the underlying mechanisms of this
NOX4-specific neurotoxicity in stroke. NOX4 can form superox-
ide or H2O2, which can interact with nitric oxide to form reactive
nitrogen species. Therefore, we stained brain sections with broad-
spectrum indicators of oxidative/nitrative stress, i.e., dihydroethi-
dium [28] and nitrotyrosine [8]. At 12 h and 24 h after tMCAO,
brains from wild-type mice exhibited a significantly larger amount
(by a factor of 2.5–3.5) of ROS in neurons than brains from sham-
operated animals, as quantified by dihydroethidium staining
(Figure 3A). Neurons from Nox42/2 mice, in contrast, showed
only very small ischemia-induced increases in ROS relative to
those in sham-operated controls (p.0.05). ROS formation from
neurons after 24 h was also significantly reduced in Nox42/2 mice
subjected to pMCAO (Figure S6). Because the dihydroethidium
stain may also indicate oxidative chemistry events, including
formation of ONOO2 and nitration of protein tyrosine residues
[8], we analyzed the extent of protein nitration in Nox42/2 and
wild-type mice subjected to tMCAO. In agreement with our
findings on the generation of ROS, tissue nitration occurred to a
lesser extent in ischemic brains from Nox42/2 mice than in those
from wild-type controls (Figure 3B). Oxidative chemistry events
such as the formation of ROS and peroxynitrite, as detected by
Figure 1. Induction of NOX4 expression after ischemic stroke in mice and humans. (A) Relative gene expression of Nox4 in the ischemic
basal ganglia (left) and cortex (right) of wild-type mice after sham operation and 4 h, 12 h, and 24 h after tMCAO (n= 5). *, p,0.05, one-way ANOVA,
Bonferroni post-hoc test, compared with sham-treated controls. (B) Immunohistochemical detection of NOX4 protein in ischemic brains of wild-type
mice (after sham operation or tMCAO, day 1) and humans (samples from stoke patients, after routine autopsy). We compared NOX4 immunolabeling
in the ischemic forebrain cortex and the unaffected contralateral side. In ischemic samples, NOX4 was predominantly expressed in neurons
(arrowheads) and endothelial cells (arrows). This distribution was confirmed by visualization of NOX4 and NeuN or NOX4 and von Willebrand Factor in
the same structures. All scale bars represent 100 mm.
doi:10.1371/journal.pbio.1000479.g001
Role of NOX4 in Stroke
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Role of NOX4 in Stroke
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